Note: Descriptions are shown in the official language in which they were submitted.
MODULATING PLANT ABIOTIC STRESS RESPONSES USING THE KANGHAN
GENE FAMILY
Field of the Invention
The present invention relates to abiotic stress-resistant plants and processes
for obtaining
them, including flowering plants and seeds thereof.
Background of the Invention
Abiotic stress is a major challenge facing the agricultural industry (see Yang
et at.,
Molecular Plant, Volume 3, Issue 3, May 2010, Pages 469-490). Abiotic stresses
such as
drought and heat not only cause a reduction in crop yield, but also cause high
variation in
crop yield. Improving crop tolerance to abiotic stresses such as heat and
drought is
essential for maintaining a stable yield under the continued threat of climate
change. It is
also a key factor for sustaining and expanding arable land areas for crop
production.
Plants have evolved various mechanisms to cope with abiotic stress at both the
physiological and biochemical levels. Many stress-induced genes have been
identified,
including those encoding key enzymes for abscisic acid (ABA) biosynthesis and
signaling
transduction components such as protein kinases, protein phosphatases and
transcription
factors. In recent years, several stress-regulated miRNAs have also been
identified in
model plants under biotic and abiotic stress conditions. Plants respond
differently to
drought and heat stress (Rizhsky et al., Plant Physiology, April 2004, Vol.
134, pp. 1683-
1696).
Summary
Methods are provided for modulating an abiotic stress response to drought or
heat in a
plant, for example by introducing a heritable change to the plant, which
alters the
expression in the plant of an endogenous or exogenous Kanghan protein.
Similarly, plants
and plants cells having such heritable changes are provided.
Plants having enhanced drought tolerance are accordingly provided, for example
by
altering selected quantitative trait loci (QTL) associated with the family of
Kanghan genes.
Suppression of Kanghan genes, for example in null mutations, confers drought
tolerance.
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Methods are accordingly provided for modulating an abiotic stress response to
drought in
a plant, comprising introducing a heritable change to the plant which alters
the expression
in the plant of an endogenous or exogenous Kanghan protein. The Kanghan
protein may
for example be at least 35% identical to, or at least 49% positively aligned
with, a protein
encoded by the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID
NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and/or SEQ ID NO: 13; and this alignment
may for
example be over an alignment length of at least 90 amino acids, with BLOSUM or
PAM
substitution matrix, with gaps permitted. Alternative degrees of sequence
similarity are
contemplated in alternative embodiments, for example 50%, 75%, 90% or 95%
identical
to, or at least 75%, 90% or 100% positively aligned with, the protein encoded
by the
nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ
ID
NO: 4, SEQ ID NO: 5, or SEQ ID NO: 13; over an alignment length of at least
90, 100 or
110 amino acids, with BLOSUM or PAM substitution matrix, and with gaps
permitted.
The Kanghan protein includes a variety conserved domains, such as domains:
identical to
hTVKDChphAhp (SEQ ID NO: 6); and/or, at least 80% identical to LTVKDCLEhAhK-
G (SEQ ID NO: 7); and/or, at least 70% identical to LTVKDCLEhAFKKG (SEQ ID NO:
8); and/or at least 80% identical to VshKGpVlEstshpEs.chhhpQs-huA+LH1FpPph
(SEQ
ID NO: 9); and/or, at least 70% identical to VsMKGEVIEspsh-EAhcL11cQP-
1GA+LH1FoPc1 (SEQ ID NO: 10); and/or, at least 80% identical to
cppDYDtStpAAhVAlpLISSAR1hLK1DuhhTEYSsQaLhDpsutpp (SEQ ID NO: 11);
and/or, at least 70% identical to
spphhpShupscGhCHPDC-
KAssEpEDYDASQpAAhVAVsLISSAR1hLKLDusaTEYSAQYLVDNAGpccs (SEQ ID
NO: 12).
In alternative embodiments, the plant may lack an endogenous Kanghan protein,
such as a
protein that has the sequence characteristics of Kanghan proteins described
above, such as
being at least 35% identical to, or at least 49% positively aligned with, a
protein encoded
by the nucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:
3,
SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 13; over an alignment length of at
least 90
amino acids, with BLOSUM or PAM substitution matrix.
The plant may be an angiosperm, and may for example belong to the family of
Brassicaceae, Fabaceae, Poaceae, or Asteraceae plants. The plant may for
example be a
Caspsella rubella, Brassica rapa, Brassica napus, Brassica carinata, Eutrema
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salsugineum, Thelhigiella parvula, Camelina sativa, Glycine max, Triticum, Zea
maize,
Oryza sativa or Helianthus annuus plant.
The heritable change may be one that sufficiently decreases the expression of
the Kanghan
protein so as to enhance drought tolerance relative to an unmodified plant,
for example
improving drought tolerance by an objective measure by 10% to 100% or more.
The heritable change may for example involve expressing in the plant an
inhibitory
polynucleotide that down-regulates the expression of the Kanghan protein, such
as an
inhibitory RNA, for example an anti-sense oligonucleotide, an RNAi
oligonucleotide, a
microRNA, a small interfering RNA, or a CRISPR guide RNA. Alternatively, the
heritable
change may be an alteration of a Kanghan gene sequence encoding the Kanghan
protein,
for example by transformation with an exogenous Kanghan gene encoding the
exogenous
Kanghan protein, or by editing or mutation of an endogenous Kanghan gene
encoding the
endogenous Kanghan protein. The editing or mutation may for example introduce
a change
to a coding sequence of the Kanghan gene which changes the amino acid sequence
of the
Kanghan protein.
In accordance with the foregoing methods, there are also provided parental
plants or plant
cells that are produced by these processes. Similarly, plant lines, varieties
or cultivars are
provided that include the parental plant or plant cell, and the plant line,
variety or cultivar
may for example be characterized by an improved drought tolerance
characteristic. Seeds
and plant parts are provided, for example from foregoing plant lines,
varieties or cultivars.
Seeds in turn may be used to provide progeny plants, such as progeny plants
that are
genetically derived from the plant line, variety or cultivar so as to retain
the improved
drought tolerance characteristic.
Methods of marker assisted selection may for example be used to introduce the
heritable
change, with subsequent screening of the plant or plant cell or progeny for
the desired
modulation of the abiotic stress response to drought.
A further embodiment is a method for producing a plant having increased
tolerance to heat
stress, comprising introducing into a plant cell an expression construct
comprising a
nucleic acid molecule encoding a polypeptide with at least 80% identity to SEQ
ID NO:
14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and/or SEQ ID
NO: 19 over an alignment length of at least 90 amino acids, operatively linked
to at least
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one regulatory element, said at least one regulatory element being effective
to direct
expression of said nucleic acid molecule in the plant; and growing the plant
cell into the
plant. In another embodiment, the nucleic acid molecule encodes a polypeptide
with at
least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%,
at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least
92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or
100% identity to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,
SEQ
ID NO: 18, and/or SEQ ID NO: 19 over an alignment length of at least 90 amino
acids, at
least 100 amino acids, at least 110 amino acids, or over the full length of
the amino acid
sequence set forth in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO:
17,
SEQ ID NO: 18, or SEQ ID NO: 19. The polypeptide encoded by the nucleic acid
molecule
will preferably have the same biological activity as the polypeptide set forth
in SEQ ID
NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID
NO: 19. In an embodiment, at least one regulatory element comprises a
promoter, for
example a constitutive promoter. In a further embodiment, the regulatory
element is a
regulatory element that is not naturally in operative linkage with the nucleic
acid molecule.
For example, the regulatory element may be a synthetic regulatory element, a
regulatory
element derived from a different species than the nucleic acid molecule, or a
regulatory
element derived from a different gene within the same species as the nucleic
acid molecule.
In an embodiment, the nucleic acid molecule is derived from a different
species than the
plant cell into which the expression construct is introduced. In a further
embodiment, the
nucleic acid molecule is derived from Arabidopsis and the plant cell is a
Triticum cell.
The method may further comprise a step of assessing the heat tolerance of the
plant relative
to a control plant of the same variety or genetic background that does not
comprise the
expression construct and identifying the plant as having increased tolerance
to heat stress
if it exhibits increased heat tolerance relative to the control plant. Tests
for heat tolerance
are known and will be understood by one skilled in the art (for example, see
Kumar et al,
Journal of Plant Biochemistry and Biotechnology, July 2013; Hatfield and
Prueger (2015),
Weather and Climate Extremes 10:4-10). In wheat, heat tolerance may be
assessed by, for
example, subjecting newly germinated seedlings, seedlings, or plants to heat
stress at a
temperature of about 27 or higher of about 30 C or higher (e.g. conditions
such as 36 C,
42/38 C (day/night), or 40/38 C (day/night)) for a period of time (typically
days or weeks,
for example two or three weeks) then allowing them to recover at a standard
growth
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temperature between about 13-25 C (e.g. growth conditions such as 25 C,
25/20
C(day/night), 24/16 C (day/night), or 18/13 C (day/night)) for a period of
time (e.g. 3-
weeks) and then measuring viability or another indicator of heat stress, such
as yield,
biomass, or canopy temperature.
5 Further provided is a plant cell, plant, seed, or plant tissue comprising
an expression
construct as described above. In an embodiment, the plant cell, plant, seed,
or plant tissue
is a Poaceae cell, plant, seed, or tissue. In a further embodiment, the plant
cell, plant, seed,
or plant tissue is a cereal plant cell, plant, seed, or tissue. Cereal plants
include
commercially important grain crops such as rice (Oryza sativa), wheat/spelt
(Triticum),
10 corn/maize (Zea mays), barley (Hordeum vulgare), Sorghum, oat (Avena
sativa), rye
(Secale cereale), and Triticale. In a further embodiment, the plant cell,
plant, seed, or
plant tissue is Triticum.
In accordance with the foregoing methods, there are also provided parental
plants or plant
cells that are produced by these processes. Seeds in turn may be used to
provide progeny
.. plants, such as progeny plants that are genetically derived from the plant
line, variety or
cultivar so as to retain the improved drought tolerance characteristic. Seeds
and plant parts
that are derived from the foregoing plant lines may be characterized by
improved drought
tolerance characteristics, for example they may be subjected to RNAseq
analyses to
identify transcripts that exhibit contrasting differential expression patterns
when compared
.. with their respective wild type controls. The combinatory profile of these
genes can be an
evaluation benchmark for drought tolerance.
Methods of marker assisted selection may for example be used to introduce the
heritable
change, with subsequent screening of the plant or plant cell or progeny for
the desired
modulation of the abiotic stress response to drought.
.. Brief Description of the Drawings and List of Sequences
In order that the invention may be more clearly understood, embodiments
thereof will now
be described in detail by way of example, with reference to the accompanying
drawings,
in which:
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Figure 1 is a graph showing segregation of a drought tolerance trait from 500
F2 individual
lines, calculated by the survival days after drought treatment (cessation of
watering). The
survival in days of Col and #95 plants are marked by arrows and legends.
Figure 2 is a diagram depicting the gene structure of four members of the
Kanghan gene
family in Arabidopsis ecotype Col and #95. The locations of premature stop
codons are
indicated as TAA;
Figure 3 roughly depicts the relative location of 4 conserved protein domains
within 5
members of the Arabidopsis Kanghan gene family: at5g18065, at5g18040,
at4g29770,
at4g29760 and at1g48180.
Figure 4 is an alternative illustration of the conserved protein domains
within the 5
members of the Arabidopsis Kanghan gene family: at5g18065, at5g18040,
at4g29770,
at4g29760 and at1g48180, with an additional sequence identified as
"lcliQuery_10001"
which is the sequence of at5g18065 plus the translation of the at5g18065 cDNA
following
what appears to be a premature stop codon in at5g18065 which truncates the
protein.
Alternative protein consensus sequences are also set out in Figure 4, with
varying degrees
of sequence consensus as illustrated (with lower case descriptors for residues
having
conserved properties based on Taylor W. R. (1986) J. Theor. Biol. 119:205-218,
as follows:
alcohol => o S, T 1, aliphatic => 1 { I, L, V }, aromatic => a { F, H, W, Y },
charged =>
c D, E, H, K, R 1, hydrophobic => h { A, C, F, G, H, I, K, L, M, R, T, V, W, Y
},
negative => - { D, E }, polar => p C, D, E, H, K, N, Q, R, S, T 1, positive =>
+ H, K,
R 1, small => s { A, C, D, G, N, P, S, T, V }, tiny => u { A, G, S }, turnlike
=> t { A, C,
D, E, G, H, K, N, Q, R, S, T }.
Figure 5 depicts conserved domain A in Kanghan proteins using a sequence logo,
using
the sequence of the 5 Kanghan proteins identified by QTL analysis as having
the greatest
contribution to drought tolerance.
Figure 6 is a continuation of Fig. 5, depicting conserved domain B in Kanghan
proteins
using a sequence logo, using the sequence of the 5 Kanghan proteins identified
by QTL
analysis as having the greatest contribution to drought tolerance.
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Figure 7 is a continuation of Fig. 6, depicting conserved domain C in Kanghan
proteins
using a sequence logo, using the sequence of the 5 Kanghan proteins identified
by QTL
analysis as having the greatest contribution to drought tolerance.
Figure 8 is a diagram depicting the construct for overexpression of
Arabidopsis Kanghanl
in wheat wild type (Fielder) based on the monocot special overexpression
vector
PANIC5E.
Figure 9A is a photograph of 3 week old wild-type (left) and transgenic
(right) wheat
seedlings grown under standard conditions (25 C). The transgenic wheat
seedlings
heterologously express At5g18040.
Figure 9B is a photograph of the plants from Figure 9A, after being incubated
for three
weeks at 40/38 C (day/night), followed by three weeks at 25 C.
Figure 9C is a photograph of the plants from Figure 9B after being grown for
an additional
seven weeks at 25 C.
Figure 10 is a near infrared leaf surface temperature image of wild-type
(left) and
transgenic (right) wheat plants grown under standard conditions. The
transgenic wheat
plant expresses At5g18040 from a heterologous At5g18040 expression construct.
Figure 11 shows a DNA neighbor phylogenetic tree of the Brassica napus Kanghan
gene
candidates and their Arabidopsis thaliana counterparts.
Figure 12 shows a protein neighbor phylogenetic tree of the Brassica napus
Kanghan gene
candidates and their Arabidopsis thaliana counterparts.
Figure 13 shows a DNA neighbor phylogenetic tree of the Brassica napus Kanghan
gene
candidates.
Figure 14 shows a map of the pGEMS-T vector (Promega, USA).
Figure 15 shows a map of the pCAMBIA 1301-35S-Int-T7 vector.
Figure 16 shows a partial map of an RNAi construct designed to target Brassica
napus
Kanghan genes.
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Figure 17 shows infrared thermal images of a wild-type Brassica napus line and
a Kanghan
RNAi Brassica napus line.
Figure 18 shows wild-type Brassica napus plants and Brassica napus plants from
two
Kanghan RNAi lines that have been subjected to drought treatment.
Figure 19 shows the survival ratio, after 35 days recovery, of wild-type
Brassica napus
plants and Brassica napus plants from two RNAi lines that have been subjected
to drought
treatment.
The following is a list of sequences appearing in this document:
SEQ ID NO: 1 is a CDS of the At4g29760 gene from Arabidopsis;
ATGGCTGAGCGATTATTACAATCTATGTCAAGGGTGGCTGGCCGATGTCATC
CAGATTGCGTAAAAGCAAGTGATGAGCAAGAAGATTACCATGCATCTCAAA
ATGCAGC _____________________________________________________________ IT!
GGTAGCTGTGAATCTGATTAGCTCTGCAAGGTTAATACTGAAA
CTCGACGCTGAGTTTACTGAGTACTCAGCTCAGTTTTTGATGGACAATGCTGG
AAAGGAAGACGACCCGGGAGAAGTGGATCAACAACGCAATCAGGTCACGAC
CGAAAACTGCCTTC GCTACTTGG CCGAAAACGTTTG GACCAAGAAG GAAAAT
GGGCAGGGAGGAATGGATCAACAACGCCCTGTGCTCACTGTCAAAGACTGCT
TGGAACTTGCTTTTAAAAAAGGGCTGCCGAGAAGAGAACACTGGGCACATTT
GGGATGTACCTTCAAGGCTCCCCCAITTGCTTGTCAGATACCTCGCGTTCCTG
TGAAAGGAGAAGTGGTTGAGGTTAAGACTTTTGATGAAGCATTCAAGCTGTT
GGTGCATCAACCCATTGGAGCAAAACTGCATTTGTTCAGTCCGCAGATTGAT
AATGTTGGAGAGGGAGTTTACAAAGGCCTCACGACAGGTAATGAAACACAC
TATGTTGGACTTAGAGATGTGCTAATAGCTIVAGTGGAGGAGTTCGAGGGAG
ATTCTGTTGCTATTGTGAAGATCTGCTACAAGAAGAAGC _____________________________ FYI
CATTTATCAAA
GTGTCTTTGAGCGTTAGGTTTCTCTCAGTAGCACATGATGGTGATAAGTCTAA
GTTCATAGCGCCAACAGGTCTGCTTGTTGACTTCTGTGTCCCGCGCTTATCTA
TCAACTAA
SEQ ID NO: 2 is a CDS of the At4g29770 gene from Arabidopsis;
ATGATGGCAATCTCAGAAAAAGGAGTCATGGCAATCTCAGAAAAAGGAGTC
ATGGCAACGAAAATTGACAAAAACGGCGTCCTTCGAGAGTTAAGGCGACATT
TCACTGAGTTTTCTCTACGCGACGTAGATCTGTGTCTCCGGAGTTCATCGCAG
ATGGAGTCATTGTTAGAATGTTTTGCAATCACGGATGGCAAATGTCATCCCG
ATTGCTTAAAAGCAAACAATGAGCAAGAAGATTACGATGCATGTCAATCTGC
AGCTTTGGTAGCTGTGAG1"1"IGATTAGCTCTGCACGTGTTATCTTCAAGATCG
ACTCTAAGTATACTGAGTACTCACCTCAGTATTTGGTGGATAACGTTGGGAA
GGAAGAAGTTGAGGGAGAAATGGATCAACCAAGCTGTCAGTACACTGTCGG
AAACCTCCITAGTTACTTGGTGGAAAACGTTTGGACCAAGAAGGAAGTTAGG
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CAGAGAGAAATG GATCAACAAC GC C GTGAGTTCACTGTCAAAGACTGCTTTG
AATT'TGCTTTTAAAAAAGG G CTTCCAAGAAATG GACATTGG GC GCATGTGG G
ATGTATATTCCCGGTTCCTCCATTTGCTTGTCAAATACCTCGCGITCCCATGA
AAGGAGAAGTGATTGAGGCTGCAAATGTGAGTGAAGCGTTGAAGCTGGGTA
TGCAACAACCAGCGGCAGCAAGGCTGCATTTGTTCAGTCCAGAGTTTGATCT
TG'FTGGAGAGGGTATTTACGATGGCCCGTCAGGTAATGAAACACGATATGTT
GGACTTAGAGATGTGCTCATGGTTGAGG CGGAGAAGATCAAGGGAGAAACT
GTTTTTACTGTGCAGATATGCTACAAGAAGAAGACTTCATTTGTCAAAGTGTC
TACGAGAAGTATGATTCTCCC GCTTAATGGTGACGACGAGTCTCAGGTCACA
GAG CCAG CATGTCTACTTGTTGACTTCTGTATCC CACGTTITTCTATCAACTA
A
SEQ ID NO: 3 is a CDS of the At5g18065 gene from Arabidopsis;
ATGGATATGAATCAGCTATTCAT GCAAT CTATTGCAAACAGTCGTGGACTCT
GTCATCCAGATTGCGAAAAAGCAAATAATGAGCGTGAAGATTATGATGCGTC
TCAACATGCCGCTATGGTAGCGGTGAATCTGATTAGCTCTGCACGGGTTATCC
TCAAGCTTGATGCTGTGTATACTGAGTACTCAGCTCAGTATTTGGTGGATAAT
GCTGGGAAGGAAGACAACCAGGGAGAAATGGATCAACAAAGCTCTCAGCTC
ACTCTCCAAAACTT GCTTCAGTATAT GGATGAAAATGTCTGGAATAAGAAGG
AAGATGTGCAGGGAGAAAGGGAGCAACCACTCACTGTCAAAGACTGCCTTG
AATGTGCTTIVAAGTAA
SEQ ID NO: 4 is a CDS of the At5g18040 gene from Arabidopsis;
ATGAATATGATTCAG CGATTCATG CAATCTATGG CAAAGACGCGTG GC CTCT
GTCATCCAGATTGC GTAAAAG CAAGTAGTGAG CAAGAAGATTACG ATG CGTC
TCAGCTCAGTATITGGTGGATAATGCTGGGAAGGAAGACGACCAGGGAGAA
ATGGATGAACCAAGCTCTCAGTTCACTATCGAAAACTTGCATCAGTATATGG
TGGAAAATGTCTGGAATAAGAGGTAAGATGTGCAGGGAGAGGGAGCAACCA
CTCACTGTCAAAGACTGCCTTGAATGTGCTITCAAGAAAGGGCTACCGAGAA
GAGAACATTGGGCACATGTGGGATGTACATTCAAGGCTCCCCCATTTGCTTG
TCACATACCCCGCGTGCCCATGAAAGGAGAAGTGATTGAGACTAAGAGYITG
GATGAAGCGTTTAAGCTGTTGATTAAACAACCGGTGGGTGCAAGACTCCATG
TGTTCAGTCCAGACCTTGATAATGTTGGAGAGGGAGTTTACGAGGGCCTGTC
TAGCCTGTCTC GTAAGGAATCACG CTATGTTGGACTTAG G GATGTCATCATA
GTTGCAGTGAATAAGTCCGAGGGAAAAACTGTTGCTACTGTGAAGATATGIT
ACAAGAAGAAGACTTCATTTGTCAAAGTGTGTTTGAGCCGTATGTTTGTCCAG
CTTG GTGGTGGCGAGGAGTCTCAG GTGAAAGAG C CAACAGGTCTGCTTGTTG
ACTIVTGTATCCCACGCTTATCTATCAACTAA
SEQ ID NO: 5 is a CDS of the At1g51670 gene from Arabidopsis;
ATGGCACTCCCTCCCTATGATCCGAATTTCACATTGGCT ___________ l'ITTCATACGGTAG
ACGCGATAATGTCTTTGAGAATGACCCAGAG CAC GATGAATCTGCTTCTGCT
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GCTATCGTAGCGGTTGAGCTGATAAGCTCTGCACGGCTTGCACTTAAGCTGG
ATAGTGTCCGCACTGAGTACTCAGCTCAGTATTTGGTGGACAAAGCTGGCTC
ACGCAACCTCAGGCGCAGGCGCAAGCTCACTGTCAAGGACTGCCT1TAACIT1
GCGTTAAAGAAAGGCGGCATACCGAGAGCAGAAGATTGGCCACCTTTGGGA
TCTGAGTCAAAGACCCCATCATCGTACGAACCTGCTCTCGTITCCATGAAAG
GAGAAGTGATTGAGCCTAAGGATATGGACGAAGTACCTGAGTTGTTGGTGCA
TCAATCAGCCGTGGGAGCAAAACTGCATGTGTTCACTCCACACATTGAACTT
CAACAAGACGCAATTTACTTGCCTCGTCAGGTGAGTATGCGCGCTACGTTGG
ACTTAGAGATGGGATAG
SEQ ID NO: 6 is a consensus sequence of Kanghan conserved domain B (100%
consensus)
hTVKDChphAhp
SEQ ID NO: 7 is a consensus sequence of Kanghan conserved domain B (80%
consensus)
LTVKDCLEhAhKXG (where X is Lys or absent)
SEQ ID NO: 8 is a consensus sequence of Kanghan conserved domain B (70%
consensus)
LTVKDCLEhAFKKG
SEQ ID NO: 9 is a consensus sequence of Kanghan conserved domain C (80%
consensus)
VshKGpVlEstshpEsXchhhpQs-huA+LH1FpPph (where X is any amino acid)
SEQ ID NO: 10 is a consensus sequence of Kanghan conserved domain C (70%
consensus)
VsMKGEVIEspsh-EAhcL11cQP1GA+LH1FoPc1
SEQ ID NO: 11 is a consensus sequence of Kanghan conserved domain A (80%
consensus)
cppDYDtStpAAhVA1pLISSAR1hLK1DuhhTEYSsQaLhDpsutpp
SEQ ID NO: 12 is a consensus sequence of Kanghan conserved domain A (70%
consensus)
spphhpShupscGhCHPDCXKA5sEpEDYDASQpAAhVAVsLISSAR1hLKLDusaTEYSA
QYLVDNAGpccs (where X is any amino acid or absent)
SEQ ID NO: 13 is a CDS of the At1g48180 gene from Arabidopsis:
ATGGCACTCCCACCCTATGATCCCAATTTCAAATTTGCATTCTCTCTTGGCAC
GATTGCGAAACACCAAGATTACGATGAATCTGCTTCTGCTGCTG'17GTAGCG
CTTGATCTGATAAGCTCTGCACGGTTTGCACTTAAGCTGGATAGTGTCTATAC
TGAGTACTCTGCTAAGTATGTGGTGGACAATGCTGCTGGCTCACACAGTGGG
CGCAAGCTCACTGTCAAAGACTGTCTTGAGTTTGCCTTAAACAAAGGCGGCA
TACCGAAAGCAGAAGATMGCCACGCTTGGGATCTGTGATAACGCCCCCATC
CA 3017921 2018-09-19
ATCGTATAAACCTGATCTCGTTTCGATGAAAGGACAAGTGATTGAG CCTCAG
ACTATTGAGGAAGCATGTGACATGGTGGTGGATCAACCAGTAGGAGCAAAA
TTGCATGTGTTCAAGCCACACATTGAACTTCAACAAGACGCAAGTGCTATAA
CTGGCATTTACTGTGGCACGTCAGGTGAGCCAGCCAGCTATGTCGGACTTAG
AGATGCCATCATCGTTGGAGTCGAGAAGATCCAAGGGAAGTCTATTGGAACT
GTGAAGGTATGGTACAAGAAGITCATATTTCTGAAAGTGGCTATGAGCAGGT
GGTTTCAGITATACTCTCCGGATGGCACACACACGGGCATAAAGCGAACAGA
TTACCTTGTTGATTTTTGTGTCCCACGCCTATCCATGGATTAA
SEQ ID NO: 14 is the polypeptide encoded by SEQ ID NO: 1
MAERLLQSMSRVAGRCHPDCVKASDEQEDYHAS QNAALVAVNLISSARLILKL
DAEFTEYSAQFLMDNAGKEDDPGEVDQQRNQVTTENCLRYLAENVWTKKENG
QGGMD QQRPVLTVKDCLELAFKKGLPRREHWAHLGCTFKAPPFACQIPRVPVK
GEVVEVKTFDEAFKLLVHQPIGAKLHLFSPQIDNVGEGVYKGLTTGNETHYVGL
RD VLIASVEEFEGD SVAIVKICY KKKLS FIKVS LSVRFLSVAHDGDKSKFIAPTGL
LVDFCVPRLSIN
SEQ ID NO: 15 is the polypeptide encoded by SEQ ID NO: 2
MMAISEKGVMAISEKGVMATKIDKNGVLRELRRHFTEFSLRDVDLCLRSSSQME
SLLECFAITDGKCHPDCLKANNEQEDYDACQSAALVAVSLISSARVIFKIDSKYTE
YSPQYLVDNVGKEEVEGEMDQPSCQYTVGNLLSYLVENVW'TKKEVRQREMDQ
QRREFTVKDCFEFAFKKGLPRNGHWAHVGCIFPVPPFACQIPRVPMKGEVIEAAN
VSEALKLGMQQPAAARLHLFSPEFDLVGEGIYDGPSGNETRYVGLRDVLMVEAE
KIKGETVFTVQICYKKKTS FVKVSTRS M ILPLNGDDES QVTEPACLLVDFCIPRFS I
SEQ ID NO: 16 is the polypeptide encoded by SEQ ID NO: 3
MD MNQ LFMQ SIANSRGLCHPDCEKANNEREDYDASQHAAMVAVNLISSARVIL
KLDAVYTEYSAQYLVDNAGKEDNQGEMDQQSSQLTLONLLQYMDENVWNKK
EDVOGEREQPLTVKDCLECAF'K
SEQ ID NO: 17 is the polypeptide encoded by SEQ ID NO: 4
MNMIQRFMQS MAKTRGLCHPD CVKAS SEQEDYDAS QLSI WWI MLGRKTTREK
WMNQALSSLSKTCISIWWKMS GIRGKMCREREQPLTVKDCLECAFKKGLPRRE
HWAHVGCTFKAPPFACHIPRVPMKGEVIETKSLDEAFKLLIKOPVGARLHVFSPD
LDNVGEGVYEGLSSLSRKESRYVGLRDVIIVAVNKSEGKTVATVKICYKKKTSF
VKVCLSRMFVQLGGGEESQVKEPTGLLVDFCIPRLSIN
SEQ ID NO: 18 is the polypeptide encoded by SEQ ID NO: 5
MALPPYDPNFTLAFSYGRRDNVFENDPEHDESASAAIVAVELIS SARLALKLDSV
RTEYSAQYLVDKAGSRNLRRRRKLTVKDCLNFALKKGGIPRAEDWPPLGSESKT
PSSYEPALVSMKGEVIEPKDMDEVPELLVHQSAVGAKLHVFTPHIELQQDAIYLP
RQVSMRATLDLEMG
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SEQ ID NO: 19 is the polypeptide encoded by SEQ ID NO: 13
MALPPYDPNFKFAFSLGTIAKHQDYDESASAAVVALDLISSARFALKLDSVYTEY
SAKYVVDNAAGSHSGRKLTVKDCLEFALNKGGIPKAEDWPRLGSVITPPSSYKP
DLVSMKGQVIEPQTIEEACDMVVDQPVGAKLHVFKPHIELQQDASAITGIYCGTS
GEPASYVGLRDAIIVGVEKIQGKSIGTVKVWYKKFIFLKVAMSRWFQLYSPDGT
HTGIKRTDYLVDFCVPRLSMD
SEQ ID NOs: 20 and 21 are a primer pair designed to target BnaCO3g77540D
(L0C106364365)
TAGATTCTGCTGAGAGAGCCGCTAC (SEQ ID NO: 20)
GGATCCGTCGACGCACCTATGGGTCCATGCTTTAAC (SEQ ID NO: 21)
SEQ ID NOs: 22 and 23 are a primer pair designed to target BnaA08g12920D
(L0C106424160)
TCATCCAGATTGCCAACGAG (SEQ ID NO: 22)
GGATCCGTCGACACGCATCCTCCAGTGTCTTAG (SEQ ID NO: 23)
SEQ ID NOs: 24 and 25 are a primer pair designed to target hygromycin
TACACAGCCATCGGTCCAGA (SEQ ID NO: 24)
GTAGGAGGGCGTGGATATGTC (SEQ ID NO: 25)
SEQ ID NOs: 26 and 27 are a primer pair designed to target BnaA07g02270D
CGCTACGAGGCACGTACTCAAT (SEQ ID NO: 26)
CTCGGTCTTCCCCGGTTTC (SEQ ID NO: 27)
SEQ ID NOs: 28 and 29 are a primer pair designed to target BnaA08g12920D
GCTTAGAGACGTGATCCTGGTAGC (SEQ ID NO: 28)
CCAGTGTGGTGAACATACGGC (SEQ ID NO: 29)
SEQ ID NOs: 30 and 31 are a primer pair designed to target BnaC01g07670D
urn TGTTGGTCTCTTCTCTTTGC (SEQ ID NO: 30)
TTCTTAAGAGGCGTTTCAGATGG (SEQ ID NO: 31)
SEQ ID NOs: 32 and 33 are a primer pair designed to target BnaCO3g77540D
TGATTTGGG'TTTTGCCTGATAC (SEQ ID NO: 32)
GAAACAAACCATAAATGAGTTGCC (SEQ ID NO: 33)
SEQ ID NOs: 34 and 35 are a primer pair designed to target BnaCO3g77550D
CATTTGGGATGTGTCGATTGAG (SEQ ID NO: 34)
CCCACGTAGCTTGTTCCGTT (SEQ ID NO: 35)
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SEQ ID NOs: 36 and 37 are a primer pair designed to target BnaA01g06470D
AACACTGTCACGCAGATTGCC (SEQ ID NO: 36)
CTGTCCAGGITAGCTACCATACGA (SEQ ID NO: 37)
SEQ ID NOs: 38 and 39 are a primer pair designed to target BnaC01g08490D
CGGTATCCAACTCATTCGAAGG (SEQ ID NO: 38)
TCAAGTATATACTGGGTTGGCTGC (SEQ ID NO: 39)
Detailed Description
In the following detailed description, various non-limiting examples are set
out of
particular embodiments, together with experimental procedures that may be used
to
implement a wide variety of modifications and variations in the practice of
the present
invention. For clarity, a variety of technical terms are used herein in
accordance with what
is understood to be the commonly understood meaning, as reflected in
definitions set out
below.
The term "line" refers to a group of plants that displays very little overall
variation among
individuals sharing that designation. A "line" generally refers to a group of
plants that
display little or no genetic variation between individuals for at least one
trait. Plants within
a group of plants that display little or no genetic variation between
individuals may also be
referred to as having the same genetic background.
A "variety" or "cultivar" includes a line that is used for commercial
production. In some
aspects, Brassica varieties may for example be derived from "doubled haploid"
(DH) lines,
which refers to a line created by the process of microspore embryogenesis, in
which a plant
is created from an individual microspore. By this process, lines are created
that are
homogeneous, i.e. all plants within the line have the same genetic makeup. The
original
DH plant is referred to as DH1, while subsequent generations are referred to
as DH2, DH3
etc. Doubled haploid procedures are well known and have been established for
several
crops. A procedure for B. juncea has been described by Thiagrarajah and
Stringham (1993)
(A comparison of genetic segregation in traditional and microspore-derived
populations of
Brassica juncea in: L. Czern and Coss. Plant Breeding 111:330-334).
New lines, varieties or plants may be produced by introducing a heritable
change in a parent
plant. In this context, a "heritable change" is any molecular alteration,
typically a genetic
change, that is capable of being passed from one generation of plant to the
next. This term
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is intended to include molecular alterations such as, but not limited to,
insertions, deletions,
point mutations, frame-shift mutations, inversions, rearrangements, and the
introduction of
transgenes. There is a wide variety of techniques available for introducing
heritable
changes to plants and plant cells.
Plant "mutagenesis" in the present context is a process in which an agent
known to cause
alterations in genetic material is applied to plant material, for example the
mutagenic agent
ethyl methylsulfonate (EMS). A range of molecular techniques such as
recombination with
foreign or heterologous nucleic acid fragments or gene editing may also be
used for
mutagenesis. All such methods of introducing nucleic acid sequence changes are
included
within the term "mutagenesis" as used herein.
Plant "regeneration" involves the selection of cells capable of regeneration
(e.g. seeds,
microspores, ovules, pollen, vegetative parts) from a selected plant or
variety. These cells
may optionally be subjected to mutagenesis, following which a plant is
developed from the
cells using regeneration, fertilization, and/or growing techniques based on
the types of cells
mutagenized. Applicable regeneration techniques are known to those skilled in
the art; see,
for example, Armstrong, C. L., and Green, C. E., Planta 165:322-332 (1985);
and Close,
K. R., and Ludeman, L. A., Planta Science 52:81-89 (1987).
"Improved characteristics" of a plant means that the characteristics in
question are altered
in a way that is desirable or beneficial or both in comparison with a
reference value or
attribute, which in the absence of an express comparator relates to the
equivalent
characteristic of a wild type strain.
Plant "progeny" means the direct and indirect descendants, offspring and
derivatives of a
plant or plants and includes the first, second, third and subsequent
generations and may be
produced by self-crossing, crossing with plants with the same or different
genotypes, and
may be modified by range of suitable genetic engineering techniques.
Plant "breeding" includes all methods of developing or propagating plants and
includes
both intra and inter species and intra and inter line crosses as well as all
suitable artificial
breeding techniques. Desired traits may be transferred to other lines through
conventional
breeding methods and can also be transferred to other species through inter-
specific
crossing. Both conventional breeding methods and inter-specific crossing
methods as well
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as all other methods of transferring genetic material between plants are
included within the
concept of "breeding".
"Molecular biological techniques" means all forms of anthropomorphic
manipulation of a
biological molecules, such as nucleic acid sequences, for example to alter the
sequence and
.. expression thereof and includes the insertion, deletion, modification or
editing of
sequences or sequence fragments and the direct or indirect introduction of new
sequences
into the genome of an organism, for example by directed or random
recombination using
suitable vectors and/or techniques.
"Marker-assisted selection" (MAS) refers to the use of molecular markers to
assist in
phenotypic selection in the context of plant breeding. A wide variety of
molecular markers,
such as single nucleotide polymorphisms (SNPs), may for example be used in MAS
plant
breeding, including the application of next-generation sequencing (NGS)
technologies.
The term "genetically derived" as used for example in the phrase "an improved
characteristic genetically derived from the parent plant or cell" means that
the
.. characteristic in question is dictated wholly or in part by an aspect of
the genetic makeup
of the parent plant or cell, applying for example to progeny of the parent
plant or cell that
retain the improved characteristic of the parent plant or cell.
Various genes and nucleic acid sequences of the invention may be recombinant
sequences.
The term "recombinant" means that something has been recombined, so that when
made
in reference to a nucleic acid construct the term refers to a molecule that is
comprised of
nucleic acid sequences that are joined together or produced by means of
molecular
biological techniques. Nucleic acid "constructs" are accordingly recombinant
nucleic
acids, which have been generally been made by aggregating interoperable
component
sequencers. The term "recombinant" when made in reference to a protein or a
polypeptide
refers to a protein or polypeptide molecule which is expressed using a
recombinant nucleic
acid construct created by means of molecular biological techniques. The term
"recombinant" when made in reference to the genetic composition or an organism
or cell
refers to a gamete or progeny with new combinations of alleles that did not
occur in the
parental genomes. Recombinant nucleic acid constructs may include a nucleotide
sequence
which is ligated to, or is manipulated to become ligated to, a nucleic acid
sequence to which
it is not ligated in nature, or to which it is ligated at a different location
in nature. Referring
CA 3017921 2018-09-19
to a nucleic acid construct as 'recombinant' therefore indicates that the
nucleic acid
molecule has been manipulated using genetic engineering, i.e. by human
intervention.
Recombinant nucleic acid constructs may for example be introduced into a host
cell by
transformation. Such recombinant nucleic acid constructs may include sequences
derived
from the same host cell species or from different host cell species, which
have been isolated
and reintroduced into cells of the host species. Recombinant nucleic acid
construct
sequences may become integrated into a host cell genome, either as a result of
the original
transformation of the host cells, or as the result of subsequent recombination
and/or repair
events.
Recombinant constructs of the invention may include a variety of functional
molecular or
genomic components, as required for example to mediate gene expression or
suppression
in a transformed plant. In this context, "DNA regulatory sequences," "control
elements,"
and "regulatory elements," refer to transcriptional and translational control
sequences, such
as promoters, enhancers, polyadenylation signals, terminators, and protein
degradation
signals that regulate gene expression. In the context of the present
disclosure, "promoter"
means a sequence sufficient to direct transcription of a gene when the
promoter is operably
linked to the gene. The promoter is accordingly the portion of a gene
containing DNA
sequences that provide for the binding of RNA polymerase and initiation of
transcription.
Promoter sequences are commonly, but not universally, located in the 5' non-
coding
regions of a gene. A promoter and a gene are "operably linked" when such
sequences are
functionally connected so as to permit gene expression mediated by the
promoter. The term
"operably linked" accordingly indicates that DNA segments are arranged so that
they
function in concert for their intended purposes, such as initiating
transcription in the
promoter to proceed through the coding segment of a gene to a terminator
portion of the
gene. Gene expression may occur in some instances when appropriate molecules
(such as
transcriptional activator proteins) are bound to the promoter. Expression is
the process of
conversion of the information of a coding sequence of a gene into mRNA by
transcription
and subsequently into polypeptide (protein) by translation, as a result of
which the protein
is said to be expressed. As the term is used herein, a gene or nucleic acid is
"expressible"
if it is capable of expression under appropriate conditions in a particular
host cell.
Promoters may for example be used that provide for preferential gene
expression within a
specific organ or tissue, or during a specific period of development. For
example,
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promoters may be used that are specific for leaf (Dunsmuir, et al Nucleic
Acids Res, (1983)
11:4177-4183), root tips (Pokalsky, et al Nucleic Acids Res, (1989) 17:4661-
4673), fruit
(Peat, et al Plant MoL Biol, (1989) 13:639-651; U.S. Pat. No. 4,943,674 issued
24 Jul.,
1990; International Patent Publication WO-A 8 809 334; U.S. Pat. No. 5,175,095
issued
29 Dec., 1992; European Patent Application EP-A 0 409 629; and European Patent
Application EP-A 0 409 625) embryogenesis (U.S. Pat. No. 5,723,765 issued 3
Mar. 1998
to Oliver et al.), or young flowers (Nilsson et al. 1998). Promoters
demonstrating
preferential transcriptional activity in plant tissues are, for example,
described in European
Patent Application EP-A 0 255 378 and International Patent Publication WO-A 9
113 980.
Promoters may be identified from genes which have a differential pattern of
expression in
a specific tissue by screening a tissue of interest, for example, using
methods described in
U.S. Pat. No. 4,943,674 and European Patent Application EP-A 0255378. The
disclosure
herein includes examples of this embodiment, showing that plant tissues and
organs can
be modified by transgenic expression of a Kanghan gene.
An "isolated" nucleic acid or polynucleotide as used herein refers to a
component that is
removed from its original environment (for example, its natural environment if
it is
naturally occurring). An isolated nucleic acid or polypeptide may contain less
than about
50%, less than about 75%, less than about 90%, less than about 99.9% or less
than any
integer value between 50 and 99.9% of the cellular or biological components
with which
it was originally associated. A polynucleotide amplified using PCR so that it
is sufficiently
distinguishable (on a gel for example) from the rest of the cellular
components is, for
example, thereby "isolated". The polynucleotides of the invention may be
"substantially
pure," i.e., having the high degree of isolation as achieved using a
purification technique.
In the context of biological molecules "endogenous" refers to a molecule such
as a nucleic
acid that is naturally found in and/or produced by a given organism or cell.
An
"endogenous" molecule may also be referred to as a "native" molecule.
Conversely, in the
context of biological molecules "exogenous" refers to a molecule, such as a
nucleic acid,
that is not normally or naturally found in and/or produced by a given organism
or cell in
nature.
As used herein to describe nucleic acid or amino acid sequences the term
"heterologous"
refers to molecules or portions of molecules, such as DNA sequences, that are
artificially
introduced into a particular host cell, for example by transformation.
Heterologous DNA
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sequences may for example be introduced into a host cell by transformation.
Such
heterologous molecules may include sequences derived from the host cell.
Heterologous
DNA sequences may become integrated into the host cell genome, either as a
result of the
original transformation of the host cells, or as the result of subsequent
recombination
events.
Transformation techniques that may be employed include plant cell membrane
disruption
by electroporation, microinjection and polyethylene glycol based
transformation (such as
are disclosed in Paszkowski et al. EMBO J. 3:2717 (1984); Fromm et al., Proc.
Natl. Acad.
Sci. USA 82:5824 (1985); Rogers et al., Methods Enzymol. 118:627 (1986); and
in U.S.
.. Pat. Nos. 4,684,611; 4,801,540; 4,743,548 and 5,231,019), biolistic
transformation such as
DNA particle bombardment (for example as disclosed in Klein, et al., Nature
327: 70
(1987); Gordon-Kamm, et al. "The Plant Cell" 2:603 (1990); and in U.S. Pat.
Nos.
4,945,050; 5,015,580; 5,149,655 and 5,466,587); Agrobacterium-mediated
transformation
methods (such as those disclosed in Horsch et al. Science 233: 496 (1984);
Fraley et al.,
Proc. Nat'l Acad. Sci. USA 80:4803 (1983); and U.S. Pat. Nos. 4,940,838 and
5,464,763).
Transformation systems adapted for use in Camelina sativa are for example
described in
US Patent Publication 20140223607. Varieties of Camelina sativa are for
example
described in US Patent Publication 20120124693, and the subject of seed
samples
deposited under ATCC Accession No. PTA-11480. Aspects of the present invention
involve altering known plant varieties, such as Camelina sativa, to alter
endogenous
Kanghan genes.
Transformed plant cells may be cultured to regenerate whole plants having the
transformed
genotype and displaying a desired phenotype, as for example modified by the
expression
of a heterologous Kanghan gene during growth or development. A variety of
plant culture
techniques may be used to regenerate whole plants, such as are described in
Gamborg and
Phillips, "Plant Cell, Tissue and Organ Culture, Fundamental Methods",
Springer Berlin,
1995); Evans etal. "Protoplasts Isolation and Culture", Handbook of Plant Cell
Culture,
Macmillian Publishing Company, New York, 1983; or Binding, "Regeneration of
Plants,
Plant Protoplasts", CRC Press, Boca Raton, 1985; or in Klee et al., Ann. Rev.
of Plant Phys.
.. 38:467 (1987).
Various aspects of the present disclosure encompass nucleic acid or amino acid
sequences
that are homologous to other sequences. As the term is used herein, an amino
acid or
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CA 3017921 2018-09-19
nucleic acid sequence is "homologous" to another sequence if the two sequences
are
substantially identical, as defined herein, and the functional activity of the
sequences is
conserved (as used herein, sequence conservation or identity does not infer
evolutionary
relatedness). Nucleic acid sequences may also be homologous if they encode
substantially
identical amino acid sequences, even if the nucleic acid sequences are not
themselves
substantially identical, for example as a result of the degeneracy of the
genetic code.
With reference to biological sequences "substantial homology" or "substantial
identity" is
meant, in the alternative, a sequence identity of greater than 70%, 71%, 72%,
73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% up to 100% sequence
identity.
Homology may refer to nucleic acid or amino acid sequences as the context
dictates. In
alternative embodiments, sequence identity may for example be at least 75%, at
least 90%
or at least 95%. Optimal alignment of sequences for comparisons of identity
may be
conducted using a variety of algorithms, such as the local homology algorithm
of Smith
and Waterman (1981) Adv. AppL Math 2: 482, the homology alignment algorithm of
Needleman and Wunsch (1970)J. MoL Biol. 48:443, the search for similarity
method of
Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, and the
computerized
implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in
the Wisconsin Genetics Software Package, Genetics Computer Group, Madison,
Wis.,
U.S.A.). Sequence identity may also be determined using the BLAST algorithm,
described
in Altschul et al. (1990), J. MoL Biol. 215:403-10 (using the published
default settings).
Software for performing BLAST analysis may be available through the National
Center
for Biotechnology Information (NCBI) at their Internet site. The BLAST
algorithm
involves first identifying high scoring sequence pairs (HSPs) by identifying
short words of
length W in the query sequence that either match or satisfy some positive-
valued threshold
score T when aligned with a word of the same length in a database sequence. T
is referred
to as the neighborhood word score threshold. Initial neighborhood word hits
act as seeds
for initiating searches to find longer HSPs. The word hits are extended in
both directions
along each sequence for as far as the cumulative alignment score can be
increased.
Extension of the word hits in each direction is halted when the following
parameters are
met: the cumulative alignment score falls off by the quantity X from its
maximum achieved
value; the cumulative score goes to zero or below, due to the accumulation of
one or more
negative-scoring residue alignments; or the end of either sequence is reached.
The BLAST
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algorithm parameters W, T and X determine the sensitivity and speed of the
alignment.
The BLAST program may use as defaults a word length (W) of 11, the BLOSUM62
scoring matrix (Henikoff and Henikoff (1992) Proc. NatL Acad. Sci. USA 89:
10915-
10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison
of both
strands. One measure of the statistical similarity between two sequences using
the BLAST
algorithm is the smallest sum probability (P(N)), which provides an indication
of the
probability by which a match between two nucleotide or amino acid sequences
would occur
by chance. In alternative embodiments, nucleotide or amino acid sequences are
considered
substantially identical if the smallest sum probability in a comparison of the
test sequences
is less than about 1, less than about 0.1, less than about 0.01, or less than
about 0.001.
An alternative indication that two amino acid sequences are substantially
identical is that
one peptide is specifically immunologically reactive with antibodies that are
also
specifically immunoreactive against the other peptide. Antibodies are
specifically
immunoreactive to a peptide if the antibodies bind preferentially to the
peptide and do not
bind in a significant amount to other proteins present in the sample, so that
the preferential
binding of the antibody to the peptide is detectable in an immunoassay and
distinguishable
from non-specific binding to other peptides. Specific immunoreactivity of
antibodies to
peptides may be assessed using a variety of immunoassay formats, such as solid-
phase
ELISA immunoassays for selecting monoclonal antibodies specifically
immunoreactive
with a protein (see Harlow and Lane (1988)Antibodies, A Laboratory Manual,
Cold Spring
Harbor Publications, New York).
An alternative indication that two nucleic acid sequences are substantially
identical is that
the two sequences hybridize to each other under moderately stringent, or
stringent,
conditions. Hybridization to filter-bound sequences under moderately stringent
conditions
may, for example, be performed in 0.5 M NaHPO4, 7% sodium dodecyl sulfate
(SDS), 1
mM EDTA at 65 C., and washing in 0.2x SSC/0.1% SDS at 42 C. (see Ausubel, et
al.
(eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing
Associates,
Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3). Alternatively,
hybridization to
filter-bound sequences under stringent conditions may, for example, be
performed in 0.5
.. M NaHPO4, 7% SDS, 1 mM EDTA at 65 C., and washing in 0.1x SSC/0.1% SDS at
68
C. (see Ausubel, et al. (eds), 1989, supra). Hybridization conditions may be
modified in
accordance with known methods depending on the sequence of interest (see
Tijssen, 1993,
CA 3017921 2018-09-19
Laboratory Techniques in Biochemistry and Molecular Biology¨Hybridization with
Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of
hybridization and the
strategy of nucleic acid probe assays", Elsevier, N.Y.). Generally, stringent
conditions are
selected to be about 5 C. lower than the thermal melting point for the
specific sequence at
a defined ionic strength and pH. The term "a polynucleotide that hybridizes
under stringent
(low, intermediate) conditions" is intended to encompass both single and
double-stranded
polynucleotides although only one strand will hybridize to the complementary
strand of
another polynucleotide. Washing in the specified solutions may be conducted
for a range
of times from several minutes to several days and those skilled in the art
will readily select
appropriate wash times to discriminate between different levels of homology in
bound
sequences.
In alternative embodiments, the invention provides nucleic acids, such as
isolated or
recombinant nucleic acid molecules, comprising the sequence of a Kanghan
allele of the
invention. Isolated nucleic acids of the invention may include coding
sequences of the
invention recombined with other sequences, such as cloning vector sequences.
Homology
to sequences of the invention may be detectable by hybridization with
appropriate nucleic
acid probes, by PCR techniques with suitable primers or by other techniques.
In particular
embodiments there are provided nucleic acid probes which may comprise
sequences
homologous to portions of the alleles of the invention. Further embodiments
may involve
the use of suitable primer pairs to amplify or detect the presence of a
sequence of the
invention, for example a sequence that is associated with an abiotic stress
response, such
as drought or heat resistance.
In alternative embodiments, the invention provides methods for identifying
plants, such as
Camelina, Brassica or Tritium plants, with a desirable abiotic stress
response, such as
drought tolerance and/or heat resistance, or a desired genomic characteristic.
Methods of
the invention may for example involve determining the presence in a genome of
particular
Kanghan alleles. In particular embodiments the methods may comprise
identifying the
presence of: a nucleic acid polymorphism associated with one of the identified
alleles; or
an antigenic determinant associated with one of the alleles. Such a
determination may for
example be achieved with a range of techniques, such as PCR amplification of
the relevant
DNA fragment, DNA fingerprinting, RNA fingerprinting, gel blotting and RFLP
analysis,
nuclease protection assays, sequencing of the relevant nucleic acid fragment,
the
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CA 3017921 2018-09-19
generation of antibodies (monoclonal or polyclonal), or alternative methods
adapted to
distinguish the protein produced by the relevant alleles from other variants
or wild type
forms of that protein.
In selected embodiments, a specific base pair change in a Kanghan allele may
for example
.. be used to design protocols for MAS, such as the use of allele-specific
probes, markers or
PCR primers. For an exemplary summary of allele-specific PCR protocols, see
Myakishev
et al., 2001, Genome Research 11: 163-169, or Tanhuanpaa et al., 1999,
Molecular
Breeding 4: 543-550. In alternative embodiments, for example, various methods
for
detecting single nucleotide polymorphisms (SNPs) may be used for identifying
Kanghan
.. alleles of the invention. Such methods may for example include TaqMan
assays or
Molecular Beacon assays (Tapp et al., BioTechniques 28: 732-738), Invader
Assays (Mein
et al., Genome Research 10: 330-343, 2000) or assays based on single strand
conformational polymorphisms (SSCP) (Orita et al., Proc. Natl. Acad. Sci.
U.S.A. 86:
2766-2770, 1989).
In alternative embodiments, the invention provides progeny of parent plant
lines having
altered endogenous or heterologous Kanghan genes, for example progeny of
Camelina
sativa parent line which is the subject of ATCC Accession number PTA-11480.
Such
progeny may for example be selected to have a desired alteration in an abiotic
stress
response compared to the parent strain, such as improved drought resistance or
heat
tolerance.
In alternative embodiments, a plant seed is provided, such as an Arabidopsis,
Camelina,
Triticum or Brassica seed. In alternative embodiments, genetically stable
plants are
provided, such as plants of the genus Arabidopsis, Camelina, Triticum or
Brassica. In
further alternative embodiments the invention provides processes of producing
genetically
stable plants, such as Arabidopsis, Camelina, Triticum or Brassica plants, for
example
plants having a desired alteration in an abiotic stress response compared to a
reference
strain that does not have a particular alteration in a Kanghan gene, such as
improved
drought resistance or heat tolerance.
In various aspects, the invention involves the modulation of the number of
copies of an
expressible Kanghan coding sequence in a plant genome. By "expressible" it is
meant that
the primary structure, i.e. sequence, of the coding sequence indicates that
the sequence
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encodes an active protein. Expressible coding sequences may nevertheless not
be
expressed as an active protein in a particular cell, for example due to gene
silencing. This
'gene silencing' may for example take place by various mechanisms of
homologous
transgene inactivation or epigenetic silencing in vivo. Homologous transgene
inactivation
and epigenetic silencing in transgenic plants has been described in plants
where a transgene
has been inserted in the sense orientation, with the result that both the gene
and the
transgene are down-regulated (Napoli et al., 1990 Plant Cell 2: 279-289;
Rajeevkum et al.,
2015 Front Plant Sci 6:693). In the present invention, the expressible coding
sequences in
a genome may accordingly not all be expressed in a particular cell, and may in
some
embodiments result in suppression of Kanghan gene expression.
In other aspects, reduction of Kanghan gene expression may include the
reduction,
including the suppression or elimination (aka knockout), of expression of a
nucleic acid
sequence that encodes a Kanghan protein, such as a nucleic acid sequence of
the invention.
By elimination of expression, it is meant herein that a functional amino acid
sequence
encoded by the nucleic acid sequence is not produced at a detectable level. By
suppression
of expression, it is meant herein that a functional polypeptide encoded by the
nucleic acid
sequence is produced at a reduced level relative to the wild type level of
expression of the
polypeptide. Reduction of Kanghan expression may include the elimination
of
transcription of a nucleic acid sequence that encodes a Kanghan protein, such
as a sequence
of the invention encoding a Kanghan protein. By elimination of transcription
it is meant
herein that the mRNA sequence encoded by the nucleic acid sequence is not
transcribed at
detectable levels. Reduction of Kanghan activity may also include the
production of a
truncated amino acid sequence from a nucleic acid sequence that encodes a
Kanghan
protein, meaning that the amino acid sequence encoded by the nucleic acid
sequence is
missing one or more amino acids of the functional amino acid sequence encoded
by a wild
type nucleic acid sequence. In addition, reduction of Kanghan activity may
include the
production of a variant Kanghan amino acid sequence, meaning that the amino
acid
sequence has one or more amino acids that are different from the amino acid
sequence
encoded by a wild type nucleic acid sequence. A variety of mutations may be
introduced
into a nucleic acid sequence for the purpose of reducing Kanghan activity,
such as frame-
shift mutations, introduction of premature stop codon(s), substitutions and
deletions. For
example, mutations in coding sequences may be made so as to introduce
substitutions
23
CA 3017921 2018-09-19
within functional motifs or conserved domains in a Kanghan protein, such as
conserved
Kanghan protein domains A, B or C.
In an alternative aspect, the down-regulation of Kanghan genes may be used to
alter a plant
response to abiotic stress, for example to enhance drought tolerance. Such
down-regulation
may be tissue-specific. For example, anti-sense oligonucleotides may be
expressed to
down-regulate expression of Kanghan genes. The expression of such anti-sense
constructs
may be made to be tissue-specific by operably linking anti-sense encoding
sequences to
tissue-specific promoters. Anti-sense oligonucleotides, including anti-sense
RNA
molecules and anti-sense DNA molecules, act to block the translation of mRNA
by binding
to targeted mRNA and inhibiting protein translation from the bound mRNA. For
example,
anti-sense oligonucleotides complementary to regions of a DNA sequence
encoding a
Kanghan protein may be expressed in transformed plant cells during development
to down-
regulate the expression of the Kanghan gene. Alternative methods of down-
regulating
Kanghan gene expression may include the use of ribozymes or other enzymatic
RNA
molecules (such as hammerhead RNA structures) that are capable of catalyzing
the
cleavage of RNA (as disclosed in U.S. Pat. Nos. 4,987,071 and 5,591,610).
Aspects of the invention involve the use of gene editing to alter Kanghan gene
sequences.
For example, CRISPR-Cas system(s) (e.g., single or multiplexed) can be used to
perform
plant gene or genome interrogation or editing or manipulation. Kanghan genes
may for
example be edited for functional investigation and/or selection and/or
interrogation and/or
comparison and/or manipulation and/or transformation of plant Kanghan genes.
This
editing may be carried out so as to create, identify, develop, optimize, or
confer trait(s) or
characteristic(s) to plant(s) or to transform a plant genome, for example to
alter an abiotic
stress response in a plant, such as a drought or heat tolerance. Gene editing
can in this way
be used to provide improved production of plants, new plants with new
combinations of
traits or characteristics or new plants with enhanced traits. Such CRISPR-Cas
system(s)
can for example be used in Site-Directed Integration (SDI) or Gene Editing
(GE) or any
Near Reverse Breeding (NRB) or Reverse Breeding (RB) techniques (see the
University
of Arizona website "CRISPR-PLANT" http://www.genome.arizona.edu/crispr/).
Embodiments of the invention can be used in genome editing in plants alone or
in
combination with other molecular biological techniques, such as RNAi or
similar genome
editing techniques (see, e.g., Nekrasov, Plant Methods 2013, 9:39; Brooks,
Plant
24
CA 3017921 2018-09-19
Physiology September 2014 pp 114.247577; Shan, Nature Biotechnology 31, 686-
688
(2013); Feng, Cell Research (2013) 23:1229-1232; Xie, Mol Plant. 2013
Nov;6(6):1975-
83; Xu, Rice 2014, 7:5 (2014); Caliando et al, Nature Communications 6:6989;
US Patent
No. 6,603,061; US Patent No. 7,868,149; US 2009/0100536; Morrell et al., Nat
Rev Genet.
2011 Dec 29;13(2):85-96). Protocols for targeted plant genome editing via
CRISPR/Cas9
are also available in volume 1284 of the series Methods in Molecular Biology
pp 239-255
February 2015.
In some embodiments, the invention provides new Kanghan polypeptide sequences,
which
may be produced from wild type Kanghan proteins by a variety of molecular
biological
10 techniques. It is well known in the art that some modifications and
changes can be made
in the structure of a polypeptide without substantially altering the
biological function of
that peptide, to obtain a biologically equivalent polypeptide. As used herein,
the term
"conserved amino acid substitutions" refers to the substitution of one amino
acid for
another at a given location in the peptide, where the substitution can be made
without any
appreciable loss or gain of function, to obtain a biologically equivalent
polypeptide. In
making such changes, substitutions of like amino acid residues can be made on
the basis
of relative similarity of side-chain substituents, for example, their size,
charge,
hydrophobicity, hydrophilicity, and the like, and such substitutions may be
assayed for
their effect on the function of the peptide by routine testing. Conversely, as
used herein,
the term "non-conserved amino acid substitutions" refers to the substitution
of one amino
acid for another at a given location in the peptide, where the substitution
causes an
appreciable loss or gain of function of the peptide, to obtain a polypeptide
that is not
biologically equivalent.
In some embodiments, conserved amino acid substitutions may be made where an
amino
acid residue is substituted for another having a similar hydrophilicity value
(e.g., within a
value of plus or minus 2.0), where the following hydrophilicity values are
assigned to
amino acid residues (as detailed in U.S. Pat. No. 4,554,101): Arg (+3.0); Lys
(+3.0); Asp
(+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); G1n (+0.2); Gly (0); Pro (-0.5);
Thr (-0.4); Ala
(-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); Ile (-
1.8); Tyr (-2.3);
Phe (-2.5); and Trp (-3.4). Non-conserved amino acid substitutions may be made
were the
hydrophilicity value of the residues is significantly different, e.g.
differing by more than
2Ø
CA 3017921 2018-09-19
In alternative embodiments, conserved amino acid substitutions may be made
where an
amino acid residue is substituted for another having a similar hydropathic
index (e.g.,
within a value of plus or minus 2.0). In such embodiments, each amino acid
residue may
be assigned a hydropathic index on the basis of its hydrophobicity and charge
.. characteristics, as follows: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe
(+2.8); Cys (+2.5); Met
(+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-
1.3); Pro (-1.6);
His (-3.2); Glu (-3.5); Gin (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and
Arg (-4.5).
Non-conserved amino acid substitutions may be made were the hydropathic index
of the
residues is significantly different, e.g. differing by more than 2Ø
In alternative embodiments, conserved amino acid substitutions may be made
where an
amino acid residue is substituted for another in the same class, where the
amino acids are
divided into non-polar, acidic, basic and neutral classes, as follows: non-
polar: Ala, Val,
Leu, Ile, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral:
Gly, Ser, Thr,
Cys, Asn, Gin, Tyr. Non-conserved amino acid substitutions may be made were
the
residues do not fall into the same class, for example substitution of a basic
amino acid for
a neutral or non-polar amino acid.
Example 1: Arabidopsis Kanghan Genes
This Example illustrates that drought tolerance in Arabidopsis is conferred by
novel QTLs
located on three different chromosomes. These genes were identified in an
extremely
.. drought tolerant Arabidopsis ecotype, designated herein as #95. The #95
ecotype was
isolated during a series of drought treatment experiments, and assessed as
follows.
In one assay, 36 plants of ecotype Col and 36 plants of ecotype #95 were used
for drought
sensitivity testing. At the outset, soil for each pot was dried and weighed to
ensure that
each pot had the same amount of soil, after which water was added to maintain
moisture.
Seeds from Col and #95 were first germinated, then sown one seedling per pot
separately.
The plants were grown in a controlled environment under long-day conditions
(16-h-
light/8-h-dark cycle) at 23 C, light intensity of 50 pmol m-2 s-1 and 70%
relative humidity
(rH). Watering was stopped for both Col and #95 plants three weeks after
germination, and
all pots were then weighed again, and additional water was supplied to keep
every pot at
the same weight. Thereafter, drought treatment was initiated and survival days
were
recorded for both ecotypes. After a period of 15 days without watering, all 36
plants of
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CA 3017921 2018-09-19
ecotype Col had died. In contrast, the plants of ecotype #95 retained
considerable vigor,
and fully recovered to maturity when water supply was resumed.
The extreme drought tolerance Arabidopsis ecotype #95 was particularly evident
after
withdrawing water for 38 days. Plants of the ecotype Col were all severely
wilted due to
drought. Ecotype #95, in contrast, still exhibited clear vigor. The Fl progeny
between Col
and # 95 were also sensitive to drought, indicating the recessive nature of
the #95 drought
resistant trait. In one assay, 27 days after water was withdrawn, the plants
were segregated
into two groups, those that had died, and those that maintained vigor and were
recoverable
to full maturity when watering was resumed. In alternative drought tolerance
tests of F2
.. progeny derived from a cross between Co/ and #95, segregation of F2
population plants
after drought treatment (50 days after water withdrawal) was much lower than
3:1. This
segregation is consistent with the involvement of major QTL in controlling the
drought
tolerance trait. Figure 1 is graph illustrating the drought tolerance
diversity of the F2
generation of these Arabidopsis plants (col x #95). Segregation of the drought
tolerance
trait from 500 F2 individual lines was calculated by the survival days after
drought
treatment (cessation of watering). In Figure 1, the survival in days of Col
and #95 plants
are marked by arrows with legends. The normal distribution for the phenotype
of F2
drought tolerance indicates that several QTLs govern the drought tolerance
trait.
Map based cloning through crossing with ecotype Col, revealed that the drought-
related
trait was governed by three major QTLs distributed on three different
chromosomes. To
delineate the underlying genetic components, an Fl generation was developed
from the
seeds of a cross between Col and #95. The Fl seeds were then used to develop a
large F2
population of 5000 lines. The F2 populations showed significant segregation of
the drought
tolerance trait, with some plants showing significant drought tolerance, and
others showing
no drought tolerance, which indicated that the drought tolerance trait of #95
was controlled
by several QTLs.
A fine mapping of the genes was further pursued using 500 lines of this
population from
which 20 extremely drought tolerant individuals and 20 extremely drought
sensitive
individuals were selected to conduct a Bulk Segregate Analysis (BSA) with 106
molecular
markers which cover all 5 chromosomes of Arabidopsis. Based on this analysis,
three
major QTLs distributed on three different chromosomes were identified.
Specifically,
QTL's were identified on chromosomes 1, 4 and 5 of the Arabidopsis genome. The
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CA 3017921 2018-09-19
contribution rates of these 3 loci to the observed drought tolerance trait
were 13.8%, 29.3%,
37.7%, respectively, explaining in the aggregate more than 80% of the drought
tolerance
variation between ecotype #95 and Col.
Fine mapping was first focused on loci on Chr.4 and Chr.5, which was carried
out using
.. 700 extremely drought tolerant individuals from a total of 5000 F2 plants.
The candidate
genes were narrowed down to two regions of 540kb on Chr.4 and 189kb on Chr.5.
Single
nucleotide polymorphism (SNP) and insertion/deletion (In/del) analysis, as
well as
expression level analysis based on the TAIR database, was carried out for all
of the genes
identified in these two regions on Chr.4 and 5.
.. The full genome sequence of ecotype #95 was compared with the full genome
sequence of
Arabidopsis ecotype Columbia (ecotype Co/). The three major QTL's associated
with
drought tolerance on Chr. 1, Chr. 4 and Chr. 5 of ecotype #95 were revealed to
harbor
members of a protein coding gene family: At1g51670, At4g29760, At4g29770,
At5g18065
and At5g18040. An additional member of the gene family was recognized by
sequence
.. similarity: At1g48180. This gene family is designated herein as the Kanghan
gene family,
the first 5 of which have very strong roles in drought tolerance (a GenBank
database
accession number for a protein encoded by each of the native Arabidopsis genes
is given
after the gene name in brackets): Kanghanl (At5g18040; NP_197305.1), Kanghan2
(At4g29770; NP_001154277.1), Kanghan3 (At1g51670; NP_175578.2), Kanghan4
(At4g29760; NP 194705.1), Kanghan5 (At5g18065; NP_680172.2), Kanghan6
(At1g48180; NP_175252.1).
Analysis of the genomic sequence of Ecotype #95 reveals that mutations within
Kanghan
family genes are associated with drought tolerance. Specifically, in ecotype
#95, all 5
members of the Kanghan family strongly associated with drought tolerance have
dramatic
mutations. Specifically, four members of the Kanghan gene family (At4g29770,
At5g18065, At5g18040 and At1g51670) contain a premature stop codon (see
Fig.2), which
is indicative of loss-of-function mutations (null) in ecotype #95 compared to
the Col
variety. A fifth member of the Kanghan gene family, At4g29760, does not
contain a
premature stop codon, but 5 amino acid substitutions occur in the coding
region of this
.. gene. Among the five Kanghan genes strongly associated with drought
tolerance,
At5g18040, At4g29770 and At1g51670 are much more highly expressed (over 10
times)
in both Col and #95 compared to At5g18065 and At4g29760, suggesting that
At5g18040,
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CA 3017921 2018-09-19
At4g29770 and At1g51670 may in some circumstances contribute more than the
other two
genes to drought tolerance trait.
Example 2: Reversing Drought Resistance
To further illustrate the role of the Kanghan genes in drought tolerance, two
full length
Kanghan genes (AT5g18040 and At4g29770) from Arabidopsis ecotype Col were used
to
transform Arabidopsis ecotype #95, including at least 2kb 5'UTR, lkb 3'UTR and
CDS.
The transformants lost their drought resistance, confirming that the
modulation of
Kanghan gene expression plays a dramatic role in drought resistance.
A further illustration of the dramatic effect of Kanghan genes on drought
tolerance was
provided by introducing five Kanghan gene alleles from ecotype #95 into
ecotype
Columbia (Col) by crossing and molecular marker based selection, generation by
generation. The 71h generation of backcrossed lines was used for self-crossing
to provide
homozygous plants which contained the five Kanghan gene alleles from #95
strongly
associated with drought tolerance. These homozygous plants were subjected to
drought
treatment. The result was that introduction of the #95 Kanghan gene alleles
rendered
ecotype Columbia drastically enhanced in its drought tolerance traits.
A further illustration of the effect of Kanghan genes on abiotic stress
response was
provided by measuring the canopy temperatures of Col, #95 and the backcrossed
lines
bearing the Kanghan alleles. Increased canopy temperature was clearly evident
in #95
plants and backcrossed lines, when compared with Col ecotype plants. Further,
subjecting
seedlings of #95 and Col to heat treatment at 45 C confirmed heat sensitivity
in ecotype
#95.
As this Example illustrates, functional expression of Kanghan gene family
proteins plays
a positive role in heat tolerance, and a negative role in drought tolerance.
The invention
accordingly provides a variety of avenues for modulating abiotic stress
response in plants.
In some embodiments, this involves balancing Kanghan gene expression to
achieve a
desired phenotype of abiotic stress response, for example balancing drought
and heat
tolerance.
The negative role of the Kanghan family of genes in drought tolerance serves
as a basis for
improving plant drought tolerance by down-regulating or silencing members of
the
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CA 3017921 2018-09-19
Kanghan gene family. This may for example be achieved through a wide variety
of
techniques, including mutagenesis (TILLing) or targeted gene editing, as
discussed above.
Example 3: Kanghan Sequence Similarity and Protein Domains
Table 1: BLAST alignments of Kanghan proteins, with AT4G29770 as reference
sequence.
Percent Percent Length of
Sequence Identitie Positive Alignmen Mismatche Gap
Accession Gene
NP 001154277. AT4G2977
1 0 100 100 329 0 0
AT4G2976
NP _194705.1
0 60.432 73.38 278 108 2
AT5G1804
NP 197305.1 0 48.227 60.99 282 112 4
AT5G1806
NP 680172.2
63.415 73.98 123 42 1
AT1G4818
NP 175252.1 0 35.907 50.19 259 120 5
AT1G5167
NP 175578.2
0 36.628 49.42 172 70 4
5
Table 2: Continuation of BLAST alignments of Kanghan proteins, with AT4G29770
as
reference sequence.
Sequence Query Query Subject Subject ..
Max
Accession Start End Start End E Value Score
NP 001154277.1
1 329 1 329 0 685
NP 194705.1 54 329 3 280 2.53E-115 347
NP 197305.1 51 329 2 252 1.06E-80 258
NP 680172.2 56 178 7 126 1.16E-42 155
NP 175252.1 77 329 21 239 1.45E-39 150
NP 175578.2 80 249 28 162 8.24E-20 95.1
Table 3: BLAST alignments of Kanghan proteins, with AT1G51670 as reference
sequence.
Percent Length of
Identitie Percent Alignmen Mismatche Gap
Positives t
CA 3017921 2018-09-19
NP 175578.2 AT1G5167
0 100 100 178 0 0
NP_175252.1 AT1G4818
0 65.625 76.25 160 48 3
NP 194705.1 AT4G2976
0 43.407 53.85 182 62 7
NP 001154277. A14G2977
1 0 36.628 49.42 172 70 4
NP 197305.1 AT5G1804
0 45.833 60.42 96 47 4
NP 680172.2 AT5G1806
43.75 51.04 96 21 2
Table 4: Continuation of BLAST alignments of Kanghan proteins, with AT1G51670
as
reference sequence.
Query Query Subject Subject Max
Start End Start End E Value Score
NP 175578.2 1 178 1 178 6.95E-127 366
NP 175252.1 1 160 1 153 5.32E-61 201
NP 194705.1 20 162 19 198 6.40E-25 108
NP1001154277 .1
28 162 80 249 4.46E-20 95.1
NP 197305.1 69 162 77 169 9.09E-13 73.6
NP_680172.2 28 90 31 126
1.36E-09 63.2
5 As set out in the tables above, which alternatively set out BLAST alignments
with
reference sequences that are the most divergent of the Kanghan genes
(AT4G29770 and
AT1G51670) the Kanghan gene family may be defined as including genes that
encode
proteins, that when optimally aligned, have at least 35% identity and/or at
least 49%
positive alignments, over a length of at least 90 amino acids, with BLOSUM or
PAM
substitution matrix, with gaps permitted.
This Example further illustrates the existence of conserved protein domains
encoded by
Kanghan family genes, as depicted in Figs. 3 through 7.
Conserved domain A is close to the amino end of the proteins, and as shown in
Figures 4
and 5, comprises a region that may be defined as having a reasonably high
degree of
consensus (80%) to the following sequence:
31
CA 3017921 2018-09-19
cppDYDtStpAAhVAlpLISSAR1hLKIDuhhTEYSsQaLhDpsutpp. Alternatively, at a
slightly reduced level of consensus, conserved domain A may be defined as
comprising a
region that is defined as having a reasonably high degree of consensus (70%)
to the
following sequence:
spphhpShupscGhCHPDC-
KAssEpEDYDASQpAAhVAVsLISSAR1hLKLDusaTEYSAQYLVDNAGpccs
Conserved domain B, as shown in Figures 4 and 6, comprises a region that may
be defined
as having a high degree of consensus (100%) to the following sequence:
hTVKDChphAhp.
Alternatively, at a reduced level of consensus, conserved domain B may be
defined as
comprising a region that is defined as having at least 80% identity to the
following
sequence: LTVKDCLEhAhK-G. Alternatively, at a further reduced level of
consensus,
conserved domain B may be defined as comprising a region that is defined as
having at
least 70% identity to the following sequence: LTVKDCLEhAFKKG.
Conserved domain C, as shown in Figures 4 and 7, comprises a region that may
be defined
as having at least 80% identity to the following sequence:
VshKGpVlEstshpEs.chhhpQs-
huA+LHIFpPph. Alternatively, at a reduced level of consensus, conserved domain
C may
be defined as comprising a region that is defined as having at least 70%
identity to the
following sequence: VsMKGEVIEspsh-EAhcL11cQP-1GA+LH1FoPcl. Figures 5, 6 and 7
illustrate consensus sequences using a sequence logo, which is a graphical
representation
of an amino acid or nucleic acid multiple sequence alignment (CLUSTL W). Each
logo
consists of stacks of symbols, one stack for each position in the sequence.
The overall
height of the stack indicates the sequence conservation at that position,
while the height of
symbols within the stack indicates the relative frequency of each amino or
nucleic acid at
that position. The width of the stack is proportional to the fraction of valid
symbols in that
position - positions with many gaps have thin stacks (Crooks et al., Genome
Research,
14:1188-1190, (2004); Schneider and Stephens, 1990, Nucleic Acids Res. 18:6097-
6100).
Shading of the weblogo images reflects amino acid chemistry (AA).
Conserved domain A is absent in Kanghan1 (At5g18040) in Columbia (Col) due to
an 82
bp deletion compared to the orthologous gene in other species of Arabidopsis.
As shown
in Figure 2, the At5g18040 gene in Ecotype #95 contains conserved domain A,
before the
premature stop codon, so that the existence of this domain on its own does not
appear to
confer drought tolerance.
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CA 3017921 2018-09-19
Conserved domain B is relatively highly conserved in all members of Kanghan
gene family
in Columbia. In contrast, the premature stop codons of Kanghanl, Kanghan2,
Kanghan3,
and Kanghan5 cause the loss of conserved domain B in #95. Accordingly, this
absence of
this domain is closely associated with the drought tolerance trait.
Conserved domain C and the tasi-RNA target site are not present in Kanghan5
(At5g18065) in both Columbia and #95.
BLAST searching reveals that Kanghan family genes are widely distributed in
Brassicaceae, in addition to the six Kanghan genes in Arabidopsis thaliana,
there are also
5 members in Arabidopsis lyrata, 6 members in Caspsella rubella, 5 members in
Brassica
rapa, 11 members in Brassica napus, 3 members in Eutrema salsugineum, 1 member
in
Thellugiella parvula, and at least 24 members in Camelina sativa. Most of
these Kanghan
genes include all three conserved domains, and all of them contain conserved
domain B.
Table 5: BLASTP search results identifying plant Kanghan proteins based on
sequence
similarity to the protein encoded by AT4G29770.
Length of
Seq Gene Identities Positives
Alignment
NP 001154277.1
AT4G29770
100 100 329
CAB43652.1 hypothetical protein [Arabidopsis
thaliana] 100 100 282
NP_567833.1 target of trans acting-siR480/255
[Arabidopsis thaliana] 100 100 277
XP 002869410.1 hypothetical protein
ARALYDRAFT_491783
[Arabidopsis lyrata subsp. lyrata] 85.56 89.89 277
XP_006293511.1 hypothetical protein
CARUB_v10023817mg [Capsella
rubella] 73.188 83.33 276
XP_010447809.1 PREDICTED: uncharacterized
protein L0C104730345 [Camelina
sativa] 71.326 82.8 279
PREDICTED: uncharacterized
XP_010438266.1 protein L0C104721886 [Camelina
sativa] 70.504 82.37 278
PREDICTED: uncharacterized
XP_010433066.1 protein L0C104717221 [Camelina
sativa] 70.922 81.56 282
PREDICTED: uncharacterized
XP_010447810.1 protein L0C104730347 [Camelina
sativa] 68.1 79.57 279
33
CA 3017921 2018-09-19
Length of
Seq Gene Identities
Positives Alignment
PREDICTED: uncharacterized
XP 010436343.1 protein L0C104720070 [Camelina
satival 74.194 82.66 248
XP 002869411.1 predicted protein [Arabidopsis
_
lyrata subsp. lyratal 65.233 76.34 279
PREDICTED: uncharacterized
XP_010441644.1 protein L0C104724792 [Camelina
saliva] 66.791 78.36 268
PREDICTED: uncharacterized
XP 010494756.1 protein LOC104771851 [Camelina
saliva] 66.045 77.99 268
PREDICTED: uncharacterized
XP_010451117.1 protein L0C104733215 [Camelina
saliva] 65.108 76.26 278
PREDICTED: uncharacterized
XP 010441643.1 protein L0C104724791 [Camelina
satival 62.816 76.9 277
XP 002871796.1 predicted protein [Arabidopsis
_
lyrata subsp. lyratal 64.234 74.09 274
hypothetical protein
XP_006280936.1 CARUB_v10026934mg [Capsella
rubella] 66.415 77.36 265
PREDICTED: uncharacterized
XP 010438262.1 protein L0C104721884 [Camelina
satival 65.556 74.81 270
XP 002871797.1 predicted protein [Arabidopsis
lyrata subsp. lyratal 63.296 72.28 267
NP 194705.1 AT4G29760 60.432 73.38 278
hypothetical protein
XP_006413298.1 EUTSA_v10026005mg [Eutrema
salsugineuml 54.373 70.72 263
PREDICTED: uncharacterized
XP_013601305.1 protein L0C106308720 [Brassica
oleracea var. oleraceal 53.409 70.45 264
PREDICTED: uncharacterized
XP 013720359.1 protein L0C106424160 [Brassica
napusl 54.444 67.78 270
hypothetical protein
XP 006412791.1 EUTSA_v10027444mg [Eutrema
salsugineuml 54.412 68.75 272
PREDICTED: uncharacterized
XPO13628081.1 protein LOC106334325 [Brassica
oleracea var. oleraceal 54.851 68.28 268
PREDICIED: uncharacterized
XP_010436344.1 protein L0C104720071 [Camelina
saliva] 62.673 70.05 217
PREDICTED: uncharacterized
XP_009108974.1 protein LOC103834660 isoform X2
Brassica rapal 53.333 66.67 270
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CA 3017921 2018-09-19
Length of
Seq Gene Identities Positives
Alignment
PREDICTED: uncharacterized
XP_010438269.1 protein L0C104721889 [Camelina
sativa] 50.158 57.41 317
CDY23253.1 BnaA08g12930D [Brassica napus] 52.239 68.28 268
hypothetical protein
XP_006294905.1 CARUB_v10023956mg [Capsella
rubella] 49.811 63.77 265
PREDICTED: uncharacterized
XP 009108973.1 protein L0C103834660 isoform X1
[Brassica rap] 51.493 67.16 268
PREDICTED: uncharacterized
XP 009127652.1 protein L0C103852500 [Brassica
rap] 51.515 68.56 264
PREDICTED: uncharacterized
XP_013659423.1 protein L0C106364376 [Brassica
napus] 52.453 65.28 265
NP 197305.1 AT5G189040 48.227 60.99 282
CDX68686.1 BnaC01g07670D [Brassica napus] 53.815 70.28 249
PREDICTED: uncharacterized
XP 013668007.1 protein L0C106372351 [Brassica
napus] 50 67.42 264
PREDICTED: uncharacterized
XP_013720313.1 protein L0C106424116 isoform X2
[Brassica napus] 50.562 66.29 267
AAM64385.1 unknown [Arabidopsis thaliana] 49.451 60.81 273
PREDICTED: uncharacterized
XP_013720312.1 protein L0C106424116 isoform X1
[Brassica napus] 50.562 66.29 267
PREDICTED: uncharacterized
XP_013659411.1 protein L0C106364365 [Brassica
napus] 52 69.6 250
CDY23252.1 BnaA08g12940D [Brassica napus] 49.064 65.17 267
CDY55618.1 BnaCO3g77520D [Brassica napus] 47.94 65.17 267
PREDICTED: uncharacterized
XP 009102300.1 protein L0C103828450 [Brassica
rapal 46.792 63.77 265
PREDICTED: uncharacterized
XP_009108975.1 protein L0C103834660 isoform X3
[Brassica rap] 47.94 62.55 267
CDY55620.1 BnaCO3g77540D [Brassica napus] 48.387 63.71 248
PREDICTED: uncharacterized
XP 010495074.1 protein L0C104772124 [Camelina
sativa] 49.434 58.11 265
PREDIC1ED: uncharacterized
XP 013674022.1 protein L0C106378439 [Brassica
napus] 47.059 65.16 221
PREDICTED: uncharacterized
XP 010433021.1 protein L0C104717183 [Camelina
sativa] 40.892 55.76 269
CA 3017921 2018-09-19
Length of
Seq Gene
Identities Positives Alignment
PREDICTED: uncharacterized
XP 010438210.1 protein L0C104721842 [Camelina
sativa] 42.804 54.98 271
PREDICTED: uncharacterized
XP_010447759.1 protein L0C104730304 [Camelina
saliva] 42.857 55.64 266
hypothetical protein
XP_006393225.1 EUTSA_v10011766mg [Eutrema
salsugineum] 42.912 52.87 261
CDY55622.1 BnaCO3g77550D [Brassica napus] 43.939 56.82
264
hypothetical protein
KFK22930.1 AALP_AAs51418U000100 [Arabis
alpina] 42.339 55.24 248
PREDICTED: uncharacterized
XP_010447760.1 protein L0C104730305 [Camelina
saliva] 42.578 55.08 256
XP 002894098.1 F21D18.8 [Arabidopsis lyrata
_
subsp. lyrata] 39.147 52.33 258
PREDICTED: uncharacterized
XP_009108976.1 protein L0C103834661 [Brassica
rapa] 47.541 64.48 183
PREDICTED: uncharacterized
XP_010479661.1 protein L0C104758482 [Camelina
saliva] 39.683 53.97 252
PREDICTED: uncharacterized
XP_010462001.1 protein L0C104742681 [Camelina
saliva] 39.044 53.39 251
hypothetical protein
KFK30349.1 AALP_AA7G249900 [Arabis
alpina] 42.387 54.73 243
hypothetical protein
XP_006304151.1 CARUB_v10010162mg [Capsella
rubella] 39.768 53.28 259
PREDICTED: uncharacterized
30_010482049.1 protein L0C104760782 [Camelina
saliva] 38.672 51.95 256
NP 680172.2 AT5G18065 63.415 73.98 123
PREDICTED: uncharacterized
XP_010479658.1 protein L0C104758479 [Camelina
sativa] 36.822 52.33 258
XP002891651.1 predicted protein [Arabidopsis
_
lyrata subsp. lyrata] 38.492 51.98 252
NP 175252.1 AT1G48180 35.907 50.19 259
hypothetical protein
XP_006304149.1 CARUB_v10010150mg [Capsella
rubella] 36.863 51.37 255
hypothetical protein
XP_002891717.1 ARALYDRAFT 892299
[Arabidopsis lyrata subsp. lyratal 37.549 51.78 253
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CA 3017921 2018-09-19
Length of
Seq Gene Identities Positives
Alignment
PREDICTED: uncharacterized
XP_010471249.1 protein LOC104751067 [Camelina
sativa] 36.957 50.87 230
AAF79518.1 F21D18.8 [Arabidopsis thaliana] 35.125 48.39 279
hypothetical protein
XP_006303339.1 CARUB_v10010206mg [Capsella
rubella] 36.8 51.6 250
PREDICTED: uncharacterized
XP_010501962.1 protein L0C104779303 [Camelina
saliva] 34.348 49.57 230
AAG50884.1 unknown protein [Arabidopsis
thaliana] 36.111 49.6 252
PREDICTED: uncharacterized
XP_010442215.1 protein L0C104725285 [Camelina
saliva] 38.095 50.6 168
PREDICTED: uncharacterized
XP_010500744.1 protein L0C104778076 [Camelina
saliva] 30.038 44.49 263
hypothetical protein
KFI(24575 .1 AALP_AAs45078U000200 [Arabis
alpina] 47.581 61.29 124
NP_175578.2 AT1G51670 36.628 49.42 172
PREDICTED: uncharacterized
XP_013684707.1 protein L0C106389038 isoform X1
[Brassica napus] 31.818 50 176
CDY43538.1 BnaA01g07060D [Brassica napus] 31.818 50 176
PREDICTED: uncharacterized
XP_013684772.1 protein L0C106389038 isoform X2
[Brassica napus] 31.818 50 176
PREDICTED: uncharacterized
XP_013596364.1 protein L0C106304487 isoform X3
[Brassica oleracea var. oleracea] 35.537 53.72 121
PREDICTED: uncharacterized
XP_013750812.1 protein LOC106453111 isoform X3
[Brassica napus] 33.871 50 124
PREDICTED: uncharacterized
XP_013750806.1 protein L0C106453111 isoform X1
[Brassica napus] 33.871 50 124
NP 001154277.1 target of trans acting-siR480/255
_
[Arabidopsis thaliana] 33.884 50.41 121
Example 4: Modulating abiotic stress response in wheat with Kanghan genes
This example illustrates a genetic modification of a wild-type wheat by gene
gun mediated
transformation using a Kanghan gene construct, to modulate an abiotic stress
response, in
this case conferring heat tolerance. Transgenic constructs for
overexpression of
37
CA 3017921 2018-09-19
Arabidopsis Kanghan family genes in wheat were produced using monocot special
overexpression vector PANIC5E. This vector was designed for stable
transformation and
overexpression of heterologous Kanghan genes in wheat. Over expression of
Arabidopsis
Kanghanl (At5g18040) in one wheat wild type (Fielder) was achieved in this way
by gene
gun mediated transformation. The construct used to perform this transformation
is shown
in Fig. 8.
To illustrate the heat tolerance of the wheat transgenic lines, three-week
seedlings of both
wild types and Ti transgenic lines were heat treated at 42/38 C (day/night).
After two
weeks of heat treatment, recovery at normal growth temperature was performed,
and
phenotypes observed. Heat tolerance was clearly observed in Ti transformants
compared
to non-transgenic plants under heat treatment. Non-transgenic plants displayed
wilt
symptoms or died. The transformants, on the other hand, recovered after
transferring to
normal growth temperature conditions, and were able to grow normally and
transit to
reproductive growth.
To further illustrate the heat tolerance of the wheat transgenic lines, three
week old
seedlings of both wild-type and Ti transgenic lines were subjected to 40/38 C
(day/night)
for three weeks, followed by a three week recovery period at 25 C. After this
recovery
period, the transgenic plants fully recovered whereas the control plants
failed to recover
(Figures 9A and 9B). After a further seven weeks at 25 C, the transgenic
plants reached
maturity and produced seeds (Figure 9C).
Under standard growth conditions of 23 C day/18 C night, 16 h photoperiod (16
h light/8
h dark), and 200 jtmol m-2s-1 light intensity wild-type and transgenic plants
are visually
indistinguishable, however as determined by infrared thermal imaging using FUR
T640
Infrared Camera, the canopy temperature of Ti transgenic wheat plants is
significantly
lower (Figure 10).
These studies illustrate the utility of the Kanghan genes in modulating
abiotic stress
response in crop species such as wheat, in this case to improve heat
tolerance.
Example 5: Identifying Kanghan homologs in Brassica napus
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CA 3017921 2018-09-19
A BLAST sequence search was carried out on available genome and transcript
data from
Brassica napus to identify potential homologues of at4g29770, at4g29760,
at5g18040,
at5g18065, at1g51670, and at1g48180. The potential candidates identified are
provided in
Table 6.
Table 6: Homologs of Arabidopsis thaliana Kanghan family genes in Brassica
napus.
Homologs of Homologs of
at4g29770, at4g29760, at1g51670 and
at5g18040, and at5g18065 at1g48180
BnaA01g06470D BnaC01g08520D
BnaA07g02270D BnaC01g08490D
BnaA08g12920D BnaA01g07060D
BnaA08g12930D
BnaA08g12940D
BnaC01g07670D
BnaCO3g77520D
BnaCO3g77540D
BnaCO3g77550D
A DNA neighbor phylogenetic tree of the Brassica napus Kanghan gene candidates
and
their Arabidopsis thaliana counterparts is provided in Figure 11 and a protein
neighbor
phylogenetic tree is provided in Figure 12. A DNA neighbor phylogenetic tree
of the
Brassica napus Kanghan gene candidates is shown in Figure 13. The candidates
indicated
by arrows were selected for targeting by RNAi.
Example 6: Targeting Brassica napus Kanghan genes by RNAi
Primer design
Two conserved fragments from 12 putative Brassica napus Kanghan genes,
identified
based on ClustalW multiple alignment, were used to design two pairs of RNAi
primers.
The reverse primers were designed to include a BamH1 restriction site and a
Sall
restriction site to facilitate cloning.
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CA 3017921 2018-09-19
The first primer pair was designed to target BnaCO3g77540D (L0C106364365):
RNAiF1 GP438: TAGATTCTGCTGAGAGAGCCGCTAC (SEQ ID NO: 20)
RNAiR1 GP439: GGATCCGTCGACGCACCTATGGGTCCATGCTTTAAC (SEQ ID
NO: 21)
The second primer pair was designed to target BnaA08g12920D (L0C106424160):
RNAiF2 GP440: TCATCCAGATTGCCAACGAG (SEQ ID NO: 22)
RNAiR2 GP441: GGATCCGTCGACACGCATCCTCCAGTGTCTTAG (SEQ ID NO:
23)
Production of BnKanghan RNAi construct and establishment of Brassica napus
RNAi
lines
To generate a cDNA library of Brassica napus, total RNA was isolated from 3-
week-old
leaves of canola wild type 'Hero' using the Plant RNeasy Mini Kit (Qiagen).
Then, RNA
samples were used for library construction using the QuantiTect Reverse
Transcription Kit
(Qiagen). The primer pairs RNAiF1 GP438 (SEQ ID NO: 20) + RNAiR1 GP439 (SEQ
ID NO: 21) and RNAiF2 (SEQ ID NO: 22) + RNAiR2 GP441 (SEQ ID NO: 23) were used
separately to amplify fragments from two target BnKanghan genes from the
obtained
cDNA library. Each of the resulting PCR products was isolated and cloned into
the
pGEMO-T vector (Promega, USA). A map of the pGEM-T vector is provided in
Figure
14. Then, two copies of the Kanghan gene fragments were subcloned into the
pCAMBIA
1301-35S-Int-T7 vector in opposite orientations using a Pstl, Sall digest and
a BamH1,
Sad l digest to generate two RNAi constructs, one for each gene fragment. A
map of the
pCAMBIA 1301-35S-Int-T7 vector is provided in Figure 15 and a partial map of
the
resulting RNAi constructs is provided in Figure 16.
Next, a genetic modification of canola wild type 'Hero' was conducted using
both of these
completed RNAi constructs through agrobacterium-mediated transformation aimed
to
obtain increased drought tolerance. Positive transformants were confirmed
using a pair of
hygromycin specific primers (HptF TACACAGCCATCGGTCCAGA (SEQ ID NO: 24)
and HptR GTAGGAGGGCGTGGATATGTC (SEQ ID NO: 25)). A cross was carried out
between Ti positive transformants from the two different constructs. In the C2
generation,
CA 3017921 2018-09-19
lines harboring both constructs together were selected for further evaluation
of silencing
of BnKanghan family genes and drought tolerance traits.
To assess the expression level of BnKanghan family genes in transgenic and
crossing lines,
a number of primer pairs were designed for qRT-PCR assays to assess the
expression levels
of seven candidate Kanghan genes from Brassica napus. The targets of these
primer pairs
are identified in Table 7. In total, 12 lines harboring both RNAi constructs
from the C2
generation were selected to detect expression level changes of BnKanghan
family genes.
Each line tested showed decreases in expression of at least three BnKanghan
genes.
Individual lines C2-83-20 and C2-83-10 each showed decreased expression of six
BnKanghan genes. These two lines were selected for further drought tolerance
measurements.
Table 7: qRT-PCR primers targeting BnKanghan family genes.
primer SEQ ID product
no. NO: primer sequence length Kanghan gene
GP635 26 CGCTACGAGGCACGTACTCAAT 103 BnaA07g02270D
GP636 27 CTCGGTCTTCCCCGGTTTC
GP637 28 GCTTAGAGACGTGATCCTGGTAGC 128 BnaA08g12920D
GP638 29 CCAGTGTGGTGAACATACGGC
GP639 30 GTTTTGTTGGTCTCTTCTCTTTGC 71 BnaC01g07670D
GP640 31 TIC1TAAGAGGCGIT1CAGATGG
GP641 32 TGA1TTGGG1-1'1'1GCCTGATAC 69 BnaCO3g77540D
GP642 33 GAAACAAACCATAAATGAGTTGCC
GP645 34 CATITGGGATGTGTCGATTGAG 165 BnaCO3g77550D
GP646 35 CCCACGTAGC1-1 GITCCGTT
GP649 36 AACACTGTCACGCAGATTGCC 124 BnaA01g06470D
GP650 37 CTGTCCAGGTTAGCTACCATACGA
GP655 38 CGGTATCCAACTCATTCGAAGG 121 BnaC01g08490D
GP656 39 TCAAGTATATACTGGGTTGGCTGC
Testing canopy temperature and drought tolerance of Brassica napus RNAi lines
To predict the potential drought tolerance of the C2-83-20 and C2-83-10 lines,
the canopy
temperatures were measured using an infrared camera. In comparison to wild
type plants,
higher canopy temperatures were observed for the transgenic plants (Figure 17)
indicating
a lower leaf water potential. These RNAi phenotypes are similar to loss-of-
function alleles
of AtKanghan genes in Arabidopsis, which suggests a similar role for the
BnKanghan
genes in canola.
41
CA 3017921 2018-09-19
To assess the drought tolerance of the transgenic plants, four weeks-old
plants of both wild
type and these two transgenic lines were subjected to drought treatment. The
same amount
of soil and water were applied to each individual plant before treatment, and
then the water
supply was stopped. After two weeks of drought treatment, recovery by re-
watering of the
plants was performed. The resulting phenotypes are shown in Figures 18 and 19.
Increased
drought tolerance was clearly observed in transgenic lines compared to wild
type plants
under drought conditions that lead to wilt symptoms or death of the wild type
plants. The
transformants, on the other hand, recovered after being transferred to normal
watering
conditions and were able to grow up normally and transit to reproductive
growth. This
demonstrates that silencing of BnKanghan family genes in crop species, such as
canola,
can improve drought tolerance.
While the present application has been described with reference to specific
examples, it is
to be understood that the application is not limited to the disclosed
examples. To the
contrary, the present application is intended to cover various modifications
and equivalent
arrangements encompassed by the scope of the appended claims viewed in light
of the
teaching of the description as a whole.
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CA 3017921 2018-09-19
