Note: Descriptions are shown in the official language in which they were submitted.
WO91/13992 PCTtGB9~/00416
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PLANT PROMOTER
The present invention relates to a plant promoter.
Extensin is the best characterised structural
protein in the walls of plant cells (Cassab and Varner, Ann.
Rev. Plant Physiol. 39, 321-353, 1988). It is a member of a
class of highly basic, hydroxyproline-rich glycoproteins
present in a wide variety of plants. Extensin has been
reported to be present at greatest abundance in sclerenchyma
tissues and a variety of functional roles have been ascribed
to the protein, including mechanical strengthening and
organisation of the cell wall. Extensins vary in their ~ -
primary structure in different plants, organs and tissues.
In some cases, there are two different extensins within the
same tissue or cell type, which may be function-related.
As part of a programme to isolate plant genes
expressed in an organ-specific manner, mRNA species present
in the roots of oilseed rape (Brassica napus L.) were
investigated, with the aim of isolating se~uences abundant
in root but not in other tissues. A family of cross-
hybridising sequences present at highly enhanced levels in
root has been isolated, characterised, and shown to encode
proteins homologous to extensin. This has enabled us to
identify a promoter capable of directing the expression of
proteins in roots.
Accordingly, the present invention provides a
promoter which is capable of directing protein expression in
roots of plants and which has:
(a) the seguence from nucleotide -1616 to
nucleotide -1 shown in ~igure 1 or a part of the said
sequence or
; (b) a said sequence (a) modified by one or more
nucleotide substitutions, insertions and/or deletions and/or
by an extension at either or each end.
The invention also provides a DNA fragment
comprisin~ such a promoter operably linked to a gene,
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WO91/13992 PCT/GB91/00416
2~78327 ~
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typically a heterologous gene encoding a protein.
Additionally provided is a vector which comprises a gene
encoding a protein under the control of a promoter as above
such that the gene is capable of being expressed in a plant
cell transformed with the vector. A suitable vector is one
in which the promoter is fused directly to the 5'-end of the
gene. The vector may further contain a region which enables
the gene and the promoter to be transferred to and stably
integrated in a plant cell genome. The vector is generally
a plasmid.
Plant cells can be transformed with such a vector.
The invention therefore further provides plant cells which
harbour a promoter as above operably linked to a gene
encoding a protein. Transgenic plants may be regenerated
from such plant cells. A transgenic plant can be obtained
which harbours in its cells a promoter as above operably
linked to a gene encoding a protein. Seed may be obtained
from the transgenic plants. Plants may in turn be grown
from this seed.
The invention additionally provides a method of
producing a transgenic plant capable of producing a desired
protein in the roots of the plant, which method comprises:
(i) transforming a plant cell with a vector
according to the invention, the protein encoded by the gene
under the control of the said promoter being the desired
protein; and
(ii) regenerating plants from the transformed
cells.
In the accompanying drawings:
Figure l shows the nucleotide sequence of gene
extA, with the predicted amino acid sequence of the rape
extensin polypeptide, and the sequence of the promoter of
the invention from nucleotide -1616 to nucleotide -l. The
complete sequence is the sequence of the 2.7kb HincII-HincII
fragment shown in Figure 2. The predicted site of cleavage
of the leader sequence is indicated by a colon (:). The
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WO91/t3992 PCT/GB91/00416
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transcription start point is indicated by a circumflex
(T^S), and a sequence similar to the consensus "TATA"
control box is also indicated. Other sequence features are
as indicated on the Figure.
Figure 2 is a restriction map of the oilseed rape
genomic clone lambdaB31 (12.7 kb) encoding gene extA, a
restriction map of pRlambdaS4, a HindIII-AvaI fragment from
lambdaB31 in pUC18, and a sequencing map of the 2.7kb
HincII-HincII fragment containing extA. ra and la, indicate
right arm and left arm of the EMBL 3 lambda vector,
respectively; ~ \ \\\~ indicates coding sequence.
Sa/H2-Al/Sm/Xl fragment is a part of EMBL 3. Key to
restriction site symbols on sequencing map: Sa, Sal I; H3,
Hind III; Sm, SmaI; H2, Hinc II; Pl, Pst I; AI, AvaI; XI,
Xma I; Ns, Nsi I; RI, Rsa I; Ha, Hae III; Ss, Ssp I; Nd, Nde
I; Pv, Pvu II.
Figure 3 shows a detailed restriction map of
pRlambdaS4, a sub-clone from ~B31 comprising a HindIII-AvaI
fragm~nt of lambdaB31 in pUC18.~ shows the location and
direction of transcription of the extensin coding sequence.
There are no sites in the insert for PvuI, XhoI, BscI,
BamHI, BglII and EcoRV.
Figure 4 shows a simplified restriction map of the
sub-clone pRlambdaS4;~ indicates the position of the
extensin coding sequence andl lindicates the region
containing the promoter conferring root expression.
Figure 5 is a map of the hybrid gene employed in
Example 2; ~ indicates the nopaline synthase (NOS)
terminator.
Figure 6 is a map of the hybrid gene from Figure 5
in the vector BIN 19.
Figure 7 shows the rape extensin promoter on a
HaeIII fragment excised from lambdaB3I, as described in
Exampl~ 3.
Figure 8 shows the fusion construct comprising the
rape extensin promoter and the glucuronidase gene coding
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WO91/13992 PCT/GB91/00416
2~783`%~
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sequence, prepared in Example 3.
The full length promoter of the invention is
composed of the sequence upstream of the oilseed rape extA
gene, from nucleotide -1616 to nucleotide -1 in Figure 1,
base 1 being the adenine (~) base of the CAT transcriptional
start codon for the extA gene. The promoter may be obtained
from a clone, lambdaB31 by excision on a 4.5kb HindIII-AvaI
fragment (Figure 3). Clone lambdaB31 was obtained from a
genomic library prepared from oilseed rape (Brassica naPus
L). The HindIII-AvaI fragment was subcloned in pUC18 as
pRlambdaS4 from which the promoter can be excised as a
0.96kb HaeIII fragment. E. coli D~5~ harbouring pRlambdaS4
and bacteriophase lambdaB31 have been deposited at the
National Collection of Industrial and Marine Bacteria,
Aberdeen, GB on 8 March 1990 under accession numbers NCIMB
40265 and NCIMB 40266.
A part of the full length sequence from nucleotide
-1616 to nucleotide -1 and modified forms of the full length
or part sequence are alternative promoters according to the
invention. The full length or part promoter sequence may be
modified by one or more nucleotide-substitutions, insertions -
and/or deletions and/or by an extension at either or both
ends. Any part or modified promoter sequence must however
still be capable of acting as a promoter in the roots of
plants. When the full length sequence or a part of the full
length sequence, i.e. an unmodified sequence, is modified
typically there is a degree of homology of at least 60
between a modified sequence and the unmodified natural
sequence. The degree of homology may be at least 75~, at
least 85~ or at least 95%.
A part of the full length promoter sequence may be
obtained by use of restriction endonucleases and/or
exonucleases. The promoter may be obtained as a 0.96 kb
fragment by treating the sequence shown in Figure 1 with
HaeIII. A modified sequence may be obtained by introducing
changes into an unmodified promoter sequence. This may be
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WO9l/13992 PCT/GB91/OW16
~ 2~783~7
achieved by any appropriate technique, including restriction
of the natural sequence with an endonuclease, insertion of
oligonucleotide linker adapters, use of an exonuclease
and/or a polymerase and site-directed mutagenesis.
Whether a part of the full length promoter sequence
or a modified sequence is capable of acting as a promoter
may be readily ascertained by experiment. The putative
promoter sequence is fused to the glucuronidase coding
sequence in the plomoter-less binary vector pBI101 at the
SmaI site as described in Example 3. Following the
procedure of Example 3, expression of glucuronidase is
investigated in the transgenic hairy roots produced.
The promoter may be operably linked to a gene,
typically a heterologous gene, encoding a protein. The
heterologous gene may encode any protein it is desired to
express. "Heterologous" means that the gene is not
naturally operably linked to the promoter, i.e. a
heterologous gene is not the oilseed rape extA gene. The
protein may additionally comprise a transit peptide sequence
at its N-terminus, encoded within the "heterologous" gene
sequence.
The prom~ter is typically used to express proteins
in the roots of plants. The protein whose expression is
controlled by the promoter may be for example a protein
conferring biological control of pests or pathogens, in
particular pests or pathogens to which the roots of plants
are susceptible.
The promoter sequence may be fused directly to a
gene or via a linker. The linker sequence may comprise an
intron. Excluding the length of any intron sequence, the
linker may be composed of up to 4S bases, for example up to
30 or up to 15 bases.
DNA fragments and vectors can be prepared in which
the promoter is operably linked to a gene, typically a
heterologous gene. The fragments and vectors may be single
or double stranded. Plant cells can be transformed by such
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WO91~1~992 PCT/GB91/00416
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fragment by direct DNA uptake, or by way of such a vector.
The vector incorporates the gene under the control of the
promoter. The vector contains regulatory elements capable
of enabling the gene to be expressed in a plant cell
transformed with the vector. Such regulatory elements
include, besides the promoter, translational initiation
and/or termination sequences. The vector typically also
contains a region which enables the gene and associated
regulatory control elements to be transferred to and be
stably integrated in the plant cell genome.
The vector is therefore typically provided with
transcriptional regulatory sequences and/or, if not present
at the 3'-end of the coding sequence of the gene, a stop
codon. A DNA fragment may therefore also incorporate a
terminator sequence and other sequences which are capable of
enabling the gene to be expressed in plant cells. An
enhancer or other element able to increase or decrease
levels of expression obtained in particular parts of a plant
or under certain conditions may be provided in the DNA
fragment and/or vector. The vector is also typically
provided with an antibiotic resistance gene which confers
resistance on transformed plant cells, allowing transformed
cells, tissues and plants to be selected by growth on
appropriate media containing the antibiotic.
Transformed cells are selected by growth in an
appropriate medium. Plant tissue can therefore be obtained
comprising a plant cell which harbours the gene under the
control of the promoter , for example in the plant cell
genome. The gene is therefore expressible in the plant
cell. Plants can then be regenerated which include the gene
and the promoter in their cells, for example integrated in
the plant cell genome, such that the gene can be expressed.
The regenerated plants can be reproduced and, for example,
seed obtained. Root-specific expression of proteins can
therefore be directed by the present promoter in plants.
Alternatively, transformed roots may be grown in culture for
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WO 91/13992 PCI/GP~9~ 416
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the purposes of production of proteins, especially plant
proteins.
A preferred way of transforming a plant cell is to
use Aqrobacterium tumefaciens containing a vector comprising
the promoter operably linked to the gene encoding a protein
it is wished to express. A hybrid plasmid vector may
therefore be employed which comprises:
(a) the gene under the control of the promoter and
other regulatory elements capable of enabling the gene to be
expressed when integrated in the genome of a plant cell;
(b) at least one DNA sequence which delineates the
DNA to be integrated into the plant genome; and
(c) a DNA sequence which enables this DNA to be
transferred to the plant genome.
Typically the DNA to be integrated into the plant
cell genome is delineated by the T-DNA border sequences of a
Ti-plasmid. If only one border sequence is present, it is
preferably the right border sequence. The DNA sequence
which enables the DNA to be transferrec to the plant cell
genome is generally the virulence (vir) region of a
Ti-plasmid.
The gene and its transcriptional and translational
control elements, including the promoter, can therefore be
provided between the T-DNA borders of a Ti-plasmid. The
plasmid may be a disarmed Ti-plasmid from which the genes
for tumorigenicity have been deleted. The gene and its
transcriptional and control elements, including the
promoter, can, however, be provided between T-DNA borders in
a binary vector ln trans with a Ti-plasmid with a vir
region. Such a binary vector therefore comprises:
(a) the gene under the control o~ the promoter and
other regulatory elements capable of enabling the gene to be
expressed when integrated in the genome of a plant cell; and
(b) at least one DNA sequence which delineates the
DNA to be integrated into the plant genome.
Aarobacterium tumefaciens, therefore, containing a
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WO91/13g92 PCT/GB91/00416
2~7~27 ~,
hybrid plasmid vector or a binary vector ln trans wlth a
Ti-plasmid possessing a vir region can be used to transform
plant cells. Tissue explants such as stems or leaf discs
may be inoculated with the bacterium. Alternatively, the
bacterium may be co-cultured with regenerating plant
protoplasts. Plant protoplasts may also be transformed by
direct introduction of DNA fragments which encode the gene
of interest and in which the promoter and appropriate other
transcriptional and translational control elements are
present or of a vector incorporating such a fragment.
Direct introduction may be achieved using electroporation,
polyethylene glycol, microinjection or particle bombardment.
Plant cells from monocotyledonous or dicotyledonous
plants can be transformed according to the present
invention. Monocotyledonous species include barley, wheat,
maize and rice. Dicotyledonous species include tobacco,
tomato, sunflower, petunia, cotton, sugarbeet, potato,
lettuce, melon, soybean, canola (rapeseed) and poplars.
Tissue cultures of transformed plant cells are propagated to
regenerate differentiated transformed whole plants. The
transformed plant cells may be cultured on a suitable
medium, preferably a selectable growth medium. Plants may
be regenerated from the resulting callus. Transgenic plants
are thereby obtained whose cells harbour the promoter
operably linked to the gene encoding the protein it is
wished to express, for example integrated in their genome.
The gene is consequently expressible in the cells. Seed
from the regenerated plants can be collected for future use,
and plants grown from this seed.
The following Examples illustrate the invention.
EXAMPLE 1
1. A family of cross-hybridising cDNA clones was
isolated from a cDNA library produced with poly (A) +RNA from
the roots of oilseed rape (Brassica naDus L.). The clones
were selected as abundantly expressed in root by
differential screening of the root CDNA library with cDNA
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WO91/13992 PCT/GB91/00416
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probes prepared from root, green leaf, etiolated leaf and
developing seed. mRNA species corresponding to the selected
abundant clones were expressed in roots at levels of at
least 400 x those in other organs, as shown by Northern blot
analysis and RNase protection assays. One of the cDNA
clones was designated pRRt566.
2. An extensin gene designated extA, was obtained from
an oilseed rape (Brassica naPus L.) genomic library screened
with pRRt566 at high stringency. The gene is a mem~er of a
multigene family, consisting of about three members per
haploid genome with strong homology to the probe, and a
further twenty or so members of weaker homology. The
isolated gene, although not identical to the cDNA probe, was
also found to be specifically expressed in roots, and was
transcribed into a mRNA species of approximately 1300
nucleotides in size. A single transcription start was
identified by Sl mapping. A complete nucleotide sequence of
the extA gene and its flanking regions was determined. This
enabled the gene promoter to be identified. In more de:ail:
Identification of extensln qenes in qenomic DNA
The cDNA species from rape root, pRRt566,
previously identified as encoding a protein homologous to
extensin, was used as a probe on Southern blots of
restrictions of rape genomic DNA. The insert of pRRt566
hybridised to a large number of genomic fragments in all
restriction digests, but the strength of hybridisation to
all fragments was not equal; 1-2 fragments in all di~ests
hybridised at an intensity at least five times that of the
other bands. Copy number reconstructions indicated that the
strong bands corresponded to approx. 2-3 gene copies per
haploid genome: the patterns can be consistently interpreted
to give a total of 3 genes per genome highly homologous to
this probe. The weakly hybridising fragments were present
at less than one estimated copy per haploid genome, and must
therefore represent genes that are only partly homologous to
the extensin probe. The total sequences detected by this
: :
~'091/13992 PCT/GB91/00416
2~7~327 - 10 - ~
probe make up a large multigene family, of which three
genes, determined to be closely homologous to pRRt566, form
a sub-family. In confirmation of these conclusions, when a
3' flanking sequence probe (240 bp) was prepared from
pRRt566, and hybridised to genomic blots as above,
hybridisation of this probe to the strongly hybridising
bands was identical to that using the whole cDNA insert as a
probe, except hybridisation to the pRRt566 weakly
hybridising bands was largely eliminated.
Isolation and characterisation of a genomic clone containinq
an extensin aene
An oilseed rape genomic library, constructed by
inserting random Sau 3A fragments of genomic DNA, into
lambda EMBL3, was screened at low stringency with a probe
prepared from the insert of pRRt566. This cDNA clone was
isolated from a library prepared from rape root mRNA, and
encoded part of an extensin polypeptide. Forty positive
phage plaques were identified, and were extensively plaque-
purified. ~he isolated clones were then re-screened under
high stringency conditions (O.l x SSC, 0.1% SDS at 65C),
when only one phage clone gave continued strong
hybridisation to the cDNA probe. This clone, lambdaB3l, was
selected for further study.
DNA from the selected phage was isolated and
characterised by restriction and Southern blotting, using
pRRt566 as a probe. The restriction map of lambdaB3l
obtained is shown in Fig. 2, and shows that the clone
contains an insert of 12.7 kb. The region of DNA
hybridising to the cDNA probe was localised to a fragment of
1.0 kb (Nsi I-Pst I) adjacent to the left arm of the vector.
To further characterise the gene present on this clone,
restriction with Dde I and Rsa I was carried out, followed
by Southern blotting and probing with the cD~A as before.
These two enzymes were chosen because they restrict the
homologous cDNA sequences at multiple sites, corresponding
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WO91/1~992 PCT/GB91/00416
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to the repeats in the coding sequence, and also generate
unique diagnostic fragments from the 3' non-coding region of
the sequence. Although the genomic clone contained the 84
bp restriction fragments generated from the region of
pRRt566 encoding the 28 amino acid repeat sequences, the
diagnostic fragments specific to pRRt566 were not present,
suggesting that the gene present was not identical to the
cDNA. DNAs from a further ten phage clones were purified
and similarly screened; however, none of the clones
contained diagnostic fragments identical to pRRt566 and were
not considered further in the present study.
To further characterise the gene present on
lambdaB31, a Southern blot of insert DNA was hybridised with
a probe prepared from a second rape root extensin cDNA
clone, pRRt592; this probe contained only 39 bp of sequence
identified as encoding a putative leader sequence of an
extensin precursor polypeptide and hybridised to the same
1.0 kb fragment as was detected by the pRRt566 probe. A 3'
flanking sequence prc~e from pRRt566 was prepared by
subcloning a Hinc II-EcoRI fragment into pUC 18. This probe
contained no coding sequence, which hybridises very strongly
due to its G-C rich nucleotide composition. The 3' probe
failed to hybridise to lambdaB31 DNA suggesting that thls
clone contained the 5' end and coding sequence of an
extensin gene, but either lacked the 3' flanking sequence,
or was divergent in the 3' flanking region.
When the 1.0 kb fragment of lambdaB31 was labelled
and used as a probe on Southern blots of restricted rape
genomic DNA, similar results (not shown) to those o~tained
when pRRt566 was used as a probe, were obtained. A 1.85 kb
Hinc II fragment of genomic DNA which was present at 1 copy
per haploid genome, corresponded to the expected Hinc II
fragment predicted from the lambdaB31 restriction map.
Se~uence of extensin aene extA
Approximately 2.7 kb of DNA sequence of the genomic
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WO9l/l3992 PCT/GB91/00416
2 ~7 ~ 3 2 1 - 12 -
clone lambdaB31 was fully determined. The 5.3kb
HindIII-AvaI fragment from lambdaB31 was subcloned into
pUC18 (Yanisch-Perron et al, Gene 33, 103-119, 1985) to give
pRlambdaS4 (Figures 2 and 3) from which the 2.7kb
HincII-HincII fragment was sequenced (Figure 2). The
sequencing map for this region is shown in Fig. 2, and the
complete determined sequence is given in Fig. 1. The
sequence contains an open reading frame predicting a
polypeptide of 299 residues. The first 23 residues of the
sequence encode a leader sequence, when compared with other
extensin sequences and as defined by the rules of von Heijne
(1985, J. Mol. Biol. 184, 99-105). The remainder of the
I coding sequence encodes a highly proline-rich polypeptide of
276 amino acids.
The promoter of the invention is the 5' flanking
sequence of the extA gene as shown in Figure 1. This
sequence contains a sequence (CTATATAAA) closely homologous
to the consensus "TATA" ~ox for plants seventy-four bases 5'
to the initiation codon. Apart from this no significant
features can be recognised. Comparison of this 5' flanking
sequence with that of the carrot extensin gene pDC5Al (Chen
and Varner, EMBO J. 4, 2145-2151, 1985) reveals no strongly
conserved sequence regions, apart from the "TATA" box.
Expression of extA
To confirm that extA represents an expressed gene,
and to determine its transcription start, a series of Sl
mapping experiments were carried out. Fragments of extA 5'
t~` end-labelled at a Dde I site (base 71) and extending in a 5'
direction to the Nsi I site (base -74) or the Dde I site
(base -237) were isolated and hybridised to poly(A)+RNA from
rape roots. ~fter treatment with Sl nuclease, the protEcted
fragments were sized by polyacrylamide gel electrophoresis.
In bo~h cases, protected fragments of 66-75 bases were
obtai~ed with the strongest band corresponding to the
underlined base in the sequence TAAGAGC_TCAAAC, which was
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W 0 91/13992 PCr/CB91/00416
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designated base +l (indiated in Figure 1 by T^S). This
sequence is in good agreement with the consensus
transcription start in plants, -CATC- and is at a congruous
distance from the "TATA" box (34 bases). No other protected
fragments that were not also present in the controls were
observed. A further probe, labelled at the Nde I site at
base -86 and extending to the Nde I site at base -453, was
prepared and hybridises as above. No protected fragments of
this probe were observed, although a small amount of intact
probe was present in both the experimental and control
hybridisations. These results suggest that the determined
transcription start is the only start site in extA, in
contrast to the carrot extensin gene where two transcription
start points were observed.
To confirm the above conclusions, and to
investigate the organ-specificity of expression of extA,
fragments of the gene were used as probes on Northern blots
of RNA from different rape organs. A probe consisting of
the complete coding sequence of the gene, and extending to
base -74, was hybridised to RNA from four rape organs: root,
green leaf, etiolated leaf and developing seed. This probe
hybridised to two mRNA species in root, of approx. 1300 and
1480 bases, and hybridised only very weakly to a mRNA
species in developing seeds, of approx. 1600 bases. This
hybridisation pattern is very similar to that given by the
cDNA species pRRt556 (and closely homologous species), and
rehybridisation of the same blot to the cDNA probe confirmed
that the same bands were detected by both probes. If the
size of 3' flanking sequence of pRRt566 is added to that of
the sequence of extA, the completed gene would be predicted
to produce an mRNA species of approx. 1250 bases, assuming a
poly(A) tail of 30-bases is added to the cDNA sequence. The
observed mRNA species of approx. 1260 bases in rape roots
can ~herefore be suggested to be the product of extA. A
very low level of hybridisation of the extA probe to a large
mRNA species (approx. 4.6 kb) pres~nt in root was also
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WO91/13992 PCT/GB91/00416
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observed.
EXAMPLE 2: Expression of the extA_in_Transgenic Tobacco
The original rape extA gene as isolated from the
lambdaB31 clone lacked a 3' terminator region due to the
cloning strategy used (Figure 4). Accordingly, a 260bp
nopaline synthase (nos) BamHI-EcoRI fragment which contains
an efficient plant terminator sequence was excised from the
clone pNOP-NEO and ligated into BamHI-EcoRI cut pUC18 and
cloned in E. coli DH5h. pNOP-NEO is a clone containing -
the NOS promoter linked to the Neomycin Phosphotransferase
(NPT) gene which encodes kanamycin resistance, linked to the
NOS terminator as published by Bevan, Nucleic Acids Research
2, 8711-8721 (1984).
The extA gene, excised from pRlambdaS4 on a 4.75kb
HindIII-SalI fragment, was then ligated into the NOS
terminator pUC18 clone, also restricted with HindIII-SalI,
and cloned in E. coli DH5~. The final extA - NOS terminator
construct is shown in Figure 5. The whole extA - NOS
terminator construct was then excised on an intact 5.01kb
HindIII-EcoRI fragment and ligated into the binary vector
pBIN19 (Bevan, 1984) restricted with HindIII-EcoRI, and
cloned into E. coli MC1022. The final hybrid gene construct
designated pBINl9:extA is shown in Figure 6 and contains
additionally the endogenous kanamycin resistance gene (NPT)
located between the T-DNA borders.
, This construct termed pBINl9:extA was triparentally
t ~ mated into A. tumefaciens LBA4404 and used to transform leaf
discs of Nicotiana tabacum (SRl). Prior to tobacco being
' used as a test transgenic system, Southern blotting
experiments were performed on tobacco DNA to show that
, hybridisation of extensin coding region to tobacco DNA did
; not occur. Even at low stringency no hybridisation of the
extensin coding region to tobacco DNA could be seen. In
detail:
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WO9l/l~992 PCT/GB91/00416
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Bacterial strains and coniuqations
The vector plasmid pBIN19 in Escherichia collstrain MC1022 (Bevan, Nucl. Acids Res. 12, 8711-~721, 1984),
the mobilising E. coli strain HB101/pRK2013 (Ditta et ai,
PNAS USA 77, 7347-7351, 1980) and the host Aarobacterium
tumefaciens strain LBA4404/pAL4404 (Hoekema et al, Nature
303, 179-180, 1983) were used. For bacterial conjugation
antibiotics were used at the following concentrations:
kanamycin acid sulphate, 50~g.ml 1 (kan); rifampicin,
lOO~g.ml 1 (rif~; streptomycin sulphate, 500 ~g.ml 1
(strep). The recombinant vector BINl9:ext A was mobilized
in a triparental mating by mixing 200~1 each of overnight
cultures of E. coli MC1022/BIN19 (kanR, strepS, rifS), E.
coli HB101/pRK2013 (kanR, strepS, rifS) and A. tumefaciens
LBA4404 (kanS, strepR, rifR), and incubating at 27C on a L3
plate without antibiotic selection for 15h. The cell
mixture was then diluted with 10 mM MgS04; plated out on
minimal medium containing kanamycin and incubated at 27C
.or 4 days. The A. tumefaciens LBA4404 colonies were
checked for the correct antibiotics resistance markers
(kanR, strepR, rifR), and further analysed by colony
hybridisation and Southern analysis to confirm that the nPt
and ext A genes were present in an intact, unrearranged form
in the BINl9:ext A plasmid.
~ .
Tobacco transformation and reqeneration
Agrobacterium tumefaciens LBA4404/pBINl9:ext A was
cultured in LB medium containing 20~g.ml 1 kanamycin at
27C, the bacteria were pelleted, washed three times in 2 m~
MgS04, and resuspended in MS salts (Flow Labs.,
Rickmansworth, Herts., UK), lOmg.ml 1 sucrose (pH 5.8) at a
density of approx 109 cells.ml 1.
Expanded leaves from well-watered N. tabacum (SRl)
plants were surface-sterilized with 70~ (v/v) ethanol (30 s)
followed by 5% (w/v) Ca(OCl)2 (15 min). After washing in
sterile water, each leaf was cut into squares (approx. 8 x
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WO91/13~92 PCT/GB9t/004t6
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m~2) avoiding major veins and then placed in the bacterial
suspension, which was intermittently agitated to wet the cut
leaf-edges. After 10-15 min. incubation (22-25C) the leaf
pleces were placed (adaxial surface upwards on agar plates
of shoot-induction medium, comprising MS salts (including
vitamins), 10 mg.ml 1 sucrose, 2 ~g.ml 1 N~
furfurylaminopurine (kinetin), 0.2 ~g/ml 1 1-naphthylacetic
acid (pH 5.8) and 8 mg.ml 1 bacto-agar, and cultured at
25C, 18-h photoperiod (120~mol.m 2.s 1 PAR). After 2 days
the leaf pieces were transferred to liquid shoot-ir,duction
medium containing 1 mg.ml 1 carbenicillin (disodium salt)
and gently agitated (60 rpm shaker) overnight. The leaf
pieces were surface-dried on sterile absorbant paper and
placed on agar plates of shoot-induction medium containing
500 ~g.ml 1 carbenicillin, 200 ~g.ml 1 kanamycin (acid
sulphate) and cultured under the conditions described above.
Shootlets which developed from the cut edges of the
leaf explants (four to six weeks from inoculation) were
excised and transferred to agar plates of half-strength MS
salts, 5 mg.ml 1 sucrose, 250 ~g.ml 1 carbenicillin, 200
~g.ml 1 kanamycin (pH 5.8). The shootlets which continued
to expand were transferred to 60-ml sterile vessels
(Sterilin, Feltham, Middlesex, UK) containing root-induction
medium comprising half-strength MS salts, lOO~g.ml 1
carbenicillin, lOO~g.ml 1 kanamycin and 8 mg.ml 1 agar.
Shootlets which developed roots were transferred to 250-ml
sterile glass jars containing half-strength MS salts, 50
~g.ml 1 carbenicillin and 8 mg.ml 1 agar. When the
plantlets had developed an extensive root system, they were
removed from culture and potted in 1:1 Levington
compost:perlite (Silvaperl Products, Harrogate, UK), and
grown in a controlled environment with daily watering.
Tissues from the transgenic plants were harvested into
liquid air for extraction of DNA, for genomic analysis to
assess the integrity of the transferred gene, and RNA, for
analyses of extensin gene expression. The potted plants
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WO91/13992 PCT/Gn91/004~6
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- 17 -
flowered after about five weeks, and following
self-pollination, the seeds from each plant were collected
at dehiscence.
Extraction of DNA and Southern Analvsis
The DNA from N. tabacum leaves was extracted and
purified. The DNA probes for hybridization were
32P-labelled to high specific activities (>8 x 1011 Bq.~g 1)
using random priming according to Feinberg and Vogelstein
(Anal. Biochem. 132, 6-13, 1983). Standard techniques were
used for Southern transfer and hybridization and high
stringency (30 mM NaCl, 3 mM Na3 citrate, 65C, 60 min) was
employed in the final post-hybridization wash. Filters were
autoradiograhed for 72 h at 80C using flashed X-ray film
(Fujimex, Swindon, UK) and intensifying screens (Du Pont,
Stevenage, UK).
Extraction of RNA and Northern AnalYsis
Total RNAs from tissues of the transgenic N.
tabacum plants were isolated by the procedure of Logemann et
al. (Anal. Biochell. 163, 16-20, 1987). To estimate the size
of RNAs specifically hybridizing to the rape extensin probe,
10~g of each sample RNA was glyoxalated, run on agarose
-- gels, and transferred to nitrocellulose. Northern blots
were washed to high stringency (15 mM NaCl, 1.5 mM Na3
` citrate, 0.1~ sodium dodecyl sulphate (SDS), 25 min, 50C),
and the filters autoradiographed for 2 weeks as described
above.
Fifteen transgenic tobacco plants were regenerated
i and grown from the initial transformation experiment.
Samples of leaf and root were collected for analyses by
Southern and Northern blotting. The majority of the
transgenic plants regenerated contained intact, unrearranged
copies of the introduced hybrid extensin gene. Estimates of
the copy number of the introduced gene in the various
independent transformants varied from one copy to five
- copies. Only trans~enic tobacco plants containing intact,
unrearranged copies of the introduced gene were used for
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WO91/1399~ PCT/GB9l/00416
~7832~ ~
- 18 -
further analysis of extensin expression.
Because of the lack of availability of specific
rape extensin antibodies, Northern hybridisations were
performed on total RNA extracted from tobacco leaf and root
tissues, using the extensin l.0 kb coding sequence from the
genomic clone lambdaB31 as a probe. No hybridisation to
leaf RNA from either control or transformed tobacco plants
was seen. Hybridisation of the extensin probe to transgenic
root RNA was seen in all the transformants tested, although
the level of hybridisation between individual transformants
varied (position effect).
Seeds were collected from the initial batch of
transformed tobacco, and were sown in compost. Plants were
grown from this seed.
EXAMPLE 3: Expression of Glucuronidase Enzyme under Control
of the Rape Promoter
In order to see the expression of the rape gene
extA in rape, the rape promoter from lambdaB31 was fused in
a translational fusion to the coding sequence of the
glucuronidase (GUS) gene in a binary vector variant of
8IN19. The clone containing the construct was mated with an
Aarobacterium strain (LBA 9402) containing an oncogenic Ri
plasmid, pRi 1885 (Constantino et al, Plasmid 5, 170-182,
1981). Inoculation of this strain into rape seedlings
produces transgenic hairy roots, which are then easily
assayable for GUS activity. Whole plants can be regenerated
from excised transgenic hairy roots.
In detail, the rape extensin promoter was excised
from pRlambdaS4 as a l.Okb HaeIII-HaeIII fragment. This
fragment was blunt-end ligated into a modified pBINl9 binary
vector, pBIlOl containing a promoterless glucuronidase (GUS)
gene, restricted with SmaI at the start of the GUS coding
sequence (see Jefferson, Plant Molecular Biology Reporter 5,
387-40S, 1987) (Figure 8). The rape promoter expression
.~ from this translational fusion will give a glucuronidase
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WO91/13992 PCT/GBgl/00416
2~7~327
enzyme with either 7 or 11 extra amino acids at the N-
terminus, depending on which rape ATG is used as the
translation initiation codon.
The extA-GUS hybrid gene in pBI101 (pBIlOl:extA-
GUS) was cloned in E. coli MC1022 and then transferred into
Aarobacterium tumefaciens or Aqrobacterium rhizogenes by
tri-parental matings with the appropriate Agrobacterium
strains. Preliminary results to test for expression from
this construct, using the X-Gluc (5-bromo~4-chloro-3-
indolyl-~-D-glucuronide) histochemical assay of Jefferson
(Plant Molecular Biology Reporter 5, 387-405, 1987) in rape
'hairy roots' transformed with Aarobacterium rhizoqenes
containing pBIlOl:extA-GUS, have clearly shown that the
promoter is active in rape root tissue.
Ra~e Tissue culture and transformation ~rotocols:
Transformation experiments usina A. rhizoqenes
Axenically growing seedlings of B. na~us cv.
Bienvenue (winter rape), Brutor and Rapid Cycling were
inoculated at their cotyledonary nodes with an A. rhizoqenes
LBA9402 strain carrying both the Ri plasmid pRi 1855
(Constantino et al, 1981) and the kanamycin resistance
fusion construct of Figure 8 (pBIlOl:extA-GUS) as separate
independent replicons. Hairy roots arising from the site of
inoculation can be excised and grown in a culture medium (1
x MS salts and vitamins, 4% sucrose, 2 g/l CaC12.H20, 0.1
mg/1 NAA, 2.5 mg/l BAP) 0.1 mg/l thiamine, 200 mg/l
cephotaxime, 50-mg/1 kanamycin, 8 g/l Bacto Difco agar, pH
5.8, to proliferate the hairy roots.
Regeneration can be achieved as follows: 0.5 cm
sections of the terminal 2 - 3 cm of hairy roots are
excised, treated with 2,4 - D at a concentration of 3 mg/l,
and plated on shooting medium containing kanamycin at 50
mg/l to select for tissues which contained the T-DNA
kanamycin gene of the fusion construct of Figure 8. Shoots
arising from these hairy roots are excised and grown on
medium F of Pelletier et al (Mol. Gen. Genet. 191, 244-250
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WO91/1399' PCr/GB91/00416
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(1983)) for 2-3 weeks. They are then transferred to rooting
medium G, and when a sufficient root system has developed
they are transferred to soil and grown to maturity in a
containment growth room.
Transgenic winter rape plants are vernalised to
induce flowering by growing them at 4C with a 8h daylight
period. Flowers are emasculated before anthesis and
outcrossed with wild type rape pollen. Seeds obtained after
pod set are collected, sown in soil and the progeny plants
grown to maturity. The progeny plants are initially scored
visually with regard to the degree of the hairy root
syndrome being exhibited. Kanamycin resistance in the
leaves of the progeny is assayed by transferrlng leaf discs
to callus inducing media (see below) containing kanamycin at
; either 50 or 150 mg/l. Both the Ri and the kanamycin
resistance traits can be further confirmed by Southern
-~ hybridisation analysis.
The media used in the Examples are:
Media
1. For seedlina arowth
MS salts 2.355 g/l
Sucrose 20 g/l
Difco Bacto agar 15 g/l pH
5.8
2. For Aarobacterium rhizoaenes liauld culture - YMB
~ Mannitol 10 g/l
,; Yeast extract 0.4 g/l
KH2P04 0.5 g/l
MgS4 2H2 0.2 g/l
NaCl 0.1 g/l
Kanamycin 50 mg/lpH 7.0
, 3. For hairv root culture (Ooms' medium: Ooms et al (1985)
Theor. Appl. Genet. 71, 325-329)
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MS salts 4.71 g/l
CaC12.2H20 2 g/l
Sucrose 40 g/l
NAA 0.18 mg/l
BAP 2.5 mg/l
Thiamine HCl 0.1 mg/l
Difco Bacto agar 8 g/l
Xanamycin 25 or 50 mg/l
Cephotaxime 200 mg/l pH 5.8
4. For shoot induction, multi~lication and rootina
D solution, RCC medium, F and G media are as
described by Pelletier et al. (1983) and Guerche et al.
(Mol. Gen. Genet. 206, 382-386, 1987). In addition, a level
of Cephotaxime of 200 mg/l is maintained in all these media
at all times. ~
5. Callusinq medium for transformed N. tabacum and rape -
leaf discs
MS3SC 100 kan.
- MS salts 4.7 g/l
~, Sucrose 30 g/l
- NAA 2 mg/l
Kinetin 0.2 mg/l
Kanamycin 100 mg/l
Agar 8 g/l
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