Note: Descriptions are shown in the official language in which they were submitted.
212~751
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Inventors: Steven J. Rothstein
Daphne R. Goring
Tracy L. Glavin
Ulrike Schafer
SELF-INCOMPATIBILITY GBNES ASSOCIATED WITH
THE A10 ~T.T.~T.~
CROSS-REFEREN OE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S.
Serial No. 08/208,909, filed on March 11, 1994, which in
turn is a continuation of U.S. Serial No. 07/847,564,
filed on March 3, 1992, now abandoned.
BACR~uN~ OF T~E lNV~..l-ION
1. Field of the Invention
This invention is directed to DNA molecules that
encode the S locus glycoprotein and the S locus serine
kinase protein associated with the A10 self-
incompatibility allele. Moreover, this invention is
directed to a method for producing a self-incompatible
plant by the use of such DNA molecules.
2. Background
Oil seed crops have significant economic importance
throughout the world. For example, the oil seed crops
Brassica napus ssp. oleifera and Brassica rapa (formerly
campestris) are the second most valuable crops in Canada
with an annual growth on 7-9 million acres. Since canola
seed is required for oil and m~a/l production, yield is
extremely important in determining the usefulness of a
Brassica line. B. napus F~ hybrid lines have been found
to be superior to B. napus established lines. Typically,
Fl hybrids produce yields that are greater than 20 to
21237~i1
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70%, compared with established lines. Thompson, Adv.
Appl. Biol. 7: 1 (1983); Johnston, Euphytica 20: 81
(1971).
The prerequisite for developing hybrid lines is the
use of a pollen-control system to prevent self-
pollination of the female line. Several approaches have
been used for pollen control in the production of hybrid
canola: dominant nuclear male-sterility genes,
cytoplasmic male-sterility genes, and the self-
incompatibility system. The most economic and flexible
approach utilizes a self-incompatibility system that is
naturally present in B. oleracea and B. campestris.
Gowers, Euphytica 24: 537 (1975).
In the Brassica family, the self-incompatibility
system is inherited as a dominant genetic locus called
the S locus. Nasrallah et al., Annu. Rev. Plant Physiol.
Plant Mol. Biol. 42: 393 (1991); Dzelzkalns et al., Dev.
Biol. 153: 70 (1992). The presence of a functional
S allele results in a barrier to fertilization when the
pollen grain originates from a plant carrying the same
S allele as the pistil. The pollen phenotype, which is
determined by the diploid parental genotype and not by
the haploid pollen, is thought to be propagated by
putative S allele products deposited in the outside wall
of the pollen grain by the surrounding tapetum during
pollen development. de Nettancourt, INCOMPATIBILITY IN
ANGIOSPERMS (Springer-Verlag, 1977). Thus, during
pollen-pistil interactions, there is a recognition of
self versus non-self leading to either a block in pollen
germination when both parents carry the same S allele or
successful fertilization when different S alleles are
present.
The diploid species, Brassica oleracea and B.
campestris are typically self Jncompatible, while B.
napus, an allotetraploid composed of both of these
genomes, generally occurs as a self-compatible plant.
Downey et al., "Rapeseed and mustard," in PRINCIPLES OF
CULTIVAR DEVELOPMENT, Fehr (ed.) pages 437-86 (Macmillan
Publishing Co. 1987). Although the S-alleles have been
21237~1
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introgressed into B. napus using traditional genetic
approaches, there are two problems that limit this
approach for the production of hybrid seed. First, there
is some environmental variability in the level of
hybridity from year to year when using self-
incompatibility as the pollen control system. Second,
and most importantly, the level of dominance of the self-
compatible alleles is not always sufficient in
heterozygotes, with the level of dominance widely
variable depending on genetic background.
This second problem is an extremely important one
because hybrid breeding schemes using self-
incompatibility require the use of heterozygotes for the
final production of the hybrid seed. Thus, it would be
useful to be able to confer the self-incompatible
phenotype by gene transformation since this would be
faster than traditional breeding approaches and would
allow the co-transfer of herbicide resistance genes which
would be useful under some breeding schemes.
Two types of genes have been found to co-segregate
with the self-incompatibility phenotype: the S locus
glycoprotein (SLG) gene and the S locus receptor kinase
(SRR) gene. Transformation experiments have shown that
the SLG gene alone is not sufficient to confer self-
incompatibility to a self-compatible B. napus. Nishio et
al., Sex. Plant Reprod. 5: 101 (1992). Nevertheless,
there is evidence that SLG genes are required for self-
incompatibility. For example, a naturally occurring
variant of B. campestris with normal SRR expression, but
low levels of SLG expression has been associated with
self-compatibility. Nasrallah et al., Plant J. 2: 497
(1992). Therefore, transformation of a self-compatible
plant to a self-incompatible plant requires the
introduction of both SRR and SL~ genes.
The expression of any foreign gene in the genome of
a plant cell may be repressed due to hypermethylation of
the foreign gene, the particular chromosomal context of
the foreign gene, or the presence of multiple copies of
the foreign gene. Such problems are compounded when a
2~237~i1
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desired phenotype of a transgenic plant is dependent upon
the expression of two foreign genes. This fact may
explain the observation that there is no report of a
transgenic self-incompatible plant which was produced by
transforming a self-compatible plant with both SRK and
SLG genes.
SUMMARY OF T~E lNV~.. llON
Accordingly, it is an object of the present invention
to provide a method for producing a self-incompatible
plant which requires transformation of a single S locus
gene.
It is a further object of this invention to provide
DNA molecules that encode a functional SRK-A10 protein.
These and other objects are achieved, in accordance
with one embodiment of the present invention, by the
provision of an isolated DNA molecule, comprised of the
nucleotide sequence of SEQ ID NO:5, wherein the DNA
molecule encodes an SRK protein. In particular, these
objects are achieved by the provision of a DNA molecule
consisting of the nucleotide sequence of SEQ ID NO:6.
In accordance with another embodiment of the present
invention, there has been provided an isolated DNA
molecule, comprised of the nucleotide sequence of SEQ ID
NO:4, wherein the DNA molecule encodes an SLG protein.
In accordance with a further embodiment of the
present invention, there has been provided an expression
vector comprising a regulatory element and a DNA molecule
that encodes either an SLG protein or an SRK protein,
wherein the regulatory element controls the production of
the SLG protein or the SRK protein. A suitable
regulatory element is selected from the group consisting
of the SLG-910 regulatory element! the SLG-A10 regulatory
element, the SRK-A10 regulatory element and the CaMV 35S
35 promoter.
In accordance with another embodiment of the present
invention, there has been provided a method of producing
a self-incompatible plant, comprising the steps of:
2123751
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(a) producing a first parent self-compatible plant
comprising an expression vector that comprises
an SRK-encoding DNA molecule, wherein the first
parent plant does not contain the A10 allele;
and
(b) cross-fertilizing the first parent plant with
a second parent plant having the A10 allele to
produce a progeny plant,
wherein the progeny plant produces SLG-A10 protein
and SRK-A10 protein, resulting in the self-
incompatibility phenotype.
In accordance with a further embodiment of the
present invention, there has been provided a method of
producing hybrid seed, comprising the steps of:
(a) producing a self-incompatible transgenic plant
comprising the nucleotide sequence of SEQ ID
NO:6; and
(b) cross-fertilizing the transgenic plant with a
second plant.
In accordance with another embodiment of the present
invention, there has been provided a method for
conferring the self-incompatible phenotype on a self-
compatible plant, comprising the steps of:
(a) preparing a first expression vector comprising
an SLG-encoding DNA molecule consisting of the
nucleotide sequence of SEQ ID NO:2;
(b) preparing a second expression vector comprising
an SRK-encoding DNA molecule consisting of the
nucleotide sequence of SEQ ID NO:6; and
(c) transferring the first expression vector and
the second expression vector into a self-
compatible plant to produce a transformed
plant,
wherein the transformed p~ant expresses SLG-A10
protein and SRK-A10 protein, resulting in a self-
incompatibility phenotype.
In accordance with a further embodiment of the
present invention, there has been provided a transformed
plant comprising an expression vector, wherein the
21237~i~
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expression vector comprises the nucleotide sequence of
SEQ ID NO:6.
In accordance with another embodiment of the present
invention, there has been provided an isolated DNA
molecule comprising the nucleotide sequence of SEQ ID
NO:8, wherein the DNA molecule is the SLG-A10 promoter.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the predicted amino acid sequence of
the SLG-A10 cDNA tSEQ ID NO:1]. The underlined section
represents a putative signal peptide. Conserved cysteine
residues are marked by stars above the amino acid
residues. Double underlining indicates potential N-
glycosylation sites.
Figure 2 shows the alignment of the SLG-A10 and SR~-
A10 cDNA sequences [SEQ ID NOs: 2 and 3]. Panel A
depicts the coding region of the SLG-A10 cDNA that was
aligned with the SRK-A10 cDNA sequence. A region of 100%
homology between the two genes is marked. The 1-bp
deletion in the SRR-A10 gene leading to premature
termination is indicated by the arrow. Underneath the
SRR-A10 cDNA is a representation of the S-receptor kinase
domains that would be translated if the deletion was not
present. Panel B shows the alignment of the SLG-A10
nucleotide sequence [SEQ ID NO:2] and the SRR-A10
nucleotide sequence [SEQ ID NO:3]. Identical nucleotides
are marked by dashes, and gaps are represented by blank
spaces. The predicted start and stop codons for both
genes are underlined. The 1-bp deletion in the SR~-A10
gene is marked by an arrow.
Figure 3 shows the nucleotide sequence of the
corrected SRR-A10 gene [SEQ'~ID NO:6], which is
constructed by inserting a nucleotide at position 948 of
the SRR-A10 cDNA sequence [SEQ ID N0:3].
Figure 4 shows the nucleotide sequence of the SLG-A10
promoter.
2123~1
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DETAIT,~D DESCRIPTION OF PR~FERRED EMBOD
1. Definitions
In the description that follows, a number of terms
are used extensively. The following definitions are
provided to facilitate understanding of the invention.
A structural gene is a DNA sequence that is
transcribed into messenger RNA (mRNA) which is then
translated into a sequence of amino acids characteristic
of a specific polypeptide.
A promoter is a DNA sequence that directs the
transcription of a structural gene. Typically, a
promoter is located in the 5' region of a gene, proximal
to the transcriptional start site of a structural gene.
If a promoter is an inducible promoter, then the rate of
transcription increases in response to an inducing agent.
In contrast, the rate of transcription is not regulated
by an inducing agent if the promoter is a constitutive
promoter.
An isolated DNA molecule is a fragment of DNA that
is not integrated in the genomic DNA of an organism. For
example, the SLG-A10 gene is a DNA fragment that has been
separated from the genomic DNA of a Brassica napus plant.
Another example of an isolated DNA molecule is a
chemically-synthesized DNA molecule that is not
integrated in the genomic DNA of an organism.
An enhancer is a DNA regulatory element that can
increase the efficiency of transcription, regardless of
the distance or orientation of the enhancer relative to
the start site of transcription.
Complementary DNA (cDNA) is a single-stranded DNA
molecule that is formed from an mRNA template by the
enzyme reverse transcriptase. Typically, a primer
complementary to portions of mRNA is employed for the
initiation of reverse transcription. Those skilled in
the art also use the term "cDNA" to refer to a double-
stranded DNA molecule consisting of such a single-
stranded DNA molecule and its complementary DNA strand.
- 21237~1
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The term expression refers to the biosynthesis of a
gene product. For example, in the case of a structural
gene, expression involves transcription of the structural
gene into mRNA and the translation of mRNA into one or
more polypeptides.
A cloninq vector is a DNA molecule, such as a
plasmid, cosmid, or bacteriophage, that has the
capability of replicating autonomously in a host cell.
Cloning vectors typically contain one or a small number
of restriction endonuclease recognition sites at which
foreign DNA sequences can be inserted in a determinable
fashion without loss of an essential biological function
of the vector, as well as a marker gene that is suitable
for use in the identification and selection of cells
transformed with the cloning vector. Marker genes
typically include genes that provide tetracycline
resistance or ampicillin resistance.
An expression vector is a DNA molecule comprising a
gene that is expressed in a host cell. Typically, gene
expression is placed under the control of certain
regulatory elements, including constitutive or inducible
promoters, tissue-specific regulatory elements, and
enhancers. Such a gene is said to be "operably linked
to" the regulatory elements.
A foreign gene refers in the present description to
a DNA sequence that is operably linked to at least one
heterologous regulatory element. For example, any gene
other than the SRK-A10 structural gene is considered to
be a foreign gene if the expression of that gene is
controlled by a regulatory element of the SRR-A10 gene.
A recombinant host may be any prokaryotic or
eukaryotic cell that contains either a cloning vector or
expression vector. This term also includes those
prokaryotic or eukaryotic célls that have been
genetically engineered to contain the cloned gene(s) in
the chromosome or genome of the host cell.
A transqenic plant is a plant having one or more
plant cells that contain an expression vector.
212~7~1
. g
Two nucleic acid molecules are considered to have a
substantial se~uence similarity if their nucleotide
sequences share a similarity of at least 50%. Sequence
similarity determinations can be performed, for example,
using the FASTA program (Genetics Computer Group;
Madison, WI). Alternatively, sequence similarity
determinations can be performed using BLASTP (Basic Local
Alignment Search Tool) of the Experimental GENIFO(R)
BLAST Network Service. See Altschul et al., J. Mol.
Biol. 215: 403 (1990). Also, see Pasternak et al.,
"Sequence Similarity Searches, Multiple Sequence
Alignments, and Molecular Tree Building," in METHODS IN
PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et al.
(eds.), pages 251-267 (CRC Press, 1993).
As described below, the nucleotide sequence [SEQ ID
NO:3] of the SRK-A10 cDNA contains a one base pair
deletion at position 948 which would cause a premature
termination of translation of the SRK-A10 protein. It is
possible to correct this defect by inserting a nucleotide
at position 948 of the SRK-A10 cDNA sequence. In the
present context, a DNA molecule containing such a
nucleotide sequence is referred to as a corrected SR~-AlO
gene. A corrected SRR-A10 gene is capable of producing
SRK-A10 protein.
2. Isolation of cDNA Molecules Encoding Genes
Associated with the Al0 Allele
cDNA molecules encoding genes associated with the A10
allele can be isolated from a Brassica pistil cDNA
library. Preferably, the RNA used to construct the cDNA
library is obtained from the pistils of the self-
incompatible Brassica napus ssp. olifera lines Topas-2,
Regent-2, or W1. The generat~on of the Topas-2 and
Regent-2 lines has been described in U.S. Serial No.
08/208,909 (filed on March 11, 1994), and in Goring et
al., The Plant Journal 2: 983 (1992), the contents of
which are hereby incorporated by reference. The
generation of the W1 line has been described in U.S.
2123751
.
--10--
Serial No. 08/208,909, as well as in Goring et al ., Mol .
Gen. Genet. 234: 185 (1992), the contents of which are
hereby incorporated by reference. The Wl line can be
obtained from the American Type Culture Collection
(Rockville, MD) as accession number 75570.
Total RNA can be prepared from pistils using
techniques well-known to those in the art. In general,
RNA isolation techniques must provide a method for
breaking plant cells, a means of inhibiting RNase-
directed degradation of RNA, and a method of separating
RNA from DNA, protein, and polysaccharide contaminants.
For example, total RNA can be isolated from pistils by
freezing plant tissue in liquid nitrogen, grinding the
frozen tissue with a mortar and pestle to lyse cells,
extracting the ground tissue with a solution of
phenol/chloroform to remove proteins, and separating RNA
from the remaining impurities by selective precipitation
with lithium chloride. See, for example, Ausubel et al.
(eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, pages
4.3.1-4.3.4 (John Wiley & Sons 1990) [hereinafter
"Ausubel"]. Also, see Sharrock et al ., Genes and
Development 3:1745 (1989).
Alternatively, total RNA can be isolated from pistils
by extracting ground tissue with guanidinium
isothiocyanate, extracting with organic solvents, and
separating RNA from contaminants using differential
centrifugation. See, for example, Strommer et al .,
"Isolation and characterization of Plant mRNA," in
METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY,
Glick et al . (eds.), pages 49-65 (CRC Press, 1993).
In order to construct a cDNA library, poly(A)+ RNA
must be isolated from a total RNA preparation. Poly(A)+
RNA can be isolated from total RNA by using the standard
tec-hnique of olgo(dT)-cellulose chromatography. See, for
example, Strommer et al ., supra .
Double-stranded cDNA molecules are synthesized from
poly(A)+ RNA using techniques well-known to those in the
art. See, for example, Ausubel at pages 5.5.2-5.6.8.
Moreover, commercially available kits can be used to
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synthesize double-stranded cDNA molecules. For example,
such kits are available from GIBCO/BRL (Gaithersburg,
MD), Clontech Laboratories, Inc. (Palo Alto, CA), Promega
Corporation (Madison, WI) and Stratagene Cloning Systems
(La Jolla, CA).
Various cloning vectors are appropriate for the
construction of a pistil cDNA library. For example, a
cDNA library can be prepared in a vector derived from
bacteriophage, such as a ~gtlO vector. Huynh et al.,
"Constructing and Screening cDNA Libraries in ~gtlO and
Agtll," in DNA Cloning: A Practical Approach, Vol . I,
Glover (ed.), pages 49-78 (IRL Press, 1985).
Alternatively, double-stranded cDNA molecules can be
inserted into a plasmid vector, such as a pBLUESCRIPT
vector (Stratagene Cloning Systems; La Jolla, CA), or
other commercially available vectors. Suitable cloning
vectors also can be obtained from the American Type
Culture Collection (Rockville, MD).
In order to amplify the cloned cDNA molecules, the
cDNA library is inserted into a procaryotic host, using
standard techniques. For example, the pistil cDNA
library can be introduced into competent E. coli DH5
cells, which can be obtained from GIBCO/BRL
(Gaithersburg, MD).
cDNA clones encoding SLG-A10 and SRK-A10 proteins can
be isolated from a cDNA library using radiolabeled
oligonucleotide probes. Suitable nucleotide sequences
for such probes are disclosed in Goring et al., The Plant
Cell 5: 531 (1993), the contents of which are hereby
incorporated by reference. Moreover, oligonucleotide
probes can be designed using the unique nucleotide
sequences of SLG-A10 and SRR-A10 cDNA molecules, which
are provided below. General techniques for screening
cDNA libraries with radiolabeIed oligonucleotide probes
are described, for example, by Ausubel at pages 2.11.1-
2.12.5, 6.1.1-6.1.23, and 6.4.1.-6.4.10.
The above-described methods can be used to obtain
SLG-A10 and SRK-A10 cDNA clones. The cDNAs can be
analyzed using a variety of techniques such as
21237~1
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restriction analysis, Northern analysis, and in situ
hybridization. See, for example, Goring et al., The
Plant Journal 2: 983 (1992), and Goring et al., The Plant
Cell 5: 531 (1993).
In the present invention, a cDNA clone encoding an
SLG protein was isolated from a B. napus oleifera cDNA
library. The nucleotide sequence of the SLG-A10 cDNA
clone [SEQ ID N0:2] was found to have a high degree of
sequence similarity with the following SLG genes: 56
[Nasrallah et al., Nature 326: 617 (1987)], S8 and S13
[Dwyer et al., Plant Nolec. Biol. 16: 481 tl991)], 529
[Trick et al., Mol. Gen. Genet. 218: 112 (1989)], and 910
[Goring et al., Mol. Gen. Genet. 234: 185 (1992)].
However, the SLG-A10 cDNA also was found to contain the
following unique nucleotide sequence [SEQ ID N0:4]:
(395) CACGAATCTT ACTAGACGTA ATGAGAGAAC (424)
GTGCTTAGAA TGATCTGCAT TACTCTCTTG.
Thus, the present invention includes an SLG-encoding DNA
molecule comprising the nucleotide sequence of SEQ ID
NO:4.
The isolation and characterization of the SRR-A10
cDNA clone is described in Example 2. The nucleotide
sequence of the SRK-A10 cDNA clone [SEQ ID NO:3] was
compared with the nucleotide sequences of the SRK-910
gene [Goring et al., Plant Cell 4: 1273 (1992)] and the
SRR6 gene [Stein et al., Proc. Natl. Acad. Sci. USA 88:
8816 (1991)]. The results of these analyses revealed
that the SRR-A10 cDNA has a high degree of nucleotide
sequence similarity with the SRR-910 and SRR6 genes, and
that the SRR-A10 cDNA contains the following unique
nucleotide sequence [SEQ ID N0:5]:
(1267) TGGAAATCTC GCTGATATGC GGAATTACGT (1296)
ACCTTTAGAG CGACTATACG CCTTAATGCA.
Thus, the present invention inclu~es an SRK-encoding DNA
molecule comprising the nucleotide sequence of SEQ ID
NO:5.
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3. Use of DNA Nolecules Encoding SLG-A10 and 8RR-A10
Protein~ to Produce Self-Incompatible Plants
Surprisingly, the nucleotide sequence of the SRR-A10
cDNA clone contains a one base pair deletion which would
lead to premature termination of the translation of SRR-
A10 mRNA and the production of a truncated SRK-A10
protein. See Example 2. As described below, plants that
contain the A10 allele express the SLG-A10 gene and yet,
such plants are self-compatible. This observation
suggests that a functional SRR gene is required for self-
incompatibility in plants that carry the A10 allele.
A DNA molecule encoding a corrected SRR-A10 gene can
be obtained by adding a nucleotide to a DNA molecule
containing the naturally occurring SRK-A10 sequence to
correct the deletion. Specifically, a "corrected SRK-A10
gene" can be obtained by adding a nucleotide at position
948 ~SEQ ID NO:6]. See Figure 3. Methods that can be
used to synthesize such a corrected SRR-A10 gene include
oligonucleotide-directed mutagenesis, linker-scanning
mutagenesis, mutagenesis using the polymerase chain
reaction, and the like. Ausubel at pages 8Ø3-8.5.9.
Also see generally, McPherson (ed.), DIRECTED
MUTAGENESIS: A PRACTICAL APPROACH, IRL Press (1991). A
specific method that has been used to correct the SRR-A10
gene is illustrated in Example 4.
Although Example 4 presents a method to add an
adenine residue, a cytosine residue or guanine residue
also may be used to correct the SRR-A10 gene. However,
the insertion of a thymine residue would result in the
presence of a stop codon.
A corrected SRK-A10 gene can be used to confer the
self-incompatible phenotype upon a self-compatible plant.
For example, a self-compatible p~ant that lacks the A10
allele can be transformed with a DNA molecule encoding
the SLG-A10 protein and a DNA molecule containing a
corrected SRR-A10 gene.
A preferred method for producing a self-incompatible
plant is to transform a self-compatible plant that
~123751
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contains an A10 allele with a DNA molecule that contains
a corrected SR~-A10 gene. This approach advantageously
allows the use of alternative schemes for breeding
hybrids since the required self-incompatibility genes
would be located at different chromosomal locations.
Therefore, one could have two self-compatible lines in
which one self-compatible line contains the A10 allele
and the other self-compatible line would be a transgenic
line that contains the corrected SRR-A10 gene, but does
not contain an A10 allele. The two self-compatible lines
can be propagated and maintained as inbred lines and when
crossed would produce self-incompatible progeny. The
self-incompatible progeny could be used as the female
line and crossed with any pollen donor to give hybrid
seed. Self-compatible lines that contain the A10 allele
include, for example, Ceres, Regent, Westar, Wl, and
certain Topas lines.
In order to express a protein that confers the self-
incompatibility phenotype, an expression vector is
constructed in which a DNA molecule encoding the SLG-A10
or SRK-A10 protein is operably linked to DNA sequences
that regulate gene transcription. The general
requirements of an expression vector are described below
in the context of a transient expression system. Here,
however, the objective is to introduce the expression
vector into plant tissue in such a manner that SLG-A10
and/or SRK-A10 proteins are expressed in the tissue of an
adult plant. Mitotic stability can be achieved using
plant viral vectors that provide epichromosomal
replication.
An alternative and preferred method of obtaining
mitotic stability is provided by the integration of
expression vector sequences into the host chromosome.
Such mitotic stability can be provided by Agrobacterium-
mediated transformation, as discussed below.
Transcription of the SLG-A10 and/or SRR-A10 genes may
be controlled by a promoter of an S-locus gene, or by a
viral promoter, such as a Cauliflower Mosaic Virus (CaMV)
promoter, a Figwort Mosaic Virus promoter, and the like.
2123751
_ -15-
Gruber et al., "Vectors for Plant Transformation," in
METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY,
Glick et al . (eds.), pages 89-119 (CRC Press, 1993).
Preferably, the promoter is the SLG-A10 promoter, the
SRK-910 promoter, or a CaMV 35S promoter.
As described in Example 1, plant tissue contains high
levels of SLG-A10 mRNA, compared with levels of SLG-910
mRNA. This observation indicates that the SLG-A10
promoter is an especially strong promoter. Thus, the
o SLG-A10 promoter is a particularly preferred promoter.
In order to select transformed cells, the expression
vector contains a selectable marker gene. For example,
such genes may confer resistance to kanamycin or
hygromycin. Although the expression vector can contain
DNA sequences encoding SLG-A10 or SRK-A10 protein under
the control of a regulatory element, as well as the
selectable marker gene under control of constitutive
promoter, the selectable marker gene is preferably
delivered to host cells in a separate selection
expression vector.
4. I~olation of SLG-A10 and SRR-A10 Genes from a
Genomic Library
The methodology, described above, can be used to
isolate cDNA clones encoding SLG-A10 and SRK-A10
proteins. The SLG-A10 and SRR-A10 genes can be isolated
from a genomic library using, for example, the
corresponding cDNA clones as probes.
A plant genomic DNA library can be prepared by means
well-known in the art. See, for example, Slightom et al.
"Construction of ~ Clone Banks," in METHODS IN PLANT
MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glick et al . (eds.),
pages 121-146 (CRC Press, 1993).J A preferred source of
plant genomic DNA is Brassica napus DNA. A more
preferred source of plant genomic DNA is the Wl canola
line. Genomic DNA can be isolated from Brassica napus
tissue, for example, by lysing plant tissue with the
detergent Sarkosyl, digesting the lysate with proteinase
21237~1
.
-16-
K, clearing insoluble debris from the lysate by
centrifugation, precipitating nucleic acid from the
lysate using isopropanol, and purifying resuspended DNA
on a cesium chloride density gradient. Ausubel et al.,
S supra, at pages 2.3.1-2.3.3.
DNA fragments that are suitable for the production
of a genomic library can be obtained by the random
shearing of genomic DNA or by the partial digestion of
genomic DNA with restriction endonucleases. See, for
example, Ausubel at pages 5.3.2-5.4.4, and Slightom et
al., supra.
Genomic DNA fragments can be inserted into a vector,
such as a bacteriophage or cosmid vector, in accordance
with conventional techniques, such as the use of
restriction enzyme digestion to provide appropriate
termini, the use of alkaline phosphatase treatment to
avoid undesirable joining of DNA molecules, and ligation
with appropriate ligases. Techniques for such
manipulation are disclosed by Slightom et al., supra, and
are well-known in the art. Also see Ausubel at pages
3Ø5-3.17.5.
Standard techniques can be used to screen a genomic
library using a cDNA clone or oligonucleotide probe.
See, for example, Ausubel at pages 6Ø3-6.6.1; Slightom
et al., supra; Raleigh et al., Genetics 122:279 (1989).
5. Identification of a Regulatory Element of the
SLG-A10 Gene or the SR~-A10 Gene
The present invention also contemplates the isolation
and use of SLG-A10 and SRR-A10 genomic regulatory
elements. In the present context, a "regulatory element"
is a DNA sequence that controls gene expression. A
regulatory element contains at least a promoter, but may
include an enhancer, as well as DNA sequences that confer
tissue-specific gene expression.
Genomic clones can be analyzed using a variety of
techniques such as restriction analysis, Southern
analysis, primer extension analysis, and DNA sequence
21~3751
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analysis. Primer extension analysis or S1 nuclease
protection analysis, for example, can be used to localize
the putative start site of transcription of the cloned
gene. Ausubel at pages 4.8.1-4.8.5; Walmsley et al .,
"Quantitative and Qualitative Analysis of Exogenous Gene
Expression by the S1 Nuclease Protection Assay," in
METHODS IN MOLECULAR BIOLOGY, VOL. 7: GENE TRANSFER AND
EXPRESSION PROTOCOLS, Murray (ed.), pages 271-281 (Humana
Press Inc. 1991). However, structural analysis per se
cannot lead to the identification of a regulatory element
associated with either the SLG-A10 gene or the SRR-A10
gene because a model for Brassica S-locus regulatory
sequences has not been developed. Thus, the regulatory
element of the SLG-A10 gene or the SRK-A10 gene must be
identified using functional analysis.
The general approach of such functional analysis
involves subcloning fragments of the genomic clone into
an expression vector which contains a reporter gene,
introducing the expression vector into various plant
tissues, and assaying the tissue to detect the transient
expression of the reporter gene. The presence of a
regulatory element in the genomic subclone is verified by
the observation of reporter gene expression in pistils,
and the low level of reporter gene expression in anther
tissue.
Methods for generating fragments of a genomic clone
are well-known. Preferably, enzymatic digestion is used
to form nested deletions of genomic DNA fragments. See,
for example, Ausubel at pages 7.2.1-7.2.20; An et al.,
"Techniques for Isolating and Characterizing
Transcription Promoters, Enhancers, and Terminators," in
METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY,
Glick et al . (eds.), pages 155-16J6 (CRC Press, 1993).
As an example, the possibility that the regulatory
element resides "upstream," or 5'-ward, of the
transcriptional start site can be tested by subcloning a
DNA fragment that contains the upstream region, digesting
the DNA fragment in either the 5' to 3' direction or in
the 3' to 5' direction to produce nested deletions, and
-18- 212 3751
subcloning the small fragments into expression vectors
for transient expression.
The selection of an appropriate expression vector
will depend upon the method of introducing the expression
vector into host cells. Typically, an expression vector
contains: (1) prokaryotic DNA elements coding for a
bacterial replication origin and an antibiotic resistance
marker to provide for the growth and selection of the
expression vector in the bacterial host; (2) a reporter
gene that is operably linked to the test DNA elements;
and (3) DNA elements that control the processing of
reporter gene transcripts, such as a transcription
termination/polyadenylation sequence. Useful reporter
genes include ~-glucuronidase, B-galactosidase,
chloramphenicol acetyl transferase, luciferase, and the
like. Preferably, the reporter gene is either the B-
glucuronidase (GUS) gene or the luciferase gene. General
descriptions of plant expression vectors and reporter
genes can be found in Gruber et al., "Vectors for Plant
Transformation," in METHODS IN PLANT MOLECULAR BIOLOGY
AND BIOTECHNOLOGY, Glick et al. (eds.), pages 89-119 (CRC
Press, 1993). Moreover, GUS expression vectors and GUS
gene cassettes are available from Clontech Laboratories,
Inc. (Palo Alto, CA), while luciferase expression vectors
and luciferase gene cassettes are available from Promega
Corporation (Madison, WI).
Expression vectors containing test genomic fragments
can be introduced into protoplasts, or into intact
tissues or isolated cells. Preferably, expression
vectors are introduced into intact tissues. General
methods of culturing plant tissues are provided, for
example, by Miki et al., "Procedures for Introducing
Foreign DNA into Plants," in METHODS IN PLANT MOLECULAR
BIOLOGY AND BIOTECHNOLOGY, Glick et al. (eds.), pages 67-
88 (CRC Press, 1993).
Methods of introducing expression vectors into plant
tissue include the direct infection or co-cultivation of
plant tissue with Agrobacterium tumefaciens. Horsch et
al., Science 227:1229 (1985). General descriptions of
2123751
_. --19--
Agrobacterium vector systems and methods for
Agrobacterium-mediated gene transfer are provided by
Gruber et al., supra, and Miki et al., supra.
Descriptions of techniques for Agrobacterium-mediated
transformation of Brassica are provided, for example by
Fry et al., Plant Cell Reports 6: 321 (1987), and
Toriyama et al., Theor. Appl. Genet. 81: 769 (1991), the
contents of which are hereby incorporated by reference.
Alternatively, expression vectors are introduced into
lo Brassica tissues using a direct gene transfer method such
as microprojectile-mediated delivery, DNA injection,
electroporation, and the like. See, for example, Gruber
et al., supra; Miki et al., supra; Klein et al.,
Biotechnology 10:268 (1992).
The above-described methods can be used to identify
DNA sequences that regulate expression of the SLG-A10
gene or the SRK-A10 gene. For example, f igure 4 shows
the nucleotide sequence of the SLG-A10 promoter tSEQ ID
NO:8]. Variants of such regulatory elements can be
produced by deleting, adding and/or substituting
nucleotides. Such variants can be obtained, for example,
by oligonucleotide-directed mutagenesis, linker-sc~nn;ng
mutagenesis, mutagenesis using the polymerase chain
reaction, and the like. Ausubel at pages 8Ø3-8.5.9.
Also see generally, McPherson (ed.), DIRECTED
MUTAGENESIS: A PRACTICAL APPROACH, IRL Press (1991).
Thus, the present invention also encompasses DNA
molecules comprising nucleotide sequences that have
substantial sequence similarity with naturally occurring
SLG-A10 or SRK-A10 gene regulatory elements.
The present invention, thus generally described, will
be understood more readily by reference to the following
examples, which are provided by-way of illustration and
are not intended to be limiting of the present invention.
- 2123751
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Example 1
Isolation and Characterization of SLG-AlO cDNA
A cDNA molecule encoding the SLG-A10 protein was
isolated from a Regent-2 cDNA library, as described in
U.S. Serial No. 08/208,909, and in Goring et al., Plant
J. 2: 983 (1992), the contents of which are hereby
incorporated by reference.
Genomic blot analyses were used to characterize the
SLG-A10 gene. In these studies, genomic DNA was
extracted from leaves using methods described by Goring
et al., Mol. Gen. Genet. 234: 185 (1992). Approximately
5 to 10 ~g of genomic DNA were digested with HindIII,
fractionated through a 0.7~ agarose gel, and transferred
to Zetabind membrane (Cuno, Inc. Laboratory Products;
Meriden, CT). The membranes were prehybridized and
hybridized as described previously. Id. Radiolabeled
probes were obtained by random priming using a full-
length SLG-A10 cDNA. Feinberg et al., Anal. Biochem.
132: 6 (1983). Following hybridization, filters were
washed using two, 30-minute washes in 0.1 x SSC (lx SSC
is O.lM sodium chloride, 0.015 M sodium citrate), 0.1%
SDS at 50C to 53C for cross-hybridization, and at 65C
to 68C for specific hybridization.
A survey of different canola cultivars revealed that
SLG-A10 probes hybridize to an 8.4 kilobase HindIII
fragment and a 9.2 kilobase HindIII fragment. Further
analysis revealed that the 8.4 kilobase ~indIII band
corresponds to the SLG-A10 gene, while the 9.2 kilobase
band results from cross-hybridization of the SLG-A10
probes to the S~R-A10 gene.
In the self-compatible canola cultivars, Ceres,
Regent, and Westar, the SLG-A10 gene is present in a
homozygous form. The A10 locuS also is occasionally
found in the self-compatible Topas line. In the self-
incompatible line W1, which was developed from a cross
between a self-incompatible B.campestris and the self-
compatible canola cultivar Westar, the SLG-A10 gene is
present in a homozygous state.
21237~1
_
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A cross between Wl and a self-compatible winter
canola line, which does not carry the A10 allele,
demonstrated the segregation pattern of the A10 allele
relative to the self-incompatibility phenotype. Of the
five genomic DNA samples from self-incompatible progeny,
only two contained the A10 allele, while genomic DNA from
four self-compatible progeny carried the A10 allele. The
allele responsible for self-incompatibility in the Wl
line has been determined to be the 910 allele which was
present only in the Wl plant and in the resulting self-
incompatible plants from the cross involving Wl. Thus,
the A10 allele does not segregate with self-
incompatibility in the Wl line.
The sequence of the SLG-A10 cDNA was analyzed to
determine if an alteration of the nucleotide sequence was
responsible for the inability of the A10 allele to
provide a self-incompatibility phenotype. To sequence
the full-length SLG-A10 cDNA clone, deletions were made
using exonuclease III and mung bean nuclease according to
the procedure in the Stratagene kit (Stratagene Cloning
Systems; La Jolla, CA). Overlapping deletions were
sequenced for both strands. All DNA and protein sequence
analyses were performed on the DNASIS and PROSIS software
(Hitachi America Ltd.; San Bruno, CA).
The results of these studies demonstrated that the
SLG-A10 sequence could be translated into a full-length
SLG protein. The predicted amino acid sequence shows
that the SLG-A10 protein contains several characteristic
features of SLG proteins, such as the signal peptide at
the 5' end, several potential sites for N-glycosylation,
and the 12 conserved cysteine residues at the carboxyl
end of the sequence. See Figure 1. The SLG-A10 sequence
shows high levels of DNA homology to a majority of
characterized SLG genes ranging ~rom 84 to 91% for DNA
and 79 to 86% similarity for the predicted amino acid
sequences. Trick et al., Mol. Gen. Genet. 218: 112
(1989); Dwyer et al., Plant Mol. Biol. 16: 481 (1991);
Goring et al., Plant J. 2: 983 (1992); Goring et al.,
Mol. Gen. Genet. 234: 185 (1992). A number of these
212~
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-22-
alleles have been associated with phenotypically strong
self-incompatibility reactions. Thus, the sequence
predictions suggest that the SLG-A10 gene should be able
to promote a strong self-incompatibility phenotype.
RNA samples from different tissues of the Wl line
were examined to determine if the lack of a self-
incompatibility phenotype was due to an altered
expression pattern of the SLG-A10 gene. The SLG-A10
steady state mRNA levels were then compared to that of
the SLG-910 allele, which is associated with Wl self-
incompatibility. In these studies, total RNA was
extracted from W1 tissues using the method of Jones et
al., EMBO J. 4: 2411 (1985). Poly(A)+ RNA samples were
extracted from Westar tissue using the MICRO-FASTRACK
mRNA isolation kits (Invitrogen; San Diego, CA). For the
RNA blots, 10 ~g of Wl total RNA was fractionated through
a 1.2% formaldehyde gel and transferred to Zetabind
membranes. See, for example, Sambrook et al., MOLECULAR
CLONING: A LABORATORY MANUAL, 2nd ed. (Cold Spring Harbor
Laboratory Press 1989). Hybridization conditions and
washing conditions for specific hybridization are
described above.
The results of these studies demonstrated that both
SLG genes were predominantly expressed in the stigma
tissue, with mRNA transcripts detected in samples before
and after anthesis. When SLG mRNA levels were compared
to plasmid controls, the steady state levels of SLG-A10
mRNA were found to be about four to eight times higher
than the SLG-910 transcripts. Similar results were also
detected in the Regent-2 line when the steady state SLG-
A10 mRNA levels were compared with steady state SLG-A14
mRNA levels. The SLG-A14 allele is associated with self-
incompatibility in Regent-2 line;
Moreover, the developmental profile of SLG-A10 mRNA
levels in stigma samples from developing Wl buds was
found to be very similar to that observed for the SLG-910
allele, as described by Goring et al., Mol. Gen. Genet.
234: 185 (1992). Finally, loss of self-incompatibility
was not associated with an absence of SLG-A10 gene
~23751
-23-
expression as high levels of SLG-A10 transcripts also
were detected in stigma RNA samples from developing buds
in the self-compatible Westar line. Thus, there are no
indications that the absence of self-incompatibility in
plants containing the A10 allele is due to an altered
expression profile, unless the higher steady state levels
of SLG-A10 RNA exerts a negative effect.
~xample 2
Isolation and Characterization of the SRR-A10 Gene
A DNA molecule encoding the SRK-A10 protein was
obtained using polymerase chain reaction (PCR) techniques
described in Goring et al., Mol. Gen. Genet. 234: 185
(1992). Briefly, W1 genomic DNA was digested with
HindIII and fractionated on an agarose gel. The HindIII
fragments were used to amplify an 800 base pair region
using PCR primers to conserved regions in published SLG
sequences. The PCR products were cloned into
pBLUESCRIPT KS+ (Stratagene Cloning Systems; La Jolla,
CA) and identified by seguencing and genomic blot
hybridization patterns. Specific primers derived from
the genomic PCR clone were then used to amplify the 5'
and 3' ends of a 2.7 kilobase SRK-A10 cDNA using the
Rapid Amplification of cDNA Ends (RACE) procedure
[Frohman et al., Proc. Natl. Acad. Sci. USA 85: 8998
(1988)] with modifications described in Goring et al.,
Mol. Gen. Genet. 234: 185 (1992).
To determine which tissues expressed the SRK-A10
gene, W1 and Westar RNA samples were analyzed by RNA-PCR.
In these studies, W1 total RNA samples and Westar
poly(A)+ RNA samples were amplified with specific SRR-A10
primers that span the kinase domain, which contains
several introns. Briefly, 2~/~g of total RNA and
approximately 1 ~g of poly(A)+ RNA were treated with
DNase I to remove any contaminating genomic DNA. The RNA
samples were then used to synthesize first strand cDNA
according to the method of Harvey et al., Nucl. Acid.
Res. 19: 4002 (1991). Controls without reverse
21237~1
-24-
transcriptase also were used for each sample. One-
quarter of each cDNA sample was amplified for 30 cycles
for the pistil samples and 35 cycles for the remaining
samples and then subjected to gel electrophoresis and DNA
gel blot analysis.
The results of these studies demonstrated that the
SRR-A10 gene is predominantly expressed in the pistils
throughout bud development in both W1 and Westar lines.
The pistils are the primary site of expression for other
SR~ genes in Brassica. Stein et al., Proc. Natl. Acad.
Sci. USA 88: 8816 (1991). Also see Goring et al., Plant
Cell 4: 1273 (1992), the contents of which are hereby
incorporated by reference. Although very weak expression
of SRK genes also has been found in the anthers, SRR-A10
transcripts were not detected in this tissue.
The nucleotide sequence of the DNA molecule encoding
the SRK-A10 protein was determined as described in
Example 1. To avoid errors that may have been introduced
during PCR, several cDNA molecules derived from separate
PCR amplifications were sequenced.
SRR genes from different S alleles encode proteins
with similar features, such as a region at the N-terminal
end that is very similar in sequence to SLGs, a
transmembrane domain, and a C-terminal kinase domain.
The SRR-A10 cDNA was found to be 86% homologous with the
SRK-910 gene [Goring et al., Plant Cell 4: 1273 (1992)~
and 87% homologous with the SRR6 [ Stein et al., Proc.
Natl. Acad. Sci. USA 88: 8816 (1991)] gene. However, the
SR~-A10 gene sequence was most similar to the SLG-A10
gene sequence in the SLG domain (92% homology). An
unusual feature of this homology is the presence of a 590
base pair region with 100% sequence identity between the
two A10 allele genes suggesting that a gene conversion
event has occurred. See Figure 2. While SLG-SRR pairs
at a particular locus are very similar to each other, the
three S loci characterized to date do not have an
analogous region of identical sequence.
A comparison of the SLG-A10 and SRR-A10 sequences
revealed a few deletions or insertions occurring in
2123751
-25-
multiples of three base pairs, which would result in the
removal or addition of amino acids in the SLG domain.
This also has been detected in the SLG-SRK pairs at the
S24 and S6 loci. However, just downstream of the region
of 100% homology at position 948 in the SRK-A10 sequence,
there is a one base pair deletion that causes a shift in
the reading frame. Translation of the DNA sequence
revealed that premature termination would occur at
nucleotide 978 and a truncated protein would be produced
(Figure 2, double underline). Except for this one base
pair deletion, the SRR-A10 gene codes for the predicted
structures of a receptor kinase, including the
transmembrane and kinase domains. The predicted kinase
domain contains all of the conserved amino acids found in
kinases, and like the other S receptor kinase genes, it
shows greatest sequence similarity to serine/threonine
kinases. Hanks et al., Science 241: 42 (1988). Thus,
the SRR-A10 gene does not encode a functional S locus
receptor kinase due to the frame shift mutation.
Example 3
Characterization of the A10 Allele
Since the A10 allele is not associated with self-
incompatibility, it was possible that the A10 allele was
not linked to the S locus. Potentially, there are two
S loci in B. napus because the plant contains the genomes
of both B. campestris and B. oleracea. In the B. napus
W1 line, both the B. campestris 910 allele and the A10
allele are homozygous, suggesting that the A10 allele is
not at the S locus position in the B. campestris
component of the B. napus genome. Furthermore, in lines
in which both the 910 and A10 alleles are present as
heterozygotes, these alleles segregate randomly in the
self-progeny. This observation is consistent with the
suggestion that the 910 and A10 alleles are not linked.
The segregation pattern of the A10 allele was
compared to a B. oleracea allele (S~) that is present in
the canola cultivar, Karat. The self-incompatible
2123751
-26-
S24Karat homozygote, which does not contain the A10 allele
was crossed to the self-compatible Westar, which is
homozygous for the A10 allele. To produce a segregating
F2 population, the F~ progeny were self-pollinated using
salt to break down self-incompatibility. The A10 allele
was detected in seedlings by PCR amplification using
primers specific to the SLG-A10 gene and by DNA blot
hybridization. Surprisingly, all 30 of the tested F2
progeny carried the A10 allele.
Since a DNA probe for the S24 allele was not
available, 19 of the F2 progeny were analyzed for the
presence of the S24 allele by testing for self-
incompatibility, and the S~ SLG gene was then identified
by cross-hybridization to the SLG-A10 cDNA. In these
studies, blots were washed at 50C in O.lX SSC, 0.1~ SDS.
Thirteen of the 19 tested plants were found to be
self-incompatible. The A10 allele was present in the
524Karat/Westar Fl plants, the Westar parental line, and
all of the F2 plants. The signal intensity of the A10
allele in the F~ and F2 populations suggested that there
was one copy of the A10 allele in self-incompatible F~
and F2 plants and two copies of the A10 allele in the
self-compatible F2 plants and the Westar parental line.
Although the S24 and A10 alleles appear to be segregating
in a 0:2:1 ratio with no S~ homozygotes, there is a
statistically significant probability that the actual
ratio is 0:1:1. In summary, these observations are
consistent with the theory that the A10 allele represents
a B. oleracea S allele in the B. napus genome.
Example 4
Construction of t~e Corrected SR~-A10 cDNA
Three overlapping fragments wére produced during the
cloning of the SRR-A10 gene. The first fragment was
synthesized using the 5' RACE procedure and contained
nucleotides 0 to 770. The second fragment was obtained
from PCR using SLG-specific primers and contained
nucleotides 510 to 1310. The third fragment was
- 2123751
.
-27-
synthesized using the 3' RACE procedure and contained
nucleotides 1280 to 2685. All three fragments were
cloned in pBLUESCRIPT (pBS).
The 5' fragment containing nucleotides 0 to 770 was
digested with BamHI and NcoI, producing a fragment
containing nucleotides 0 to 719. The cloning vector that
contained nucleotides 510 to 1310 also was digested with
BamHI and NcoI and then ligated with the 719 base pair
fragment. The resultant vector contained nucleotides o
to 1310 of the SRR-A10 DNA in pBS.
The following PCR primer was designed to correct the
deletion in SRR-A10 DNA by inserting an adenine residue
at nucleotide 948: 5' TATGTGCAAGATGTGTGG 3' [SEQ ID
NO:7]. Adenine was used to correct the one base pair
deletion because the SLG-A10 cDNA contains an adenine
residue at the corresponding position. The PCR primer,
representing nucleotides 941 to 968 of the SRR-A10 gene,
was used in a PCR with a second primer containing
nucleotides 420 to 440 of the SRK-A10 gene to produce a
DNA fragment containing nucleotides 420 to 968 of the
SRK-A10 gene in which the one base pair deletion had been
corrected.
A second PCR product was obtained using different
primers that amplified DNA from nucleotides 740 to 1230
of the SRR-A10 gene. The 1310 base pair construct was
used as the template DNA in both PCR mixtures. The two
PCR products were mixed, and the two outside primers were
used in another round of PCR. The result of this PCR
reaction was the amplification of a DNA fragment from
nucleotide 420 to nucleotide 1230 of the SRR-A10 gene.
Half of the fragments produced in this manner were
expected to carry the additional adenine at position 948.
The PCR fragment was then cleaved at nucleotide 507 with
EcoRI, and at nucleotide 1163 w'i~h SalI. The resultant
fragment was cloned into pBS.
Several clones were sequenced to verify that the SRR-
A10 gene had been corrected by the addition of adenine.
One of the corrected clones was then digested with EcoRI
and SalI and the DNA fragment containing nucleotides 507
~2~7Si
-28-
to 1163 was subcloned into the 0-1310 bp fragment of the
SRR-A10 gene. This resulted in the correction of the
deletion in SRR-A10 gene in a fragment from nucleotide 0
to nucleotide 1310.
To produce a DNA molecule encoding a complete and
corrected SRR-A10 gene, a PCR product was synthesized
from nucleotides 1000 to 1970. A 3' fragment from
nucleotide 1422 to 2685 was produced by 3' RACE and was
ligated with the PCR product in pBS. The resulting
clones contained nucleotides 1000 to 2685 of the SRR-A10
gene.
A 5' fragment containing nucleotides 0 to 1163,
including the correction at nucleotide 948, was then
isolated from the corrected 1310 base pair fragment,
described above and ligated to the remainder (i.e.
nucleotides 1163-2685) of the SRR-A10 gene. The
resultant DNA fragment contained the full length DNA
(i.e., nucleotides 0-2685) encoding the SRK-A10 protein
in pBS. The region from nucleotides 1163 to 1422, which
was derived from PCR, was sequenced to ensure that no
mistakes were introduced during amplification.
Example 5
Production of Transgenic Plants COntA ining
a Corrected SR~-Al O Gene
The corrected SRR-A10 gene was inserted in pBS behind
the SRR-910 promoter, and the resulting construct was
subcloned into the plant transformation vector, DP1741.
The vector was introduced into self-compatible B. napus
cultivars using the method of Moloney et al., Plant Cell
Reports 8: 238 (1989), the contents of which are hereby
incorporated by reference.
(,
Although the foregoing refers to particular preferred
embodiments, it will be understood that the present
2~237Si
-29-
invention is not so limited. It will occur to those of
ordinary skill in the art that various modifications may
be made to the disclosed embodiments and that such
modifications are intended to be within the scope of the
present invention, which is defined by the following
claims.
All publications and patent applications mentioned
in this specification are indicative of the level of
skill of those in the art to which the invention
pertains. All publications and patent applications are
herein incorporated by reference to the same extent as if
each individual publication or patent application were
specifically and individually indicated to be
incorporated by reference in its entirety.
