U.S. patent application number 15/849717 was filed with the patent office on 2018-07-12 for brassica rod1 gene sequences and uses thereof.
The applicant listed for this patent is Bayer CropScience NV. Invention is credited to Stephane BOUROT, Peter DENOLF, Michel VAN THOURNOUT.
Application Number | 20180195080 15/849717 |
Document ID | / |
Family ID | 49881388 |
Filed Date | 2018-07-12 |
United States Patent
Application |
20180195080 |
Kind Code |
A1 |
DENOLF; Peter ; et
al. |
July 12, 2018 |
BRASSICA ROD1 GENE SEQUENCES AND USES THEREOF
Abstract
The present invention relates to Brassica juncea ROD1 nucleic
acid sequences and proteins and the use thereof to create plants
with increased levels of C18:1 and reduced levels of saturated
fatty acids in the seeds.
Inventors: |
DENOLF; Peter; (Velzeke,
BE) ; VAN THOURNOUT; Michel; (Sint-Michiels, BE)
; BOUROT; Stephane; (Comines, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bayer CropScience NV |
Diegem |
|
BE |
|
|
Family ID: |
49881388 |
Appl. No.: |
15/849717 |
Filed: |
December 21, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14409711 |
Dec 19, 2014 |
9873886 |
|
|
PCT/EP2013/064186 |
Jul 4, 2013 |
|
|
|
15849717 |
|
|
|
|
61669370 |
Jul 9, 2012 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01H 5/10 20130101; C12N
9/1288 20130101; C12Q 1/6895 20130101; A23K 10/30 20160501; C11B
1/00 20130101; C12Y 207/08002 20130101; C12N 15/8247 20130101; A01H
1/04 20130101; C12N 15/8218 20130101; C11B 1/10 20130101; C07K
14/415 20130101; C11C 3/00 20130101; C11C 3/003 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C11C 3/00 20060101 C11C003/00; C07K 14/415 20060101
C07K014/415; A23K 10/30 20060101 A23K010/30; C11B 1/00 20060101
C11B001/00; C11B 1/10 20060101 C11B001/10; A01H 1/04 20060101
A01H001/04; C12N 9/12 20060101 C12N009/12; C12Q 1/6895 20060101
C12Q001/6895; A01H 5/10 20060101 A01H005/10 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 6, 2012 |
EP |
12175303.2 |
Claims
1. A Brassica juncea plant, or a cell, part, seed or progeny
thereof, comprising at least one ROD1 gene, characterized in that
at least one ROD1 gene is a knock-out rod1 gene.
2. The plant, or a cell, part, seed or progeny thereof, of claim 1,
comprising two knock-out rod1 genes.
3. The plant, or a cell, part, seed or progeny thereof, of claim 1
or 2, wherein said knock-out rod1 gene is a knock-out allele of the
ROD1 gene encoding a protein having at least 90% sequence identity
to SEQ ID No. 2 or SEQ ID No. 4.
4. The plant of any one of claims 1-3 which is homozygous for the
knock-out ROD1 gene.
5. A transgenic Brassica juncea plant, or a cell, part, seed or
progeny thereof, comprising a chimeric gene, said chimeric gene
comprising the following operably linked DNA fragments: a) a
plant-expressible promoter; b) a DNA region, which when transcribed
yields an RNA molecule inhibitory to at least one ROD1 gene; and
optionally c) a transcription termination and polyadenylation
region functional in plant cells.
6. The transgenic plant, or a cell, part, seed or progeny thereof,
according to claim 5 wherein said RNA molecule is inhibitory to a
ROD1 gene encoding a protein having at least 90% sequence identity
to SEQ ID No. 2 and to a ROD1 gene encoding a protein having at
least 90% sequence identity to SEQ ID No. 4.
7. Seeds from the plants of any one of claims 1-6.
8. Oil from the seeds according to claim 7.
9. A method for increasing the levels of C18:1 and/or decreasing
the levels of saturated fatty acids in Brassica juncea seed oil,
comprising modulating the expression of a ROD1 gene.
10. The method according to claim 9, comprising the steps of: a)
introducing or providing a chimeric gene to a Brassica juncea plant
cell, to create transgenic cells, said chimeric gene comprising the
following operably linked DNA fragments: a plant-expressible
promoter a DNA region, which when transcribed yields an RNA
molecule inhibitory to at least one ROD1 gene; and optionally a
transcription termination and polyadenylation region functional in
plant cells; and b) regenerating transgenic plants from said
transgenic cells.
11. The method according to claim 9, comprising the steps of: a)
treating seeds or plant material with a mutagenic chemical
substance or with ionizing radiation; b) identifying plants with a
mutated ROD1 gene, wherein the ROD1 gene, prior to being mutated,
encodes a polypeptide having at least 90% sequence identity to SEQ
ID No. 2, or to SEQ ID No. 4; and c) selecting a plant with an
increased level of C18:1 in the seeds compared to a plant in which
the ROD1 gene is not mutated.
12. A method for obtaining a Brassica juncea plant with increased
levels of C18:1 and/or decreased levels of saturated fatty acids in
the seeds comprising the step of introducing a knock-out allele of
a ROD1 gene in said Brassica juncea plant, and selecting said
Brassica juncea plant with increased levels of C18:1 in the seeds
for the presence of said knock-out allele of a ROD1 gene by
analyzing genomic DNA from said plant for the presence of at least
one molecular marker, wherein said at least one molecular marker is
linked to said knock-out allele of a ROD1 gene.
13. A method to determine the presence or absence of a knock-out
allele of a ROD1 gene in a biological sample, comprising providing
genomic DNA from said biological sample, and analyzing said DNA for
the presence of at least one molecular marker, wherein the at least
one molecular marker is linked to said knock-out allele of a ROD1
gene.
14. A kit for the detection of a knock-out allele of a ROD1 gene in
Brassica juncea DNA samples, wherein said kit comprises one or more
PCR primer pairs, which are able to amplify a DNA marker linked to
said knock-out allele of a ROD1 gene.
15. A method for determining the zygosity status of a mutant ROD1
allele in a Brassica juncea plant, or a cell, part, seed or progeny
thereof, comprising determining the presence of a mutant and/or a
corresponding wild type ROD1 specific region in the genomic DNA of
said Brassica juncea plant, or a cell, part, seed or progeny
thereof.
16. A method for transferring at least one knock-out ROD1 allele
from one Brassica juncea plant to another Brassica juncea plant
comprising the steps of: (a) identifying a first Brassica juncea
plant comprising at least one knock-out ROD1 allele, (b) crossing
the first Brassica juncea plant with a second Brassica juncea plant
not comprising the at least one knock-out ROD1 allele and
collecting F1 hybrid seeds from the cross, (c) optionally,
identifying F1 Brassica juncea plants comprising the at least one
knock-out ROD1 allele, (d) backcrossing F1 Brassica juncea plants
comprising the at least one knock-out ROD1 allele with the second
Brassica juncea plant not comprising the at least one knock-out
ROD1 allele for at least one generation (x) and collecting BCx
seeds from the crosses, (e) identifying in every generation BCx
Brassica juncea plants comprising the at least one knock-out ROD1
allele by analyzing genomic DNA of said BCx plants for the presence
of at least one molecular marker, wherein the at least one
molecular marker is linked to said knock-out ROD1 allele.
17. A chimeric gene comprising the following operably linked
elements: a) a plant-expressible promoter; b) a DNA region, which
when transcribed yields an RNA molecule inhibitory to at least one
ROD1 gene, said ROD1 gene encoding a protein having at least 90%
sequence identity to SEQ ID No. 2 or SEQ ID No. 4; and optionally
c) a transcription termination and polyadenylation region
functional in plant cells.
18. A knock-out allele of a ROD1 gene, wherein the knock-out ROD1
allele is a mutated version of the native ROD1 gene selected from
the group consisting of: (a) a nucleic acid molecule which
comprises at least 90% sequence identity to SEQ ID No. 1 or SEQ ID
No. 3; (b) a nucleic acid molecule encoding an amino acid sequence
comprising at least 90% sequence identity to SEQ ID No. 2 or SEQ ID
No. 4, wherein said mutant rod1 allele comprises a mutated DNA
region consisting of one or more inserted, deleted or substituted
nucleotides compared to a corresponding wild-type DNA region in the
functional ROD1 gene and wherein said mutant rod1 allele encodes no
functional ROD1 protein or encodes a ROD1 protein with reduced
activity.
19. Method for producing oil, comprising harvesting seeds from the
Brassica juncea plants according to any one of claims 1-6 and
extracting the oil from said seeds.
20. A method of producing food, feed, or an industrial product
comprising (a) obtaining the plant or a part thereof, of any one of
claims 1-6; and (b) preparing the food, feed or industrial product
from the plant or part thereof
21. The method of claim 20 wherein (a) the food or feed is oil,
meal, grain, starch, flour or protein; or (b) the industrial
product is biofuel, fiber, industrial chemicals, a pharmaceutical
or a nutraceutical.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 61/669,370, filed Jul. 9, 2012 and European
Patent Application Serial No. 12175303.2, filed Jul. 6, 2012, the
contents of which are herein incorporated by reference in their
entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] The official copy of the sequence listing is submitted
electronically via EFS-Web as an ASCII formatted sequence listing
with a file named "BCS12-2011_ST25 sequence listing," created on
Jul. 11, 2013 and having a size of 48 kilobytes and is filed
concurrently with the specification. The sequence listing contained
in this ASCII formatted document is part of the specification and
is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The invention relates to the field of agronomy. Methods and
means are provided to modulate fatty acid composition in Brassica
juncea, such as to increase levels of unsaturated fatty acids in
Brassica juncea by modulation of expression of ROD1 genes in
various manners, including provision of knock-out ROD1 alleles or
providing inhibitory RNAs to the ROD1 genes.
BACKGROUND OF THE INVENTION
[0004] Many plant species store triacylglycerols (TAGs) in their
seeds as a carbon reserve. These TAGs are the major source of
energy and carbon material that supports seedling development
during the early stages of plant life. Vegetable oils from soybean
(Glycine max), Brassica (Brassica napus or B. rapa), sunflower
(Helianthus annuus) and many other oilseed crops are also an
important source of oil for the human diet or industrial
applications including, but not limited to biofuels, biolubricants,
nylon precursors, and detergent feedstocks. The degree and/or
amount of polyunsaturated fatty acids of vegetable oils are
characteristic and determinative properties with respect to oil
uses in food or non-food industries. More specifically, the
characteristic properties and utilities of vegetable oils are
largely determined by their fatty acyl compositions in TAG. Major
vegetable oils are comprised primarily of palmitic (16:0), stearic
(18:0), oleic (18:1cis .DELTA..sup.9), linoleic (18:2cis
.DELTA..sup.9, 12), and .alpha.-linolenic (18:3cis .DELTA..sup.9,
12, 15 or C18:3) acids. Palmitic and stearic acids are,
respectively, 16 and 18 carbon-long, saturated fatty acids. Oleic,
linoleic, and linolenic acids are 18-carbon-long, unsaturated fatty
acids containing one, two, and three double bonds, respectively.
Oleic acid is referred to as a mono-unsaturated fatty acid, while
linoleic and linolenic acids are referred to as poly-unsaturated
fatty acids. Modifications of the fatty acid compositions have been
sought after for at least a century in order to provide optimal oil
products for human nutrition and chemical (e.g., oleochemical) uses
(Gunstone, 1998, Prog Lipid Res 37:277; Broun et al., 1999, Annu
Rev Nutr 19:107; Jaworski et al, 2003, Curr Opin Plant Biol 6:178).
In particular, the polyunsaturated fatty acids (18:2 and 18:3) have
received considerable attention because they are major factors that
affect nutritional value and oil stability. However, while these
two fatty acids provide essential nutrients for humans and animals,
they increase oil instability because they comprise multiple double
bonds that may be easily oxidized during processing and
storage.
[0005] The desaturation of 18:1 into 18:2 is a critical step for
synthesizing polyunsaturated fatty acids. During storage lipid
biosynthesis, this reaction is known to be catalyzed by the fatty
acid desaturase, FAD2, a membrane-bound enzyme located on the
endoplasmic reticulum (ER) (Browse and Somerville, 1991, Annu Rev
Plant Physiol Plant Mol Biol 42:467). The FAD2 substrate 18:1 must
be esterified on the sn-2 position of phosphatidylcholine (PC)
(Miguel and Browse, 1992, J Biol Chem 267:1502; Okuley et al.,
1994, Plant Cell 6:147), which is the major membrane phospholipid
of plant cells. Not surprisingly, therefore, down-regulation of
FAD2 (and FAD3) genes has become a preferred strategy for avoiding
the need to hydrogenate vegetable oils and the concomitant
production of undesirable trans fatty acids. For example, soybean
has both seed-specific and constitutive FAD2 desaturases, so that
gene silencing of the seed-specific isoform has allowed the
production of high-oleate cultivars (>88% 18:1 in the oil) in
which membrane unsaturation and plant performance are largely
unaffected. Significantly, however, such FAD2 gene-silencing
strategies are substantially limited because, for example, canola
and other oilseed plants have only constitutive FAD2 enzymes.
Therefore, in canola and other such constitutive FAD2 crops,
silencing or down-regulation of FAD2 not only alters the fatty acid
composition of the storage triacylglycerol (TAG) in seeds, but also
of the cellular membranes, which severely compromises growth and
yield of the plant. For example, the defective FAD2 in the
Arabidopsis mutant fad2 alters fatty acid compositions of seeds as
well as vegetable tissues, and severely compromises plant growth
(Browse and Somerville, supra). FAD2 mutations and silencing that
produce the highest 18:1 levels in the oil also reduce membrane
unsaturation in vegetative and seed tissues, resulting in plants
that germinate and grow poorly. As a result, only partial
downregulation of FAD2 expression is possible, producing
approximately 70-75% 18:1 in the oil of commercial cultivars such
as Nexera/Natreon (Dow AgroSciences) and Clear Valley 75
(Cargill).
[0006] Lu et al (2009, Proc Natl Acad Sci USA 106:18837) and
WO2009/111587 describe the identification of
phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT)
from Arabidopsis, which is endoced by the ROD1 gene, which is
involved in the transfer of 18:1 into phosphatidylcholine for
desaturation and also for the reverse transfer of 18:2 and 18:3
into the triacylglycerol synthesis pathway. The PDCT enzyme
catalyzes transfer of 18:2 and 18:3 into the triacylglycerol
synthesis pathway. Seeds of an Arabidopsis rod1 mutant have a
decrease in 18:2 and 18:3 polyunsaturated fatty acids and a
concomitant increase in 18:1 relative to wild-type, whereas there
is no effect on the fatty acid compositions of leaf or root
tissues. identified in Arabidopsis. WO2009/111587 further describes
ROD1 homologs from Brassica napus, Brassica rapa, and Brassica
oleracea.
[0007] In order to use the ROD1 gene to increase 18:1 levels and
reduce 18:2 and 18:3 levels in Brassica juncea, a need remains for
knowing all ROD1 gene sequences and the functionality of the
encoded proteins in the Brassica juncea genome. The isolation of
mutant alleles corresponding to rod1 in Brassica juncea may be
complicated by the amphidiploidy and the consequent functional
redundancy of the corresponding genes.
[0008] Thus, the prior art is deficient in teaching the ROD1 gene
sequences and the number of ROD1 genes in Brassica juncea, and
which of the ROD1 genes encode a functional protein or need to be
inactivated in order to increase the levels of 18:1 in Brassica
juncea. As described hereinafter, this problem has been solved,
allowing to modulate expression of PDCT with the aim to modulate
the 18:1 levels in Brassica juncea, as will become apparent from
the different embodiments and the claims.
SUMMARY OF THE INVENTION
[0009] It is a first embodiment of the invention to provide a
Brassica juncea plant or plant cell, part, seed or progeny thereof,
comprising at least one ROD1 gene, characterized in that at least
one ROD1 gene is an inactivated or a knock-out rod1 gene. In a
further embodiment, said plant comprises two knock-out rod1 genes.
In yet a further embodiment, said knock-out gene is a knock-out
allele of the ROD1 gene encoding a protein having at least 90%
sequence identity to SEQ ID No. 2 or SEQ ID No. 4. In a further
embodiment, said Brassica juncea plant is homozygous for said
knock-out rod1 gene.
[0010] In a further embodiment, a transgenic Brassica juncea plant
is provided comprising a chimeric gene, said chimeric gene
comprising the following operably linked DNA fragments: a
plant-expressible promoter, a DNA region, which when transcribed
yields an RNA molecule inhibitory to at least one ROD1 gene; and
optionally a transcription termination and polyadenylation region
functional in plant cells. In another embodiment, said RNA molecule
is inhibitory to a ROD1 gene encoding a protein having at least 90%
sequence identity to SEQ ID No. 2 or SEQ ID No. 4.
[0011] In a further embodiment, seeds are provided from the plants
according to the invention, i.e. plants comprising a knock-out ROD1
gene or an RNA inhibitory to a ROD1 gene. In yet another
embodiment, oil from the seeds of the plants according to the
invention is provided.
[0012] In another embodiment, a method is provided for increasing
the C18:1 levels in Brassica juncea seed oil, comprising modulating
the expression of a ROD1 gene. In yet another embodiment, a method
is provided for increasing the C18:1 levels in Brassica juncea seed
oil, comprising the steps of introducing or providing an chimeric
gene to a Brassica juncea plant cell, to create transgenic cells,
said chimeric gene comprising the following operably linked DNA
fragments: a plant-expressible promoter, a DNA region, which when
transcribed yields an RNA molecule inhibitory to at least one ROD1
gene; and optionally a transcription termination and
polyadenylation region functional in plant cells; and regenerating
transgenic plants from said transgenic cells.
[0013] In again another embodiment, a method is provided for
increasing the C18:1 levels in seed oil, comprising the steps of
treating seeds or plant material with a mutagenic chemical
substance or with ionizing radiation; identifying plants with a
mutated ROD1 gene, wherein the ROD1 gene, prior to being mutated,
encodes a polypeptide having at least 90% sequence identity to SEQ
ID No. 2 or to SEQ ID No. 4; and selecting a plant with an
increased level of C18:1 in the seeds compared to a plant in which
the ROD1 gene is not mutated.
[0014] In a further embodiment, a method is provided for obtaining
a Brassica juncea plant with increased levels of C18:1 in the seeds
comprising the step of introducing a knock-out allele of a ROD1
gene in said Brassica juncea plant, and selecting said Brassica
juncea plant with increased levels of C18:1 levels in the seeds for
the presence of said knock-out allele of a ROD1 gene by analyzing
genomic DNA from said plant for the presence of at least one
molecular marker, wherein said at least one molecular marker is
linked to said knock-out allele of a ROD1 gene.
[0015] In another embodiment, a method is provided to determine the
presence or absence of a knock-out allele of a ROD1 gene in a
biological sample, comprising providing genomic DNA from said
biological sample, and analyzing said DNA for the presence of at
least one molecular marker, wherein the at least one molecular
marker is linked to said knock-out allele of a ROD1 gene.
[0016] Yet another embodiment provides a kit for the detection of a
knock-out allele of a ROD1 gene in Brassica juncea DNA samples,
wherein said kit comprises one or more PCR primer pairs, which are
able to amplify a DNA marker linked to said knock-out allele of a
ROD1 gene.
[0017] In a further embodiment, a method is provided for
determining the zygosity status of a mutant ROD1 allele in a
Brassica juncea plant, or a cell, part, seed or progeny thereof,
comprising determining the presence of a mutant and/or a
corresponding wild type ROD1 specific region in the genomic DNA of
said plant, or a cell, part, seed or progeny thereof.
[0018] Yet a further embodiment provides a method for transferring
at least one knock-out ROD1 allele from one Brassica juncea plant
to another Brassica juncea plant comprising the steps of:
identifying a first Brassica juncea plant comprising at least one
knock-out ROD1 allele; crossing the first Brassica juncea plant
with a second Brassica juncea plant not comprising the at least one
knock-out ROD1 allele and collecting F1 hybrid seeds from the
cross; optionally, identifying F1 Brassica juncea plants comprising
the at least one knock-out ROD1 allele; backcrossing F1 Brassica
juncea plants comprising the at least one knock-out ROD1 allele
with the second plant not comprising the at least one knock-out
ROD1 allele for at least one generation (x) and collecting BCx
seeds from the crosses; identifying in every generation BCx
Brassica juncea plants comprising the at least one knock-out ROD1
allele by analyzing genomic DNA of said BCx plants for the presence
of at least one molecular marker, wherein the at least one
molecular marker is linked to said knock-out ROD1 allele.
[0019] Another embodiment provides a chimeric gene comprising the
following operably linked elements: a plant-expressible promoter; a
DNA region, which when transcribed yields an RNA molecule
inhibitory to at least one ROD1 gene, said ROD1 gene encoding a
protein having at least 90% sequence identity to SEQ ID No. 2 or
SEQ ID No. 4; and optionally a transcription termination and
polyadenylation region functional in plant cells.
[0020] In again another embodiment, a knock-out allele of an ROD1
gene is provided, wherein the knock-out ROD1 allele is a mutated
version of the native ROD1 gene selected from the group consisting
of: a nucleic acid molecule which comprises at least 90% sequence
identity to SEQ ID No. 1 or SEQ ID No. 3; or a nucleic acid
molecule encoding an amino acid sequence comprising at least 90%
sequence identity to SEQ ID No. 2 or SEQ ID No. 4, wherein said
mutant rod1 allele comprises a mutated DNA region consisting of one
or more inserted, deleted or substituted nucleotides compared to a
corresponding wild-type DNA region in the functional ROD1 gene and
wherein said mutant rod1 allele encodes no functional ROD1 protein
or encodes a ROD1 protein with reduced activity.
[0021] In a further embodiment, a method is provided for producing
oil, comprising harvesting seeds from the plants according to the
invention, i.e. Brassica juncea plants comprising an inactivated or
a knock-out ROD1 gene or an RNA inhibitory to a ROD1 gene, and
extracting the oil from said seeds.
[0022] In yet a further embodiment, a method is provided of
producing food or feed, such as oil, meal, grain, starch, flour or
protein, or an industrial product, such as biofuel, fiber,
industrial chemicals, a pharmaceutical or a neutraceutical,
comprising obtaining the Brassica juncea plant or a part thereof
according to the invention, and preparing the food, feed or
industrial product from the plant or part thereof.
General Definitions
[0023] A "ROD1 gene" or "ROD1 allele", as used herein, is a gene or
allele comprising a sequence having at least 60% sequence identity
to the coding sequence of the ROD1 gene of Arabidopsis thaliana, as
described in WO2009/111587.
[0024] A ROD1 gene or ROD1 allele can, but does not need to encode
a functional ROD1 protein. Functionality of the ROD1 protein can be
tested, for example, in yeast as described in example 4 or as
described by Lu et al. (2009) Proc Natl Acad Sci USA 106:18839.
[0025] A "knock-out rod1 gene" or "knock-out rod1 allele" as used
herein is a rod1 gene or a rod1 allele which encodes no functional
ROD1 protein, or which encodes a ROD1 protein with reduced
activity. Said "knock-out rod1 gene" can be a full knock-out rod1
gene, encoding no functional ROD1 protein, or can be a partial
knock-out rod1 gene, encoding a ROD1 protein with reduced activity.
Said "knock-out rod1 gene" or "knock-out rod1 allele" can be a
mutant rod1 allele or a mutant rod1 gene, which may encode no
functional ROD1 protein, or which may encode a mutant ROD1 protein
with reduced activity. The gene or allele may also be referred to
as an inactivated gene or allele.
[0026] A "functional ROD1 gene" or "functional ROD1 allele" as used
herein is a ROD1 gene or a ROD1 allele which encodes a functional
ROD1 protein.
[0027] A "mutant rod1 gene" or "mutant rod1 allele" as used herein
refers to any rod1 gene or rod1 allele which is not found in plants
in the natural population or breeding population, but which is
produced by human intervention such as mutagenesis or gene
targeting. A mutant rod1 allele comprises knock-out rod1 alleles,
and functional rod1 alleles.
[0028] Functional ROD1 protein is a ROD1 protein which has at least
5%, or at least 10%, or at least 15%, or at least 20%, or at least
25%, or at least 30% of the activity of the protein encoded by the
Arabidopsis ROD1 gene as described in WO2009/111587, as tested, for
example, in yeast as described in example 3.
[0029] A mutant ROD1 protein with reduced functionality is a ROD1
protein encoded by a mutant rod1 gene which has reduced activity as
compared to the corresponding wild-type ROD1 protein encoded by the
wild-type ROD1 gene. Said activity may be reduced with at least
10%, or at least 20%, or at least 30%, or at least 40%, or at least
50%, or at least 60%, or at least 70%, or at least 80%, or at least
90%.
[0030] The term "nucleic acid sequence" (or nucleic acid molecule)
refers to a DNA or RNA molecule in single or double stranded form,
particularly a DNA encoding a protein or protein fragment according
to the invention. An "endogenous nucleic acid sequence" refers to a
nucleic acid sequence within a plant cell, e.g. an endogenous
allele of an ROD1 gene present within the nuclear genome of a
Brassica juncea cell. An "isolated nucleic acid sequence" is used
to refer to a nucleic acid sequence that is no longer in its
natural environment, for example in vitro or in a recombinant
bacterial or plant host cell.
[0031] The term "gene" means a DNA sequence comprising a region
(transcribed region), which is transcribed into an RNA molecule
(e.g. into a pre-mRNA, comprising intron sequences, which is then
spliced into a mature mRNA, or directly into a mRNA without intron
sequences) in a cell, operably linked to regulatory regions (e.g. a
promoter). A gene may thus comprise several operably linked
sequences, such as a promoter, a 5' leader sequence comprising e.g.
sequences involved in translation initiation, a (protein) coding
region (cDNA or genomic DNA) and a 3' non-translated sequence
comprising e.g. transcription termination sites. "Endogenous gene"
is used to differentiate from a "foreign gene", "transgene" or
"chimeric gene", and refers to a gene from a plant of a certain
plant genus, species or variety, which has not been introduced into
that plant by transformation (i.e. it is not a "transgene"), but
which is normally present in plants of that genus, species or
variety, or which is introduced in that plant from plants of
another plant genus, species or variety, in which it is normally
present, by normal breeding techniques or by somatic hybridization,
e.g., by protoplast fusion. Similarly, an "endogenous allele" of a
gene is not introduced into a plant or plant tissue by plant
transformation, but is, for example, generated by plant mutagenesis
and/or selection or obtained by screening natural populations of
plants.
[0032] "Expression of a gene" or "gene expression" refers to the
process wherein a DNA region, which is operably linked to
appropriate regulatory regions, particularly a promoter, is
transcribed into an RNA molecule. The RNA molecule is then
processed further (by post-transcriptional processes) within the
cell, e.g. by RNA splicing and translation initiation and
translation into an amino acid chain (polypeptide), and translation
termination by translation stop codons. The term "functionally
expressed" is used herein to indicate that a functional protein is
produced; the term "not functionally expressed" to indicate that a
protein with significantly reduced or no functionality (biological
activity) is produced or that no protein is produced (see further
below).
[0033] The terms "protein" or "polypeptide" are used
interchangeably and refer to molecules consisting of a chain of
amino acids, without reference to a specific mode of action, size,
3-dimensional structure or origin. A "fragment" or "portion" of an
ROD1 protein may thus still be referred to as a "protein". An
"isolated protein" is used to refer to a protein that is no longer
in its natural environment, for example in vitro or in a
recombinant bacterial or plant host cell.
[0034] As used herein, the term "allele(s)" means any of one or
more alternative forms of a gene at a particular locus. In a
diploid (or amphidiploid) cell of an organism, alleles of a given
gene are located at a specific location or locus (loci plural) on a
chromosome. One allele is present on each chromosome of the pair of
homologous chromosomes.
[0035] As used herein, the term "homologous chromosomes" means
chromosomes that contain information for the same biological
features and contain the same genes at the same loci but possibly
different alleles of those genes. Homologous chromosomes are
chromosomes that pair during meiosis. "Non-homologous chromosomes",
representing all the biological features of an organism, form a
set, and the number of sets in a cell is called ploidy. Diploid
organisms contain two sets of non-homologous chromosomes, wherein
each homologous chromosome is inherited from a different parent. In
amphidiploid species, essentially two sets of diploid genomes
exist, whereby the chromosomes of the two genomes are referred to
as "homeologous chromosomes" (and similarly, the loci or genes of
the two genomes are referred to as homeologous loci or genes). A
diploid, or amphidiploid, plant species may comprise a large number
of different alleles at a particular locus.
[0036] As used herein, the term "heterozygous" means a genetic
condition existing when two different alleles reside at a specific
locus, but are positioned individually on corresponding pairs of
homologous chromosomes in the cell. Conversely, as used herein, the
term "homozygous" means a genetic condition existing when two
identical alleles reside at a specific locus, but are positioned
individually on corresponding pairs of homologous chromosomes in
the cell.
[0037] As used herein, the term "locus" (loci plural) means a
specific place or places or a site on a chromosome where for
example a gene or genetic marker is found.
[0038] "Wild type" (also written "wildtype" or "wild-type"), as
used herein, refers to a typical form of a plant or a gene as it
most commonly occurs in nature. A "wild type plant" refers to a
plant in the natural population or in a breeding population. A
"wild type allele" refers to an allele of a gene occurring in
wild-type plants.
[0039] Whenever reference to a "plant" or "plants" according to the
invention is made, it is understood that also plant parts (cells,
tissues or organs, seed pods, seeds, severed parts such as roots,
leaves, flowers, pollen, etc.), progeny of the plants which retain
the distinguishing characteristics of the parents (especially the
fruit dehiscence properties), such as seed obtained by selfing or
crossing, e.g. hybrid seed (obtained by crossing two inbred
parental lines), hybrid plants and plant parts derived there from
are encompassed herein, unless otherwise indicated.
[0040] "Creating propagating material", as used herein, relates to
any means know in the art to produce further plants, plant parts or
seeds and includes inter alia vegetative reproduction methods (e.g.
air or ground layering, division, (bud) grafting, micropropagation,
stolons or runners, storage organs such as bulbs, corms, tubers and
rhizomes, striking or cutting, twin-scaling), sexual reproduction
(crossing with another plant) and asexual reproduction (e.g.
apomixis, somatic hybridization).
[0041] "Mutagenesis", as used herein, refers to the process in
which plant cells (e.g., a plurality of seeds or other parts, such
as pollen, etc.) are subjected to a technique which induces
mutations in the DNA of the cells, such as contact with a mutagenic
agent, such as a chemical substance (such as ethylmethylsulfonate
(EMS), ethylnitrosourea (ENU), etc.) or ionizing radiation
(neutrons (such as in fast neutron mutagenesis, etc.), alpha rays,
gamma rays (such as that supplied by a Cobalt 60 source), X-rays,
UV-radiation, etc.), T-DNA insertion mutagenesis (Azpiroz-Leehan et
al. (1997) Trends Genet 13:152-156), transposon mutagenesis
(McKenzie et al. (2002) Theor Appl Genet 105:23-33), or tissue
culture mutagenesis (induction of somaclonal variations), or a
combination of two or more of these. Thus, the desired mutagenesis
of one or more ROD1 alleles may be accomplished by one of the above
methods. While mutations created by irradiation are often large
deletions or other gross lesions such as translocations or complex
rearrangements, mutations created by chemical mutagens are often
more discrete lesions such as point mutations. For example, EMS
alkylates guanine bases, which results in base mispairing: an
alkylated guanine will pair with a thymine base, resulting
primarily in G/C to A/T transitions. Following mutagenesis, plants
are regenerated from the treated cells using known techniques. For
instance, the resulting seeds may be planted in accordance with
conventional growing procedures and following self-pollination seed
is formed on the plants. Alternatively, doubled haploid plantlets
may be extracted to immediately form homozygous plants, for example
as described by Coventry et al. (1988, Manual for Microspore
Culture Technique for Brassica napus. Dep. Crop Sci. Techn. Bull.
OAC Publication 0489. Univ. of Guelph, Guelph, Ontario, Canada).
Additional seed that is formed as a result of such self-pollination
in the present or a subsequent generation may be harvested and
screened for the presence of mutant rod1 alleles. Several
techniques are known to screen for specific mutant alleles, e.g.,
Deleteagene.TM. (Delete-a-gene; Li et al., 2001, Plant J 27:
235-242) uses polymerase chain reaction (PCR) assays to screen for
deletion mutants generated by fast neutron mutagenesis, TILLING
(targeted induced local lesions in genomes; McCallum et al., 2000,
Nat Biotechnol 18:455-457) identifies EMS-induced point mutations,
etc. Additional techniques to screen for the presence of specific
mutant rod1 alleles are described in the Examples below.
[0042] The term "gene targeting" refers herein to directed gene
modification that uses mechanisms such as homologous recombination,
mismatch repair or site-directed mutagenesis. The method can be
used to replace, insert and delete endogenous sequences or
sequences previously introduced in plant cells. Methods for gene
targeting can be found in, for example, WO 2006/105946 or
WO2009/002150. Gene targeting can be used to create mutant rod1
alleles, such as knock-out rod1 alleles.
[0043] A "variety" is used herein in conformity with the UPOV
convention and refers to a plant grouping within a single botanical
taxon of the lowest known rank, which grouping can be defined by
the expression of the characteristics resulting from a given
genotype or combination of genotypes, can be distinguished from any
other plant grouping by the expression of at least one of the said
characteristics and is considered as a unit with regard to its
suitability for being propagated unchanged (stable).
[0044] The term "comprising" is to be interpreted as specifying the
presence of the stated parts, steps or components, but does not
exclude the presence of one or more additional parts, steps or
components. A plant comprising a certain trait may thus comprise
additional traits.
[0045] It is understood that when referring to a word in the
singular (e.g. plant or root), the plural is also included herein
(e.g. a plurality of plants, a plurality of roots). Thus, reference
to an element by the indefinite article "a" or "an" does not
exclude the possibility that more than one of the element is
present, unless the context clearly requires that there be one and
only one of the elements. The indefinite article "a" or "an" thus
usually means "at least one".
[0046] For the purpose of this invention, the "sequence identity"
of two related nucleotide or amino acid sequences, expressed as a
percentage, refers to the number of positions in the two optimally
aligned sequences which have identical residues (.times.100)
divided by the number of positions compared. A gap, i.e., a
position in an alignment where a residue is present in one sequence
but not in the other, is regarded as a position with non-identical
residues. The "optimal alignment" of two sequences is found by
aligning the two sequences over the entire length according to the
Needleman and Wunsch global alignment algorithm (Needleman and
Wunsch, 1970, J Mol Biol 48(3):443-53) in The European Molecular
Biology Open Software Suite (EMBOSS, Rice et al., 2000, Trends in
Genetics 16(6): 276-277; see e.g.
http://www.ebi.ac.uk/emboss/align/index.html) using default
settings (gap opening penalty=10 (for nucleotides)/10 (for
proteins) and gap extension penalty=0.5 (for nucleotides)/0.5 (for
proteins)). For nucleotides the default scoring matrix used is
EDNAFULL and for proteins the default scoring matrix is
EBLOSUM62.
[0047] "Substantially identical" or "essentially similar", as used
herein, refers to sequences, which, when optimally aligned as
defined above, share at least a certain minimal percentage of
sequence identity (as defined further below).
[0048] "Stringent hybridization conditions" can be used to identify
nucleotide sequences, which are substantially identical to a given
nucleotide sequence. Stringent conditions are sequence dependent
and will be different in different circumstances. Generally,
stringent conditions are selected to be about 5.degree. C. lower
than the thermal melting point (T.sub.m) for the specific sequences
at a defined ionic strength and pH. The T.sub.m is the temperature
(under defined ionic strength and pH) at which 50% of the target
sequence hybridizes to a perfectly matched probe. Typically
stringent conditions will be chosen in which the salt concentration
is about 0.02 molar at pH 7 and the temperature is at least
60.degree. C. Lowering the salt concentration and/or increasing the
temperature increases stringency. Stringent conditions for RNA-DNA
hybridizations (Northern blots using a probe of e.g. 100 nt) are
for example those which include at least one wash in 0.2.times.SSC
at 63.degree. C. for 20 min, or equivalent conditions.
[0049] "High stringency conditions" can be provided, for example,
by hybridization at 65.degree. C. in an aqueous solution containing
6.times.SSC (20.times.SSC contains 3.0 M NaCl, 0.3 M Na-citrate, pH
7.0), 5.times.Denhardt's (100.times.Denhardt's contains 2% Ficoll,
2% Polyvinyl pyrollidone, 2% Bovine Serum Albumin), 0.5% sodium
dodecyl sulphate (SDS), and 20 .mu.g/ml denaturated carrier DNA
(single-stranded fish sperm DNA, with an average length of 120-3000
nucleotides) as non-specific competitor. Following hybridization,
high stringency washing may be done in several steps, with a final
wash (about 30 min) at the hybridization temperature in
0.2-0.1.times.SSC, 0.1% SDS.
[0050] "Moderate stringency conditions" refers to conditions
equivalent to hybridization in the above described solution but at
about 60-62.degree. C. Moderate stringency washing may be done at
the hybridization temperature in 1.times.SSC, 0.1% SDS.
[0051] "Low stringency" refers to conditions equivalent to
hybridization in the above described solution at about
50-52.degree. C. Low stringency washing may be done at the
hybridization temperature in 2.times.SSC, 0.1% SDS. See also
Sambrook et al. (1989) and Sambrook and Russell (2001).
DETAILED DESCRIPTION
[0052] The current invention is based on the identification of
seven ROD1 genes in Brassica juncea.
[0053] It is a first embodiment of the invention to provide a
Brassica juncea plant or plant cell, part, seed or progeny thereof,
comprising at least one ROD1 gene, characterized in that at least
one ROD1 gene is an inactivated or a knock-out rod1 gene. Said at
least one ROD1 gene can be, for example, two ROD1 genes, or four
ROD1 genes, or seven ROD1 genes, or eight ROD1 genes. In a further
embodiment, said plant comprises two knock-out rod1 genes. In yet a
further embodiment, said knock-out gene is a knock-out allele of
the ROD1 gene encoding a protein having at least 90% sequence
identity to SEQ ID No. 2 or SEQ ID No. 4. In a further embodiment,
said Brassica juncea plant is homozygous for said knock-out rod1
gene.
[0054] Said at least one, or two, or four, or seven ROD1 genes can
be selected from the group consisting of BjROD1-A1, BjROD1-B1,
BjROD1-A2, BjROD1-B2, BjROD1-A3, BjROD1-B3, and BjROD1-B4 or
variants thereof. Said eight ROD1 genes can be selected from the
group consisting of BjROD1-A1, BjROD1-B1, BjROD1-A2, BjROD1-B2,
BjROD1-A3, BjROD1-B3, and BjROD1-B4 or variants thereof and an
eighth ROD1 gene which can be a BjROD1-A4 gene.
[0055] At least 90% sequence identity as used herein can be at
least 90% sequence identity, or at least 95% sequence identity, or
at least 98% sequence identity, or can be 100% sequence
identity.
[0056] A knock-out allele of the ROD1 gene encoding a protein
having at least 90% sequence identity to SEQ ID No. 2 or to SEQ ID
No. 4 can be a knock-out allele of the ROD1 gene having at least
90% sequence identity, or at least 95% sequence identity, or at
least 98% sequence identity, or having 100% sequence identity to
SEQ ID No. 1, SEQ ID No. 3, respectively.
[0057] Said knock-out allele of said ROD1 gene can be a mutant ROD1
gene comprising one or more nucleotide deletions, insertions or
substitutions relative to the wild type nucleic acid sequences. The
mutation(s) can result in one or more changes (deletions,
insertions and/or substitutions) in the amino acid sequence of the
encoded protein is not a functional ROD1 protein.
[0058] Nucleic Acid Sequences According to the Invention
[0059] Provided are both wild type ROD1 nucleic acid sequences
encoding functional ROD1 proteins and mutant rod1 nucleic acid
sequences (comprising one or more mutations, preferably mutations
which result in no or a significantly reduced biological activity
of the encoded ROD1 protein or in no ROD1 protein being produced)
of ROD1 genes from Brassica juncea.
[0060] However, isolated ROD1 and rod1 nucleic acid sequences (e.g.
isolated from the plant by cloning or made synthetically by DNA
synthesis), as well as variants thereof and fragments of any of
these are also provided herein, as these can be used to determine
which sequence is present endogenously in a plant or plant part,
whether the sequence encodes a functional, a non-functional or no
protein (e.g. by expression in a recombinant host cell as described
below) and for selection and transfer of specific alleles from one
plant into another, in order to generate a plant having the desired
combination of functional and mutant alleles.
[0061] Nucleic acid sequences of ROD1-A1, ROD1-B1, ROD1-A2,
ROD1-B2, ROD1-A3, ROD1-B3, and ROD1-B4 have been isolated from
Brassica juncea, as depicted in the sequence listing. The wild type
ROD1 cDNA sequences are depicted, while the mutant rod1 sequences
of these sequences, and of sequences essentially similar to these,
are described herein below and in the Examples, with reference to
the wild type ROD1 sequences.
[0062] A "Brassica juncea ROD1-A1 gene", "BjROD1-A1 gene",
"Brassica juncea ROD1-A1 allele", "BjROD1-A1 allele" or "ROD1-A1
from Brassica juncea", or variant nucleic acid sequences thereof as
used herein refers to a gene, allele or a sequence of which the
cDNA sequence has at least 90%, or at least 95%, or at least 98%,
or at least 99%, or 100% sequence identity SEQ ID No. 1.
[0063] A "Brassica juncea ROD1-B1 gene", "BjROD1-B1 gene",
"Brassica juncea ROD1-B1 allele", "BjROD1-B1 allele" or "ROD1-B1
from Brassica juncea", or variant nucleic acid sequences thereof as
used herein refers to a gene, allele or a sequence of which the
cDNA sequence has at least 90%, or at least 95%, or at least 98%,
or at least 99%, or 100% sequence identity SEQ ID No 3.
[0064] A "Brassica juncea ROD1-A2 gene", "BjROD1-A2 gene",
"Brassica juncea ROD1-A2 allele", "BjROD1-A2 allele" or "ROD1-A2
from Brassica juncea", or variant nucleic acid sequences thereof as
used herein refers to a gene, allele or a sequence of which the
cDNA sequence has at least 90%, or at least 95%, or at least 98%,
or at least 99%, or 100% sequence identity SEQ ID No 5.
[0065] A "Brassica juncea ROD1-B2 gene", "BjROD1-B2 gene",
"Brassica juncea ROD1-B2 allele", "BjROD1-B2 allele" or "ROD1-B2
from Brassica juncea", or variant nucleic acid sequences thereof as
used herein refers to a gene, allele or a sequence of which the
cDNA sequence has at least 90%, or at least 95%, or at least 98%,
or at least 99%, or 100% sequence identity SEQ ID No 7.
[0066] A "Brassica juncea ROD1-A3 gene", "BjROD1-A3 gene",
"Brassica juncea ROD1-A3 allele", "BjROD1-A3 allele" or "ROD1-A3
from Brassica juncea", or variant nucleic acid sequences thereof as
used herein refers to a gene, allele or a sequence of which the
cDNA sequence has at least 90%, or at least 95%, or at least 98%,
or at least 99%, or 100% sequence identity SEQ ID No 9.
[0067] A "Brassica juncea ROD1-B3 gene", "BjROD1-B3 gene",
"Brassica juncea ROD1-B3 allele", "BjROD1-B3 allele" or "ROD1-B3
from Brassica juncea", or variant nucleic acid sequences thereof as
used herein refers to a gene, allele or a sequence of which the
cDNA sequence has at least 90%, or at least 95%, or at least 98%,
or at least 99%, or 100% sequence identity SEQ ID No 11.
[0068] A "Brassica juncea ROD1-B4 gene", "BjROD1-B4 gene",
"Brassica juncea ROD1-B4 allele", "BjROD1-B4 allele" or "ROD1-B4
from Brassica juncea", or variant nucleic acid sequences thereof as
used herein refers to a gene, allele or a sequence of which the
cDNA sequence has at least 90%, or at least 95%, or at least 98%,
or at least 99%, or 100% sequence identity SEQ ID No 13.
[0069] A BjROD1-A4 gene is a ROD1 gene which is annotated on the A
genome of Brassica juncea and homeologous to the BjROD1-B4
gene.
[0070] Thus the invention provides both nucleic acid sequences
encoding wild type, functional ROD1 proteins, including variants
and fragments thereof (as defined further below), as well as mutant
nucleic acid sequences of any of these, whereby the mutation in the
nucleic acid sequence preferably results in one or more amino acids
being inserted, deleted or substituted in comparison to the wild
type ROD1 protein. Preferably the mutation(s) in the nucleic acid
sequence result in one or more amino acid changes (i.e. in relation
to the wild type amino acid sequence one or more amino acids are
inserted, deleted and/or substituted) whereby the biological
activity of the ROD1 protein is significantly reduced or completely
abolished.
[0071] Functionality of the ROD1 protein can be tested, for
example, in yeast as described in example 3 or as described by Lu
et al. (2009) Proc Natl Acad Sci USA 106:18839.
[0072] Both endogenous and isolated nucleic acid sequences are
provided herein. Also provided are fragments of the ROD1 sequences
and ROD1 variant nucleic acid sequences defined above, for use as
primers or probes and as components of kits according to another
aspect of the invention (see further below). A "fragment" of a ROD1
or rod1 nucleic acid sequence or variant thereof (as defined) may
be of various lengths, such as at least 10, 12, 15, 18, 20, 50,
100, 200, 500, 600 contiguous nucleotides of the ROD1 or rod1
sequence (or of the variant sequence).
[0073] Wild-Type Nucleic Acid Sequences Encoding Wild-Type ROD1
Proteins
[0074] The nucleic acid sequences depicted in the sequence listing
encode wild type ROD1 proteins from Brassica juncea. Thus, these
sequences are endogenous to the Brassica juncea plants from which
they were isolated.
[0075] Other Brassica juncea varieties, breeding lines or wild
accessions may be screened for other ROD1 alleles, encoding the
same ROD1 proteins or variants thereof. For example, nucleic acid
hybridization techniques (e.g. Southern blot analysis, using for
example stringent hybridization conditions) or nucleic acid
amplification-based techniques such as PCR techniques may be used
to identify ROD1 alleles endogenous to other Brassica juncea
varieties, lines or accessions. To screen such plants, plant organs
or tissues for the presence of ROD1 alleles, the ROD1 nucleic acid
sequences provided in the sequence listing, or variants or
fragments of any of these, may be used. For example whole sequences
or fragments may be used as probes or primers. For example specific
or degenerate primers may be used to amplify nucleic acid sequences
encoding ROD1 proteins from the genomic DNA of the plant, plant
organ or tissue. These ROD1 nucleic acid sequences may be isolated
and sequenced using standard molecular biology techniques.
Bioinformatics analysis may then be used to characterize the
allele(s), for example in order to determine which ROD1 allele the
sequence corresponds to and which ROD1 protein or protein variant
is encoded by the sequence.
[0076] In addition, it is understood that ROD1 nucleic acid
sequences and variants thereof (or fragments of any of these) may
be identified in silico, by screening nucleic acid databases for
essentially similar sequences. Likewise, a nucleic acid sequence
may be synthesized chemically. Fragments of nucleic acid molecules
according to the invention are also provided, which are described
further below.
[0077] Mutant Nucleic Acid Sequences Encoding Mutant ROD1
Proteins
[0078] Nucleic acid sequences comprising one or more nucleotide
deletions, insertions or substitutions relative to the wild type
nucleic acid sequences are another embodiment of the invention, as
are fragments of such mutant nucleic acid molecules. Such mutant
nucleic acid sequences (referred to as rod1 sequences) can be
generated and/or identified using various known methods, as
described further below. Again, such nucleic acid molecules are
provided both in endogenous form and in isolated form. In one
embodiment, the mutation(s) result in one or more changes
(deletions, insertions and/or substitutions) in the amino acid
sequence of the encoded ROD1 protein (i.e. it is not a "silent
mutation"). In another embodiment, the mutation(s) in the nucleic
acid sequence result in a significantly reduced or completely
abolished biological activity of the encoded ROD1 protein relative
to the wild type protein.
[0079] The knock-out ROD1 genes may, thus, comprise one or more
mutations, such as:
[0080] (a) a "missense mutation", which is a change in the nucleic
acid sequence that results in the substitution of an amino acid for
another amino acid;
[0081] (b) a "nonsense mutation" or "STOP codon mutation", which is
a change in the nucleic acid sequence that results in the
introduction of a premature STOP codon and thus the termination of
translation (resulting in a truncated protein); plant genes contain
the translation stop codons "TGA" (UGA in RNA), "TAA" (UAA in RNA)
and "TAG" (UAG in RNA); thus any nucleotide substitution,
insertion, deletion which results in one of these codons to be in
the mature mRNA being translated (in the reading frame) will
terminate translation;
[0082] (c) an "insertion mutation" of one or more amino acids, due
to one or more codons having been added in the coding sequence of
the nucleic acid;
[0083] (d) a "deletion mutation" of one or more amino acids, due to
one or more codons having been deleted in the coding sequence of
the nucleic acid;
[0084] (e) a "frameshift mutation", resulting in the nucleic acid
sequence being translated in a different frame downstream of the
mutation. A frameshift mutation can have various causes, such as
the insertion, deletion or duplication of one or more
nucleotides;
[0085] (f) a splice site mutation, resulting in altered splicing,
which results in an altered mRNA processing and, consequently, in
an altered encoded protein which contains either deletions,
substitutions or insertions of various lengths, possibly combined
with premature translation termination.
[0086] Thus in one embodiment, nucleic acid sequences comprising
one or more of any of the types of mutations described above are
provided. In another embodiment, rod1 sequences comprising one or
more stop codon (nonsense) mutations, one or more missense
mutations, one or more frameshift mutations, and/or one or more
splice site mutations are provided. Any of the above mutant nucleic
acid sequences are provided per se (in isolated form), as are
plants and plant parts comprising such sequences endogenously. In
the tables herein below the most preferred rod1 alleles are
described.
[0087] A range of possible EMS stop codon mutations in the
BjROD1-A1, BjROD1-B1, BjROD1-A2, BjROD1-B2, BjROD1-A3, BjROD1-B3
and BjROD1-B4 genes are shown in Tables 1a-g, respectively.
TABLE-US-00001 TABLE 1a possible stop codon mutations in BjROD1-A1
position relative to the genomic position sequence (SEQ WT relative
to the stop codon ID No. 1) codon AA protein codon AA 397-399 TGG
TRP 54 TAG STOP TAA STOP TGA STOP 436-438 TGG TRP 67 TAA STOP TGA
STOP TAG STOP 496-498 CAG GLN 87 TAG STOP TAA STOP 628-630 CAA GLN
131 TAA STOP 646-648 TGG TRP 137 TAG STOP TGA STOP TAA STOP 652-654
TGG TRP 139 TGA STOP TAA STOP TAG STOP 673-675 CGA ARG 146 TGA STOP
TAA STOP 733-735 CAG GLN 166 TAA STOP TAG STOP 748-750 CAG GLN 171
TAA STOP TAG STOP 862-864 CAG GLN 209 TAG STOP TAA STOP 907-909 CAA
GLN 224 TAA STOP
TABLE-US-00002 TABLE 1b possible stop codon mutations in BjROD1-B1
position relative to the genomic position sequence (SEQ WT relative
to the stop codon ID No. 3) codon AA protein codon AA 224-226 TGG
TRP 54 TAG STOP TAA STOP TGA STOP 263-265 TGG TRP 67 TAA STOP TGA
STOP TAG STOP 323-325 CAG GLN 87 TAG STOP TAA STOP 689-691 CGG ARG
163 TAA STOP TAG STOP TGA STOP 734-736 CAA GLN 178 TAA STOP
TABLE-US-00003 TABLE 1c possible stop codon mutations in BjROD1-A2
position relative to the genomic position sequence (SEQ WT relative
to the stop codon ID No. 5) codon AA protein codon AA 412-414 TGG
TRP 57 TAA STOP TAG STOP TGA STOP 451-453 TGG TRP 70 TAA STOP TAG
STOP TGA STOP 511-513 CAA GLN 90 TAA STOP 643-645 CAA GLN 134 TAA
STOP 661-663 TGG TRP 140 TAG STOP TAA STOP TGA STOP 667-669 TGG TRP
142 TAA STOP TGA STOP TAG STOP 688-690 CGG ARG 149 TGA STOP TAG
STOP TAA STOP 736-738 CAG GLN 165 TAA STOP TAG STOP 751-753 CAG GLN
170 TAG STOP TAA STOP 865-867 CAG GLN 208 TAG STOP TAA STOP 910-912
CAA GLN 223 TAA STOP
TABLE-US-00004 TABLE 1d possible stop codon mutations in BjROD1-B2
position relative to the genomic position sequence (SEQ WT relative
to the stop codon ID No. 7) codon AA protein codon AA 298-300 TGG
TRP 42 TAG STOP TAA STOP TGA STOP 337-339 TGG TRP 55 TAA STOP TAG
STOP TGA STOP 397-399 CAG GLN 75 TAA STOP TAG STOP 529-531 CAA GLN
119 TAA STOP 547-549 TGG TRP 125 TAG STOP TGA STOP TAA STOP 553-555
TGG TRP 127 TAG STOP TGA STOP TAA STOP 574-576 CGG ARG 134 TGA STOP
TAG STOP TAA STOP 634-636 CAG GLN 154 TAG STOP TAA STOP 649-651 CAG
GLN 159 TAA STOP TAG STOP 763-765 CAG GLN 197 TAG STOP TAA STOP
808-810 CAA GLN 212 TAA STOP
TABLE-US-00005 TABLE 1e possible stop codon mutations in BjROD1-A3
position relative to the genomic position sequence (SEQ WT relative
to the stop codon ID No. 9) codon AA protein codon AA 161-163 CAA
GLN 37 TAA STOP 182-184 CAA GLN 44 TAA STOP 248-250 TGG TRP 66 TAA
STOP TGA STOP TAG STOP 287-289 TGG TRP 79 TGA STOP TAA STOP TAG
STOP 350-352 CAG GLN 100 TAA STOP TAG STOP 482-484 CAA GLN 144 TAA
STOP 500-502 TGG TRP 150 TAA STOP TAG STOP TGA STOP 506-508 TGG TRP
152 TAG STOP TAA STOP TGA STOP 521-523 CGA ARG 157 TGA STOP TAA
STOP 527-529 CGA ARG 159 TAA STOP TGA STOP 587-589 CAG GLN 179 TAG
STOP TAA STOP 602-604 CAG GLN 184 TAG STOP TAA STOP 761-763 CAA GLN
237 TAA STOP 791-793 CAA GLN 247 TAA STOP
TABLE-US-00006 TABLE 1f possible stop codon mutations in BjROD1-B3
position relative to the genomic position sequence (SEQ WT relative
to the stop codon ID No. 11) codon AA protein codon AA 100-102 CGG
ARG 11 TAA STOP TAG STOP TGA STOP 178-180 CAA GLN 37 TAA STOP
199-201 CAA GLN 44 TAA STOP 265-267 TGG TRP 66 TAG STOP TGA STOP
TAA STOP 304-306 TGG TRP 79 TAA STOP TAG STOP TGA STOP 367-369 CAG
GLN 100 TAG STOP TAA STOP 499-501 CAA GLN 144 TAA STOP 517-519 TGG
TRP 150 TAA STOP TAG STOP TGA STOP 523-525 TGG TRP 152 TAA STOP TGA
STOP TAG STOP 538-540 CGA ARG 157 TGA STOP TAA STOP 544-546 CGA ARG
159 TAA STOP TGA STOP 604-606 CAG GLN 179 TAA STOP TAG STOP 619-621
CAG GLN 184 TAG STOP TAA STOP 778-780 CAA GLN 237 TAA STOP 808-810
CAA GLN 247 TAA STOP
TABLE-US-00007 TABLE 1g possible stop codon mutations in BjROD1-B4
position relative to the genomic position sequence (SEQ WT relative
to the stop codon ID No. 13) codon AA protein codon AA 29-31 CAA
GLN 3 TAA STOP 65-67 TGG TRP 15 TAG STOP TAA STOP TGA STOP 92-94
TGG TRP 24 TAA STOP TGA STOP TAG STOP 131-133 TGG TRP 37 TGA STOP
TAA STOP TAG STOP 323-325 CAA GLN 101 TAA STOP 341-343 TGG TRP 107
TAG STOP TGA STOP TAA STOP 347-349 TGG TRP 109 TAG STOP TGA STOP
TAA STOP 362-364 CGG ARG 114 TAG STOP TAA STOP TGA STOP 368-370 CGA
ARG 116 TAA STOP TGA STOP 428-430 CAG GLN 136 TAG STOP TAA STOP
557-559 CAG GLN 179 TAA STOP TAG STOP 602-604 CAG GLN 194 TAA STOP
TAG STOP 722-724 CAA GLN 234 TAA STOP
[0088] Obviously, mutations are not limited to the ones shown in
the above tables and it is understood that analogous STOP mutations
may be present in rod1 alleles other than those depicted in the
sequence listing and referred to in the tables above. Not only
stopcodon mutations, but also mutations resulting in an amino acid
substitution may lead to proteins with reduced functionality or
with no detectable activity. Amino acids that, when substituted,
may lead to proteins with reduced activity are Glu at position 144,
Thr at position 150, Arg at position 160, Gly at position 161, and
Pro at position 172 of the BjROD1-A1 protein, or Glu at position
142, Thr at position 148, Arg at position 158, and Pro at position
169 of the BjROD1-B1 protein.
[0089] Wild-type and mutant ROD1 nucleic acid sequences from the
A-genome as described herein, such as BjROD1-A1, BjROD1-A2, and
BjROD1-A3 are also suitable to use in other Brassica species
comprising an A genome, such as Brassica napus and Brassica
rapa.
[0090] Wild-type and mutant ROD1 nucleic acid sequences from the
B-genome as described herein, such as BnROD1-B1, BnROD1-B2,
BnROD1-B3, and BnROD1-B4 are also suitable to use in other Brassica
species comprising an B genome, such as Brassica carinata and
Brassica nigra.
[0091] Amino Acid Sequences According to the Invention
[0092] Provided are both wild type ROD1 amino acid sequences and
mutant ROD1 amino acid sequences (comprising one or more mutations,
preferably mutations which result in a significantly reduced or no
biological activity of the ROD1 protein) from Brassica juncea. In
addition, mutagenesis methods can be used to generate mutations in
wild type ROD1 alleles, thereby generating mutant alleles which can
encode further mutant ROD1 proteins. In one embodiment the wild
type and/or mutant ROD1 amino acid sequences are provided within a
Brassica juncea plant (i.e. endogenously). However, isolated ROD1
amino acid sequences (e.g. isolated from the plant or made
synthetically), as well as variants thereof and fragments of any of
these are also provided herein.
[0093] Amino acid sequences of Brassica juncea ROD1-1 and ROD1-2
proteins have been isolated as depicted in the sequence listing.
The wild type ROD1 sequences are depicted, while the mutant ROD1
sequences of these sequences, and of sequences essentially similar
to these, are described herein below, with reference to the wild
type ROD1 sequences.
[0094] "Brassica juncea ROD1-A1 amino acid sequences" or "BjROD1-A1
amino acid sequences" or variant amino acid sequences thereof
according to the invention are amino acid sequences having at least
95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 2. These
amino acid sequences may also be referred to as being "essentially
similar" or "essentially identical" to the ROD1 sequences provided
in the sequence listing.
[0095] "Brassica juncea ROD1-B1 amino acid sequences" or "BjROD1-B1
amino acid sequences" or variant amino acid sequences thereof
according to the invention are amino acid sequences having at least
95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 4. These
amino acid sequences may also be referred to as being "essentially
similar" or "essentially identical" to the ROD1 sequences provided
in the sequence listing.
[0096] "Brassica juncea ROD1-A2 amino acid sequences" or "BjROD1-A2
amino acid sequences" or variant amino acid sequences thereof
according to the invention are amino acid sequences having at least
95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 6. These
amino acid sequences may also be referred to as being "essentially
similar" or "essentially identical" to the ROD1 sequences provided
in the sequence listing.
[0097] "Brassica juncea ROD1-B2 amino acid sequences" or "BjROD1-B2
amino acid sequences" or variant amino acid sequences thereof
according to the invention are amino acid sequences having at least
95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 8. These
amino acid sequences may also be referred to as being "essentially
similar" or "essentially identical" to the ROD1 sequences provided
in the sequence listing.
[0098] "Brassica juncea ROD1-A3 amino acid sequences" or "BjROD1-A3
amino acid sequences" or variant amino acid sequences thereof
according to the invention are amino acid sequences having at least
95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 10. These
amino acid sequences may also be referred to as being "essentially
similar" or "essentially identical" to the ROD1 sequences provided
in the sequence listing.
[0099] "Brassica juncea ROD1-B3 amino acid sequences" or "BjROD1-B3
amino acid sequences" or variant amino acid sequences thereof
according to the invention are amino acid sequences having at least
95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 12. These
amino acid sequences may also be referred to as being "essentially
similar" or "essentially identical" to the ROD1 sequences provided
in the sequence listing.
[0100] "Brassica juncea ROD1-B4 amino acid sequences" or "BjROD1-B4
amino acid sequences" or variant amino acid sequences thereof
according to the invention are amino acid sequences having at least
95%, 98%, 99% or 100% sequence identity with SEQ ID NO: 14. These
amino acid sequences may also be referred to as being "essentially
similar" or "essentially identical" to the ROD1 sequences provided
in the sequence listing.
[0101] "Brassica juncea ROD1-A4 amino acid sequences" or "BjROD1-A4
amino acid sequences" or variant amino acid sequences thereof
according to the invention are amino acid sequences encoded by the
BjROD1-A4 gene. These amino acid sequences may also be referred to
as being "essentially similar" or "essentially identical" to
ROD1-A4.
[0102] Thus, the invention provides both amino acid sequences of
wild type proteins, including variants and fragments thereof (as
defined further below), as well as mutant amino acid sequences of
any of these, whereby the mutation in the amino acid sequence
preferably results in a significant reduction in or a complete
abolishment of the biological activity of the ROD1 protein as
compared to the biological activity of the corresponding wild type
ROD1 protein.
[0103] Both endogenous and isolated amino acid sequences are
provided herein. Also provided are fragments of the ROD1 amino acid
sequences and ROD1 variant amino acid sequences defined above. A
"fragment" of a ROD1 amino acid sequence or variant thereof (as
defined) may be of various lengths, such as at least 10, 12, 15,
18, 20, 50, 100, 150, 175, 180 contiguous amino acids of the ROD1
sequence (or of the variant sequence).
[0104] Amino Acid Sequences of Wild-Type ROD1 Proteins
[0105] The amino acid sequences depicted in the sequence listing
are wild type ROD1 proteins from Brassica juncea. Thus, these
sequences are endogenous to the Brassica juncea plants from which
they were isolated. Other Brassica juncea varieties, breeding lines
or wild accessions may be screened for other functional ROD1
proteins with the same amino acid sequences or variants thereof, as
described above.
[0106] In addition, it is understood that ROD1 amino acid sequences
and variants thereof (or fragments of any of these) may be
identified in silico, by screening amino acid databases for
essentially similar sequences. Fragments of amino acid molecules
according to the invention are also provided.
[0107] Amino Acid Sequences of Mutant ROD1 Proteins
[0108] Amino acid sequences comprising one or more amino acid
deletions, insertions or substitutions relative to the wild type
amino acid sequences are another embodiment of the invention, as
are fragments of such mutant amino acid molecules. Such mutant
amino acid sequences can be generated and/or identified using
various known methods, as described above. Again, such amino acid
molecules are provided both in endogenous form and in isolated
form.
[0109] In one embodiment, the mutation(s) in the amino acid
sequence result in a significantly reduced or completely abolished
biological activity of the ROD1 protein relative to the wild type
protein. As described above, basically, any mutation which results
in a protein comprising at least one amino acid insertion, deletion
and/or substitution relative to the wild type protein can lead to
significantly reduced or no biological activity.
[0110] Thus in one embodiment, mutant ROD1 proteins are provided
comprising one or more deletion or insertion mutations, whereby the
deletion(s) or insertion(s) result(s) in a mutant protein which has
significantly reduced or no activity. Such mutant ROD1 proteins are
ROD1 proteins wherein at least 1, at least 2, 3, 4, 5, 10, 20, 30,
50, 100, 150, 200 or more amino acids are deleted, inserted or
substituted as compared to the wild type ROD1 protein, whereby the
deletion(s) or insertion(s) result(s) in a mutant protein which has
significantly reduced or no activity.
[0111] In another embodiment, mutant ROD1 proteins are provided
which are truncated whereby the truncation results in a mutant
protein that has significantly reduced or no activity.
[0112] In yet another embodiment, mutant ROD1 proteins are provided
comprising one or more substitution mutations, whereby the
substitution(s) result(s) in a mutant protein that has
significantly reduced or no activity.
[0113] In a further embodiment, a transgenic Brassica juncea plant
is provided comprising a chimeric gene, said chimeric gene
comprising the following operably linked DNA fragments: a
plant-expressible promoter, a DNA region, which when transcribed
yields an RNA molecule inhibitory to at least one ROD1 gene; and
optionally a transcription termination and polyadenylation region
functional in plant cells.
[0114] Said at least one ROD1 gene can be, for example, two ROD1
genes, or four ROD1 genes, or seven ROD1 genes, or eight ROD1
genes.
[0115] Said at least one, or two, or four, or seven ROD1 genes can
be selected from the group consisting of BjROD1-A1, BjROD1-B1,
BjROD1-A2, BjROD1-B2, BjROD1-A3, BjROD1-B3, and BjROD1-B4 or
variants thereof. Said eight ROD1 genes can be selected from the
group consisting of BjROD1-A1, BjROD1-B1, BjROD1-A2, BjROD1-B2,
BjROD1-A3, BjROD1-B3, and BjROD1-B4 or variants thereof and an
eighth ROD1 gene which can be a BjROD1-A4 gene.
[0116] In another embodiment, said RNA molecule is inhibitory to a
ROD1 gene encoding a protein having at least 90% sequence identity
to SEQ ID No. 2, or is inhibitory to a ROD1 gene encoding a protein
having at least 90% sequence identity to SEQ ID No. 4, is
inhibitory to both a ROD1 gene encoding a protein having at least
90% sequence identity to SEQ ID No. 2 and to a ROD1 gene encoding a
protein having at least 90% sequence identity to SEQ ID No. 4.
[0117] An RNA molecule inhibitory to at least one ROD1 gene can be
an RNA that downregulates ROD1 gene expression by decreasing the
levels of ROD1 mRNAs available for translation. Said RNA can
downregulate ROD1 gene expression through, for example,
co-suppression (sense RNA suppression), antisense RNA,
double-stranded RNA (dsRNA) or microRNA (miRNA), or ta-siRNA.
[0118] Said RNA molecule inhibitory to at least one ROD1 gene is
characterized tin that said RNA molecule comprises a region with
sufficient homology to said ROD1 genes to be downregulated.
[0119] Sufficient homology to the ROD1 genes to be downregulated as
used herein means that the transcribed DNA region (and resulting
RNA molecule) comprises at least 20 consecutive nucleotides having
at least 95% sequence identity to the nucleotide sequence or the
complement of the nucleotide of the ROD1 gene to be
downregulated.
[0120] Said RNA molecule inhibitory to at least one ROD1 gene may
be a sense RNA molecule capable of down-regulating expression of
one or more functional ROD1 genes by co-suppression. Said RNA
molecule comprises at least 20 consecutive nucleotides having at
least 95% sequence identity to the nucleotide sequence of one or
more ROD1 genes present in the plant cell or plant.
[0121] Said RNA molecule inhibitory to at least one ROD1 gene may
further be an antisense RNA molecule capable of down-regulating
expression of one or more functional ROD1 genes. Said RNA molecule
comprises at least 20 consecutive nucleotides having at least 95%
sequence identity to the complement of the nucleotide sequence of
one or more functional ROD1 genes present in the plant cell or
plant.
[0122] The minimum nucleotide sequence of the antisense or sense
RNA region of about 20 nt of the ROD1 gene may be comprised within
a larger RNA molecule, varying in size from 20 nt to a length equal
to the size of the target gene. The mentioned antisense or sense
nucleotide regions may thus be about from about 21 nt to about 1300
nt long, such as 21 nt, 40 nt, 50 nt, 100 nt, 200 nt, 300 nt, 500
nt, 1000 nt, or even about 1300 nt or larger in length. Moreover,
it is not required for the purpose of the invention that the
nucleotide sequence of the used inhibitory ROD1 RNA molecule or the
encoding region of the transgene, is completely identical or
complementary to the endogenous ROD1 gene the expression of which
is targeted to be reduced in the plant cell. The longer the
sequence, the less stringent the requirement for the overall
sequence identity is.
[0123] Thus, the sense or antisense regions may have an overall
sequence identity of about 40% or 50% or 60% or 70% or 80% or 90%
or 100% to the nucleotide sequence of the endogenous ROD1 gene or
the complement thereof. However, as mentioned, antisense or sense
regions should comprise a nucleotide sequence of 20 consecutive
nucleotides having about 95 to about 100% sequence identity to the
nucleotide sequence of the endogenous ROD1 gene. The stretch of
about 95 to about 100% sequence identity may be about 50, 75 or 100
nt. It will be clear that all combinations between mentioned length
and sequence identity can be made, both in sense and/or antisense
orientation.
[0124] The abovementioned chimeric gene may further comprise DNA
elements which result in the expression of aberrant,
non-polyadenylated ROD1 inhibitory RNA molecules. One such DNA
element suitable for that purpose is a DNA region encoding a
self-splicing ribozyme, as described in WO 00/01133. The efficiency
may also be enhanced by providing the generated RNA molecules with
nuclear localization or retention signals as described in WO
03/076619.
[0125] Said RNA molecule inhibitory to at least one ROD1 gene may
further be a double-stranded RNA molecule capable of
down-regulating ROD1 gene expression. Upon transcription of the DNA
region the RNA is able to form dsRNA molecule through conventional
base paring between a sense and antisense region, whereby the sense
and antisense region are nucleotide sequences as hereinbefore
described. dsRNA-encoding ROD1 expression-reducing chimeric genes
according to the invention may further comprise an intron, such as
a heterologous intron, located e.g. in the spacer sequence between
the sense and antisense RNA regions in accordance with the
disclosure of WO 99/53050. To achieve the construction of such a
transgene, use can be made of the vectors described in WO 02/059294
A1.
[0126] Said RNA molecule inhibitory to at least one ROD1 gene may
further be a pre-miRNA molecule which is processed into a miRNA
capable of guiding the cleavage of ROD1 mRNA. miRNAs are small
endogenous RNAs that regulate gene expression in plants, but also
in other eukaryotes. In plants, these about 21 nucleotide long RNAs
are processed from the stem-loop regions of long endogenous
pre-miRNAs by the cleavage activity of DICERLIKE1 (DCL1). Plant
miRNAs are highly complementary to conserved target mRNAs, and
guide the cleavage of their targets. miRNAs appear to be key
components in regulating the gene expression of complex networks of
pathways involved inter alia in development.
[0127] As used herein, a "miRNA" is an RNA molecule of about 20 to
22 nucleotides in length which can be loaded into a RISC complex
and direct the cleavage of a target RNA molecule, wherein the
target RNA molecule comprises a nucleotide sequence essentially
complementary to the nucleotide sequence of the miRNA molecule
whereby one or more of the following mismatches may occur: [0128] A
mismatch between the nucleotide at the 5' end of said miRNA and the
corresponding nucleotide sequence in the target RNA molecule;
[0129] A mismatch between any one of the nucleotides in position 1
to position 9 of said miRNA and the corresponding nucleotide
sequence in the target RNA molecule; [0130] Three mismatches
between any one of the nucleotides in position 12 to position 21 of
said miRNA and the corresponding nucleotide sequence in the target
RNA molecule provided that there are no more than two consecutive
mismatches.
[0131] No mismatch is allowed at positions 10 and 11 of the miRNA
(all miRNA positions are indicated starting from the 5' end of the
miRNA molecule).
[0132] As used herein, a "pre-miRNA" molecule is an RNA molecule of
about 100 to about 200 nucleotides, preferably about 100 to about
130 nucleotides which can adopt a secondary structure comprising a
dsRNA stem and a single stranded RNA loop and further comprising
the nucleotide sequence of the miRNA and its complement sequence of
the miRNA* in the double-stranded RNA stem. Preferably, the miRNA
and its complement are located about 10 to about 20 nucleotides
from the free ends of the miRNA dsRNA stem. The length and sequence
of the single stranded loop region are not critical and may vary
considerably, e.g. between 30 and 50 nt in length. Preferably, the
difference in free energy between unpaired and paired RNA structure
is between -20 and -60 kcal/mole, particularly around -40
kcal/mole. The complementarity between the miRNA and the miRNA* do
not need to be perfect and about 1 to 3 bulges of unpaired
nucleotides can be tolerated. The secondary structure adopted by an
RNA molecule can be predicted by computer algorithms conventional
in the art such as mFold, UNAFold and RNAFold. The particular
strand of the dsRNA stem from the pre-miRNA which is released by
DCL activity and loaded onto the RISC complex is determined by the
degree of complementarity at the 5' end, whereby the strand which
at its 5' end is the least involved in hydrogen bounding between
the nucleotides of the different strands of the cleaved dsRNA stem
is loaded onto the RISC complex and will determine the sequence
specificity of the target RNA molecule degradation. However, if
empirically the miRNA molecule from a particular synthetic
pre-miRNA molecule is not functional because the "wrong" strand is
loaded on the RISC complex, it will be immediately evident that
this problem can be solved by exchanging the position of the miRNA
molecule and its complement on the respective strands of the dsRNA
stem of the pre-miRNA molecule. As is known in the art, binding
between A and U involving two hydrogen bounds, or G and U involving
two hydrogen bounds is less strong that between G and C involving
three hydrogen bounds.
[0133] miRNA molecules may be comprised within their naturally
occurring pre-miRNA molecules but they can also be introduced into
existing pre-miRNA molecule scaffolds by exchanging the nucleotide
sequence of the miRNA molecule normally processed from such
existing pre-miRNA molecule for the nucleotide sequence of another
miRNA of interest. The scaffold of the pre-miRNA can also be
completely synthetic. Likewise, synthetic miRNA molecules may be
comprised within, and processed from, existing pre-miRNA molecule
scaffolds or synthetic pre-miRNA scaffolds.
[0134] Said RNA molecule inhibitory to at least one ROD1 gene may
further be a ta-siRNAs as described in WO2006/074400.
[0135] Said RNA molecule may be inhibitory to all ROD1 genes
present in said Brassica juncea plant. For example, said RNA
molecule is inhibitory to a ROD1 gene encoding a protein having at
least 90% sequence identity to SEQ ID No. 2 and SEQ ID No. 4, such
as a ROD1 gene having at least 90% sequence identity, or at least
95% sequence identity, or at least 98% sequence identity or having
100% sequence identity to SEQ ID No. 1 or SEQ ID No. 3,
respectively.
[0136] Said RNA molecule may further be inhibitory to only one ROD1
gene, such as the ROD1 genes encoding a protein having at least 90%
sequence identity to SEQ ID No. 2 only, such as a ROD1 gene having
at least 90% sequence identity, or at least 95% sequence identity,
or at least 98% sequence identity or having 100% sequence identity
to SEQ ID No. 1, or to the ROD1 gene encoding a protein having at
least 90% sequence identity to SEQ ID No. 4 only, such as a ROD1
gene having at least 90% sequence identity, or at least 95%
sequence identity, or at least 98% sequence identity or having 100%
sequence identity to SEQ ID No. 3.
[0137] As used herein, the term "plant-expressible promoter" means
a DNA sequence that is capable of controlling (initiating)
transcription in a plant cell. This includes any promoter of plant
origin, but also any promoter of non-plant origin which is capable
of directing transcription in a plant cell, i.e., certain promoters
of viral or bacterial origin such as the CaMV35S (Harpster et al.
(1988) Mol Gen Genet. 212(1):182-90, the subterranean clover virus
promoter No 4 or No 7 (WO9606932), or T-DNA gene promoters but also
tissue-specific or organ-specific promoters including but not
limited to seed-specific promoters (e.g., WO89/03887),
organ-primordia specific promoters (An et al. (1996) Plant Cell
8(1):15-30), stem-specific promoters (Keller et al., (1988) EMBO J.
7(12): 3625-3633), leaf specific promoters (Hudspeth et al. (1989)
Plant Mol Biol. 12: 579-589), mesophyl-specific promoters (such as
the light-inducible Rubisco promoters), root-specific promoters
(Keller et al. (1989) Genes Dev. 3: 1639-1646), tuber-specific
promoters (Keil et al. (1989) EMBO J. 8(5): 1323-1330), vascular
tissue specific promoters (Peleman et al. (1989) Gene 84: 359-369),
stamen-selective promoters (WO 89/10396, WO 92/13956), dehiscence
zone specific promoters (WO 97/13865) and the like.
[0138] A "heterologous promoter" as used herein refers to a
promoter which is not normally associated in its natural context
with the coding DNA region operably linked to it in the DNA
molecules according to the invention.
[0139] Said plant-expressible promoter can, for example, be a
constitutive promoter, such as the CaMV35S promoter (Harpster et
al. (1988) Mol Gen Genet. 212(1):182-90), or a seed-specific
promoter, such as the Arabidopsis oleosin promoter
(WO1998/045461).
[0140] Constitutive promoters are well known in the art, and
include the CaMV35S promoter (Harpster et al. (1988) Mol Gen Genet.
212(1):182-90), Actin promoters, such as, for example, the promoter
from the Rice Actin gene (McElroy et al., 1990, Plant Cell 2:163),
the promoter of the Cassava Vein Mosaic Virus (Verdaguer et al.,
1996 Plant Mol. Biol. 31: 1129), the GOS promoter (de Pater et al.,
1992, Plant J. 2:837), the Histone H3 promoter (Chaubet et al.,
1986, Plant Mol Biol 6:253), the Agrobacterium tumefaciens Nopaline
Synthase (Nos) promoter (Depicker et al., 1982, J. Mol. Appl.
Genet. 1: 561), or Ubiquitin promoters, such as, for example, the
promoter of the maize Ubiquitin-1 gene (Christensen et al., 1992,
Plant Mol. Biol. 18:675).
[0141] Seed specific promoters are well known in the art, including
the Arabidopsis oleosin promoter (WO1998/045461), the USP promoter
from Vicia faba described in DE10211617; the promoter sequences
described in WO2009/073738; promoters from Brassica napus for seed
specific gene expression as described in WO2009/077478; the plant
seed specific promoters described in US2007/0022502; the plant seed
specific promoters described in WO03/014347; the seed specific
promoter described in WO2009/125826; the promoters of the omega_3
fatty acid desaturase family described in WO2006/005807 and the
like.
[0142] A "transcription termination and polyadenylation region" as
used herein is a sequence that drives the cleavage of the nascent
RNA, whereafter a poly(A) tail is added at the resulting RNA 3'
end, functional in plants. Transcription termination and
polyadenylation signals functional in plants include, but are not
limited to, 3' nos, 3'35S, 3'his and 3'g7.
[0143] In a further embodiment, the seeds of the plants according
to the invention have increased levels of C18:1, or increased
levels of C18:1 and decreased levels of C18:2, or increased levels
of C18:1 and decreased levels of SATS.
[0144] In a further embodiment, seeds are provided from the plants
according to the invention, i.e. plants comprising a knock-out ROD1
gene or an RNA inhibitory to a ROD1 gene. In yet another
embodiment, oil from the seeds of the plants according to the
invention is provided.
[0145] In another embodiment, a method is provided for increasing
the C18:1 levels in Brassica juncea seed oil, comprising modulating
the expression of a ROD1 gene. In yet another embodiment, a method
is provided for increasing the C18:1 levels in Brassica juncea seed
oil, comprising the steps of introducing or providing an chimeric
gene to a Brassica juncea plant cell, to create transgenic cells,
said chimeric gene comprising the following operably linked DNA
fragments: a plant-expressible promoter, a DNA region, which when
transcribed yields an RNA molecule inhibitory to at least one ROD1
gene; and optionally a transcription termination and
polyadenylation region functional in plant cells; and regenerating
transgenic plants from said transgenic cells.
[0146] "C18:1", also referred to as "oleic acid",
"cis-9-octadecenoic", "18:1", "18:1 (n-9)", "9c-18:1" or "18:1cis
.DELTA..sup.9" as used herein, refers to a monounsaturated omega-9
fatty acid, with the IUPAC name (9Z)-Octadec-9-enoic acid.
[0147] "C18:2", also referred to as "linoleic acid",
"cis-9,12-octadecadienoic acid", "18:2", "18:2 (n-6)", "9c12c-18:1
or "18:2cis .DELTA..sup.9, 12", as used herein, refers to a
carboxylic acid with an 18-carbon chain and two double bonds with
the IUPAC name cis, cis-9,12-Octadecadienoic acid.
[0148] SATS, as used herein, refers to saturated fatty acids, which
refers to the sum of the levels of C12:0, C14:0, C16:0, C18:0,
C20:0, C22:0 and C24:0.
[0149] Increasing the C18:1 levels or increased C18:1 levels in
seed oil can be an increase of C18:1 levels with at least 2%, or at
least 5%, or at least 8%, or at least 10%, or at least 12%. Said
increase is an increase with respect to C18:1 levels as obtained in
control plants.
[0150] Decreased levels of C18:2 can be a decrease of C18:2 levels
in seed oil with at least 2%, or at least 5%, or at least 8%, or at
least 10%, or at least 20%, or at least 30%.
[0151] Decreased levels of SATS can be a decrease in the levels of
SATS in seed oil with at least 2%, or at least 3%, or at least 5%.
A decrease in the levels of SATS refers to a decrease in the total
levels of the sum of C16:0, C18:0, C20:0, C22:0 and C24:0. As such,
a decrease in the levels of SATS can be a decrease in the levels of
only one of the saturated fatty acids, or of more than one of the
saturated fatty acids.
[0152] Optionally, the increase of the C18:1 levels or decrease of
the C18:2 or SATS in seeds or in seed oil is higher than an
increase in C18:1 levels or decrease of the C18:2 or SATS in
membrane lipids. For example, the levels of C18:1 are increased, or
the C18:2 levels or SATS are increased in the seeds, but the C18:1,
C18:2 and SATS levels are unchanged in membrane lipids.
[0153] C18:1, C18:2 and SATS levels can be measured as described
herein, such as, for example, using the methods as described in
Examples 4 and 5.
[0154] The "control plant" as used herein is generally a plant of
the same species which has wild-type levels of ROD1. "Wild-type
levels of ROD1" as used herein refers to the typical levels of ROD1
protein in a plant as it most commonly occurs in nature. Said
control plant does contain an RNA molecule inhibitory to ROD1, and
in which the ROD1 genes are wild-type ROD1 genes.
[0155] A chimeric gene can be provided to a plant or plant cell
using methods well-known in the art. Methods to provide plant cells
with a chimeric are not deemed critical for the current invention
and any method to provide plant cells with a chimeric gene suitable
for a particular plant species can be used. Such methods are well
known in the art and include Agrobacterium-mediated transformation,
particle gun delivery, microinjection, electroporation of intact
cells, polyethyleneglycol-mediated protoplast transformation,
electroporation of protoplasts, liposome-mediated transformation,
silicon-whiskers mediated transformation etc. Said chimeric can be
transiently introduced into the plant cell or plant cell nucleus.
Said chimeric may be stably integrated into the genome of said
plant cell, resulting in a transformed plant cell. The transformed
plant cells obtained in this way may then be regenerated into
mature fertile transformed plants.
[0156] The obtained transformed plant, comprising the RNA molecule
inhibitory to at least one ROD1 gene, can be used in a conventional
breeding scheme to produce more transformed plants with the same
characteristics or to introduce the transgene according to the
invention in other varieties of the same or related plant species,
or in hybrid plants. Seeds obtained from the transformed plants
contain the chimeric genes of the invention as a stable genomic
insert and are also encompassed by the invention.
[0157] In again another embodiment, a method is provided for
increasing the C18:1 levels in seed oil, comprising the steps of
treating seeds or plant material with a mutagenic chemical
substance or with ionizing radiation; identifying plants with a
mutated rod1 gene, wherein the ROD1 gene, prior to being mutated,
encodes a polypeptide having at least 90% sequence identity to SEQ
ID No. 2 or to SEQ ID No. 4; and selecting a plant with an
increased level of C18:1 in the seeds compared to a plant in which
the ROD1 gene is not mutated.
[0158] Said ROD1 gene, prior to being mutated, can be, for example,
a ROD1 gene having at least 90% sequence identity, or at least 95%
sequence identity, or at least 98% sequence identity or having 100%
sequence identity to SEQ ID No. 1, or SEQ ID No. 3.
[0159] In a further embodiment, a method is provided for obtaining
a Brassica juncea plant with increased levels of C18:1 in the seeds
comprising the step of introducing a knock-out allele of a ROD1
gene in said Brassica juncea plant, and selecting said Brassica
juncea plant with increased levels of C18:1 in the seeds for the
presence of said knock-out allele of a ROD1 gene by analyzing
genomic DNA from said plant for the presence of at least one
molecular marker, wherein said at least one molecular marker is
linked to said knock-out allele of a ROD1 gene.
[0160] Introducing said knock-out allele of ROD1 can occur through
mutagenesis or gene targeting as described above. Introducing said
knock-out allele can also occur through introduction of a knock-out
ROD1 allele from one plant into another.
[0161] In another embodiment, a method is provided to determine the
presence or absence of a knock-out allele of a ROD1 gene in a
biological sample, comprising providing genomic DNA from said
biological sample, and analyzing said DNA for the presence of at
least one molecular marker, wherein the at least one molecular
marker is linked to said knock-out allele of a ROD1 gene.
[0162] Said genomic DNA can be provided by isolating genomic DNA
from said biological sample. Isolating genomic DNA refers to
isolating a biological sample comprising genomic DNA from, such as
isolating part of a tissue, such as, for example part of a leaf.
Isolating genomic DNA from said biological sample can, but does not
need to comprise, purification of genomic DNA from said sample.
[0163] Yet another embodiment provides a kit for the detection of a
knock-out allele of a ROD1 gene in Brassica juncea DNA samples,
wherein said kit comprises one or more PCR primer pairs, which are
able to amplify a DNA marker linked to said knock-out allele of a
ROD1 gene. In yet another embodiment, said kit further comprises
one or more probes.
[0164] In a specific embodiment, said knock-out allele of a ROD1
gene is a mutant ROD1 allele.
[0165] In a further embodiment, a method is provided for
determining the zygosity status of a mutant ROD1 allele in a
Brassica juncea plant, or a cell, part, seed or progeny thereof,
comprising determining the presence of a mutant and/or a
corresponding wild type ROD1 specific region in the genomic DNA of
said plant, or a cell, part, seed or progeny thereof.
[0166] Yet a further embodiment provides method for transferring at
least one knock-out ROD1 allele from one Brassica juncea plant to
another Brassica juncea plant comprising the steps of: identifying
a first Brassica juncea plant comprising at least one knock-out
ROD1 allele; crossing the first Brassica juncea plant with a second
Brassica juncea plant not comprising the at least one knock-out
ROD1 allele and collecting F1 hybrid seeds from the cross;
optionally, identifying F1 Brassica juncea plants comprising the at
least one knock-out ROD1 allele; backcrossing F1 Brassica juncea
plants comprising the at least one knock-out ROD1 allele with the
second plant not comprising the at least one knock-out ROD1 allele
for at least one generation (x) and collecting BCx seeds from the
crosses; identifying in every generation BCx Brassica juncea plants
comprising the at least one knock-out ROD1 allele by analyzing
genomic DNA of said BCx plants for the presence of at least one
molecular marker, wherein the at least one molecular marker is
linked to said knock-out ROD1 allele.
[0167] A molecular marker which is linked to said knock-out allele
of a ROD1 gene or said mutant ROD1 allele can comprise on or more
primers or probes that specifically detect said knock-out allele of
said ROD1 gene as described herein below.
[0168] N
[0169] Methods According to the Invention
[0170] Mutant rod1 alleles may be generated (for example induced by
mutagenesis) and/or identified using a range of methods, which are
conventional in the art, for example using nucleic acid
amplification based methods to amplify part or all of the rod1
genomic or cDNA.
[0171] Following mutagenesis, plants are grown from the treated
seeds, or regenerated from the treated cells using known
techniques. For instance, mutagenized seeds may be planted in
accordance with conventional growing procedures and following
self-pollination seed is formed on the plants. Alternatively,
doubled haploid plantlets may be extracted from treated microspore
or pollen cells to immediately form homozygous plants, for example
as described by Coventry et al. (1988, Manual for Microspore
Culture Technique for Brassica napus. Dep. Crop Sci. Techn. Bull.
OAC Publication 0489. Univ. of Guelph, Guelph, Ontario, Canada).
Additional seed which is formed as a result of such
self-pollination in the present or a subsequent generation may be
harvested and screened for the presence of mutant ROD1 alleles,
using techniques which are conventional in the art, for example
nucleic acid amplification based techniques, such as polymerase
chain reaction (PCR) based techniques (amplification of the rod1
alleles) or hybridization based techniques, e.g. Southern blot
analysis, BAC library screening, and the like, and/or direct
sequencing of rod1 alleles. To screen for the presence of point
mutations (so called Single Nucleotide Polymorphisms or SNPs) in
mutant ROD1 alleles, SNP detection methods conventional in the art
can be used, for example oligoligation-based techniques, single
base extension-based techniques or techniques based on differences
in restriction sites, such as TILLING.
[0172] As described above, mutagenization (spontaneous as well as
induced) of a specific wild-type ROD1 allele results in the
presence of one or more deleted, inserted, or substituted
nucleotides (hereinafter called "mutation region") in the resulting
mutant ROD1 allele. The mutant ROD1 allele can thus be
characterized by the location and the configuration of the one or
more deleted, inserted, or substituted nucleotides in the wild type
ROD1 allele. The site in the wild type ROD1 allele where the one or
more nucleotides have been inserted, deleted, or substituted,
respectively, is herein also referred to as the "mutation region or
sequence". A "5' or 3' flanking region or sequence" as used herein
refers to a DNA region or sequence in the mutant (or the
corresponding wild type) ROD1 allele of at least 20 bp, preferably
at least 50 bp, at least 750 bp, at least 1500 bp, and up to 5000
bp of DNA different from the DNA containing the one or more
deleted, inserted, or substituted nucleotides, preferably DNA from
the mutant (or the corresponding wild type) ROD1 allele which is
located either immediately upstream of and contiguous with (5'
flanking region or sequence") or immediately downstream of and
contiguous with (3' flanking region or sequence") the mutation
region in the mutant ROD1 allele (or in the corresponding wild type
ROD1 allele). A "joining region" as used herein refers to a DNA
region in the mutant (or the corresponding wild type) ROD1 allele
where the mutation region and the 5' or 3' flanking region are
linked to each other. A "sequence spanning the joining region
between the mutation region and the 5' or 3' flanking region thus
comprises a mutation sequence as well as the flanking sequence
contiguous therewith.
[0173] The tools developed to identify a specific mutant ROD1
allele or the plant or plant material comprising a specific mutant
ROD1 allele, or products which comprise plant material comprising a
specific mutant ROD1 allele are based on the specific genomic
characteristics of the specific mutant ROD1 allele as compared to
the genomic characteristics of the corresponding wild type ROD1
allele, such as, a specific restriction map of the genomic region
comprising the mutation region, molecular markers comprising
primers and/or probes as described below, or the sequence of the
flanking and/or mutation regions.
[0174] Once a specific mutant ROD1 allele has been sequenced,
molecular markers, such as primers and probes can be developed
which specifically recognize a sequence within the 5' flanking, 3'
flanking and/or mutation regions of the mutant ROD1 allele in the
nucleic acid (DNA or RNA) of a sample by way of a molecular
biological technique. For instance an amplification method can be
developed to identify the mutant ROD1 allele in biological samples
(such as samples of plants, plant material or products comprising
plant material). Such an amplification is based on at least two
specific "primers": one recognizing a sequence within the 5' or 3'
flanking region of the mutant ROD1 allele and the other recognizing
a sequence within the 3' or 5' flanking region of the mutant ROD1
allele, respectively; or one recognizing a sequence within the 5'
or 3' flanking region of the mutant ROD1 allele and the other
recognizing a sequence within the mutation region of the mutant
ROD1 allele; or one recognizing a sequence within the 5' or 3'
flanking region of the mutant ROD1 allele and the other recognizing
a sequence spanning the joining region between the 3' or 5'
flanking region and the mutation region of the specific mutant ROD1
allele (as described further below), respectively.
[0175] The primers preferably have a sequence of between 15 and 35
nucleotides which under optimized amplification conditions
"specifically recognize" a sequence within the 5' or 3' flanking
region, a sequence within the mutation region, or a sequence
spanning the joining region between the 3' or 5' flanking and
mutation regions of the specific mutant ROD1 allele, so that a
specific fragment ("mutant ROD1 specific fragment" or
discriminating amplicon) is amplified from a nucleic acid sample
comprising the specific mutant ROD1 allele. This means that only
the targeted mutant ROD1 allele, and no other sequence in the plant
genome, is amplified under optimized amplification conditions.
[0176] PCR primers suitable for the invention may be the following:
[0177] oligonucleotides ranging in length from 17 nt to about 200
nt, comprising a nucleotide sequence of at least 17 consecutive
nucleotides, preferably 20 consecutive nucleotides selected from
the 5' or 3' flanking sequence of a specific mutant ROD1 allele or
the complement thereof (i.e., for example, the sequence 5' or 3'
flanking the one or more nucleotides deleted, inserted or
substituted in the mutant ROD1 alleles of the invention, such as
the sequence 5' or 3' flanking the non-sense, mis-sense, frameshift
or splice site mutations described above or the sequence 5' or 3'
flanking the STOP codon mutations indicated in the above Tables or
the substitution mutations indicated above or the complement
thereof) (primers recognizing 5' flanking sequences); or [0178]
oligonucleotides ranging in length from 17 nt to about 200 nt,
comprising a nucleotide sequence of at least 17 consecutive
nucleotides, preferably 20 nucleotides selected from the sequence
of the mutation region of a specific mutant ROD1 allele or the
complement thereof (i.e., for example, the sequence of nucleotides
inserted or substituted in the ROD1 genes of the invention or the
complement thereof) (primers recognizing mutation sequences).
[0179] The primers may of course be longer than the mentioned 17
consecutive nucleotides, and may e.g. be 18, 19, 20, 21, 30, 35,
50, 75, 100, 150, 200 nt long or even longer. The primers may
entirely consist of nucleotide sequence selected from the mentioned
nucleotide sequences of flanking and mutation sequences. However,
the nucleotide sequence of the primers at their 5' end (i.e.
outside of the 3'-located 17 consecutive nucleotides) is less
critical. Thus, the 5' sequence of the primers may consist of a
nucleotide sequence selected from the flanking or mutation
sequences, as appropriate, but may contain several (e.g. 1, 2, 5,
10) mismatches. The 5' sequence of the primers may even entirely
consist of a nucleotide sequence unrelated to the flanking or
mutation sequences, such as e.g. a nucleotide sequence representing
restriction enzyme recognition sites. Such unrelated sequences or
flanking DNA sequences with mismatches should preferably be no
longer than 100, more preferably not longer than 50 or even 25
nucleotides.
[0180] Moreover, suitable primers may comprise or consist of a
nucleotide sequence spanning the joining region between flanking
and mutation sequences (i.e., for example, the joining region
between a sequence 5' or 3' flanking one or more nucleotides
deleted, inserted or substituted in the mutant ROD1 alleles of the
invention and the sequence of the one or more nucleotides inserted
or substituted or the sequence 3' or 5', respectively, flanking the
one or more nucleotides deleted, such as the joining region between
a sequence 5' or 3' flanking non-sense, missense, frameshift or
splice site mutations in the ROD1 genes of the invention described
above and the sequence of the non-sense, missense, frameshift or
splice site mutations, or the joining region between a sequence 5'
or 3' flanking a potential STOP codon mutation as indicated in the
above Tables or the substitution mutations indicated above and the
sequence of the potential STOP codon mutation or the substitution
mutations, respectively), provided the nucleotide sequence is not
derived exclusively from either the mutation region or flanking
regions.
[0181] It will also be immediately clear to the skilled artisan
that properly selected PCR primer pairs should also not comprise
sequences complementary to each other.
[0182] For the purpose of the invention, the "complement of a
nucleotide sequence represented in SEQ ID No: X" is the nucleotide
sequence which can be derived from the represented nucleotide
sequence by replacing the nucleotides through their complementary
nucleotide according to Chargaff's rules (A<->T; G<->C)
and reading the sequence in the 5' to 3' direction, i.e. in
opposite direction of the represented nucleotide sequence.
[0183] As used herein, "the nucleotide sequence of SEQ ID No. Z
from position X to position Y" indicates the nucleotide sequence
including both nucleotide endpoints.
[0184] Preferably, the amplified fragment has a length of between
50 and 1000 nucleotides, such as a length between 50 and 500
nucleotides, or a length between 100 and 350 nucleotides. The
specific primers may have a sequence which is between 80 and 100%
identical to a sequence within the 5' or 3' flanking region, to a
sequence within the mutation region, or to a sequence spanning the
joining region between the 3' or 5' flanking and mutation regions
of the specific mutant ROD1 allele, provided the mismatches still
allow specific identification of the specific mutant ROD1 allele
with these primers under optimized amplification conditions. The
range of allowable mismatches however, can easily be determined
experimentally and are known to a person skilled in the art.
[0185] Detection and/or identification of a "mutant ROD1 specific
fragment" can occur in various ways, e.g., via size estimation
after gel or capillary electrophoresis or via fluorescence-based
detection methods. The mutant ROD1 specific fragments may also be
directly sequenced. Other sequence specific methods for detection
of amplified DNA fragments are also known in the art.
[0186] Standard nucleic acid amplification protocols, such as PCR
protocols are described in the art, such as in `PCR Applications
Manual" (Roche Molecular Biochemicals, 2nd Edition, 1999) and other
references. The optimal conditions for the amplification, including
the sequence of the specific primers, is specified in a "PCR
identification protocol" for each specific mutant ROD1 allele. It
is however understood that a number of parameters in the PCR
identification protocol may need to be adjusted to specific
laboratory conditions, and may be modified slightly to obtain
similar results. For instance, use of a different method for
preparation of DNA may require adjustment of, for instance, the
amount of primers, polymerase, MgCl.sub.2 concentration or
annealing conditions used. Similarly, the selection of other
primers may dictate other optimal conditions for the PCR
identification protocol. These adjustments will however be apparent
to a person skilled in the art, and are furthermore detailed in
current PCR application manuals such as the one cited above.
[0187] Alternatively, specific primers can be used to amplify a
mutant ROD1 specific fragment that can be used as a "specific
probe" for identifying a specific mutant ROD1 allele in biological
samples. Contacting nucleic acid of a biological sample, with the
probe, under conditions that allow hybridization of the probe with
its corresponding fragment in the nucleic acid, results in the
formation of a nucleic acid/probe hybrid. The formation of this
hybrid can be detected (e.g. labeling of the nucleic acid or
probe), whereby the formation of this hybrid indicates the presence
of the specific mutant ROD1 allele. Such identification methods
based on hybridization with a specific probe (either on a solid
phase carrier or in solution) have been described in the art. The
specific probe is preferably a sequence that, under optimized
conditions, hybridizes specifically to a region within the 5' or 3'
flanking region and/or within the mutation region of the specific
mutant ROD1 allele (hereinafter referred to as "mutant ROD1
specific region"). Preferably, the specific probe comprises a
sequence of between 10 and 1000 bp, 50 and 600 bp, between 100 to
500 bp, between 150 to 350 bp, which is at least 80%, preferably
between 80 and 85%, more preferably between 85 and 90%, especially
preferably between 90 and 95%, most preferably between 95% and 100%
identical (or complementary) to the nucleotide sequence of a
specific region. Preferably, the specific probe will comprise a
sequence of about 13 to about 100 contiguous nucleotides identical
(or complementary) to a specific region of the specific mutant ROD1
allele.
[0188] Specific probes suitable for the invention may be the
following: [0189] oligonucleotides ranging in length from 13 nt to
about 1000 nt, comprising a nucleotide sequence of at least 13
consecutive nucleotides selected from the 5' or 3' flanking
sequence of a specific mutant ROD1 allele or the complement thereof
(i.e., for example, the sequence 5' or 3' flanking the one or more
nucleotides deleted, inserted or substituted in the mutant ROD1
alleles of the invention, such as the sequence 5' or 3' flanking
the non-sense, mis-sense, frameshift or splice site mutations
described above or the sequence 5' or 3' flanking the potential
STOP codon mutations indicated in the above Tables or the
substitution mutations indicated above), or a sequence having at
least 80% sequence identity therewith (probes recognizing 5'
flanking sequences); or [0190] oligonucleotides ranging in length
from 13 nt to about 1000 nt, comprising a nucleotide sequence of at
least 13 consecutive nucleotides selected from the mutation
sequence of a specific mutant ROD1 allele or the complement thereof
(i.e., for example, the sequence of nucleotides inserted or
substituted in the ROD1 genes of the invention, or the complement
thereof), or a sequence having at least 80% sequence identity
therewith (probes recognizing mutation sequences).
[0191] The probes may entirely consist of nucleotide sequence
selected from the mentioned nucleotide sequences of flanking and
mutation sequences. However, the nucleotide sequence of the probes
at their 5' or 3' ends is less critical. Thus, the 5' or 3'
sequences of the probes may consist of a nucleotide sequence
selected from the flanking or mutation sequences, as appropriate,
but may consist of a nucleotide sequence unrelated to the flanking
or mutation sequences. Such unrelated sequences should preferably
be no longer than 50, more preferably not longer than 25 or even no
longer than 20 or 15 nucleotides.
[0192] Moreover, suitable probes may comprise or consist of a
nucleotide sequence spanning the joining region between flanking
and mutation sequences (i.e., for example, the joining region
between a sequence 5' or 3' flanking one or more nucleotides
deleted, inserted or substituted in the mutant ROD1 alleles of the
invention and the sequence of the one or more nucleotides inserted
or substituted or the sequence 3' or 5', respectively, flanking the
one or more nucleotides deleted, such as the joining region between
a sequence 5' or 3' flanking non-sense, mis-sense, frameshift or
splice site mutations in the ROD1 genes of the invention described
above and the sequence of the non-sense, mis-sense, frameshift or
splice site mutations, or the joining region between a sequence 5'
or 3' flanking a potential STOP codon mutation as indicated in the
above Tables or the substitution mutations indicated above and the
sequence of the potential STOP codon or substitution mutation,
respectively), provided the mentioned nucleotide sequence is not
derived exclusively from either the mutation region or flanking
regions.
[0193] Detection and/or identification of a "mutant ROD1 specific
region" hybridizing to a specific probe can occur in various ways,
e.g., via size estimation after gel electrophoresis or via
fluorescence-based detection methods. Other sequence specific
methods for detection of a "mutant ROD1 specific region"
hybridizing to a specific probe are also known in the art.
[0194] Alternatively, plants or plant parts comprising one or more
mutant rod1 alleles can be generated and identified using other
methods, such as the "Delete-a-gene.TM." method which uses PCR to
screen for deletion mutants generated by fast neutron mutagenesis
(reviewed by Li and Zhang, 2002, Funct Integr Genomics 2:254-258),
by the TILLING (Targeting Induced Local Lesions IN Genomes) method
which identifies EMS-induced point mutations using denaturing
high-performance liquid chromatography (DHPLC) to detect base pair
changes by heteroduplex analysis (McCallum et al., 2000, Nat
Biotech 18:455, and McCallum et al. 2000, Plant Physiol. 123,
439-442), etc. As mentioned, TILLING uses high-throughput screening
for mutations (e.g. using Cel 1 cleavage of mutant-wildtype DNA
heteroduplexes and detection using a sequencing gel system). Thus,
the use of TILLING to identify plants or plant parts comprising one
or more mutant rod1 alleles and methods for generating and
identifying such plants, plant organs, tissues and seeds is
encompassed herein. Thus in one embodiment, the method according to
the invention comprises the steps of mutagenizing plant seeds (e.g.
EMS mutagenesis), pooling of plant individuals or DNA, PCR
amplification of a region of interest, heteroduplex formation and
high-throughput detection, identification of the mutant plant,
sequencing of the mutant PCR product. It is understood that other
mutagenesis and selection methods may equally be used to generate
such mutant plants.
[0195] Instead of inducing mutations in ROD1 alleles, natural
(spontaneous) mutant alleles may be identified by methods known in
the art. For example, ECOTILLING may be used (Henikoff et al. 2004,
Plant Physiology 135(2):630-6) to screen a plurality of plants or
plant parts for the presence of natural mutant rod1 alleles. As for
the mutagenesis techniques above, preferably Brassica species are
screened which comprise an A and/or a B genome, so that the
identified rod1 allele can subsequently be introduced into other
Brassica species, such as Brassica juncea, by crossing (inter- or
intraspecific crosses) and selection. In ECOTILLING natural
polymorphisms in breeding lines or related species are screened for
by the TILLING methodology described above, in which individual or
pools of plants are used for PCR amplification of the rod1 target,
heteroduplex formation and high-throughput analysis. This can be
followed by selecting individual plants having a required mutation
that can be used subsequently in a breeding program to incorporate
the desired mutant allele.
[0196] The identified mutant alleles can then be sequenced and the
sequence can be compared to the wild type allele to identify the
mutation(s). Optionally functionality can be tested as indicated
above. Using this approach a plurality of mutant rod1 alleles (and
plants comprising one or more of these) can be identified. The
desired mutant alleles can then be combined with the desired wild
type alleles by crossing and selection methods as described further
below. Finally a single plant comprising the desired number of
mutant rod1 and the desired number of wild type ROD1 alleles is
generated.
[0197] Oligonucleotides suitable as PCR primers or specific probes
for detection of a specific mutant ROD1 allele can also be used to
develop methods to determine the zygosity status of the specific
mutant ROD1 allele.
[0198] To determine the zygosity status of a specific mutant ROD1
allele, a nucleic acid amplification-based assay can be developed
to determine the presence of a mutant and/or corresponding wild
type ROD1 specific allele:
[0199] To determine the zygosity status of a specific mutant ROD1
allele, two primers specifically recognizing the wild-type ROD1
allele can be designed in such a way that they are directed towards
each other and have the mutation region located in between the
primers. These primers may be primers specifically recognizing the
5' and 3' flanking sequences, respectively. This set of primers
allows simultaneous diagnostic amplification of the mutant, as well
as of the corresponding wild type ROD1 allele.
[0200] Alternatively, to determine the zygosity status of a
specific mutant ROD1 allele, two primers specifically recognizing
the wild-type ROD1 allele can be designed in such a way that they
are directed towards each other and that one of them specifically
recognizes the mutation region.
[0201] These primers may be primers specifically recognizing the
sequence of the 5' or 3' flanking region and the mutation region of
the wild type ROD1 allele, respectively. This set of primers,
together with a third primer which specifically recognizes the
sequence of the mutation region in the mutant ROD1 allele, allow
simultaneous diagnostic amplification of the mutant ROD1 gene, as
well as of the wild type ROD1 gene.
[0202] Alternatively, to determine the zygosity status of a
specific mutant ROD1 allele, two primers specifically recognizing
the wild-type ROD1 allele can be designed in such a way that they
are directed towards each other and that one of them specifically
recognizes the joining region between the 5' or 3' flanking region
and the mutation region. These primers may be primers specifically
recognizing the 5' or 3' flanking sequence and the joining region
between the mutation region and the 3' or 5' flanking region of the
wild type ROD1 allele, respectively. This set of primers, together
with a third primer which specifically recognizes the joining
region between the mutation region and the 3' or 5' flanking region
of the mutant ROD1 allele, respectively, allow simultaneous
diagnostic amplification of the mutant ROD1 gene, as well as of the
wild type ROD1 gene.
[0203] Alternatively, the zygosity status of a specific mutant ROD1
allele can be determined by using alternative primer sets that
specifically recognize mutant and wild type ROD1 alleles.
[0204] If the plant is homozygous for the mutant ROD1 gene or the
corresponding wild type ROD1 gene, the diagnostic amplification
assays described above will give rise to a single amplification
product typical, preferably typical in length, for either the
mutant or wild type ROD1 allele. If the plant is heterozygous for
the mutant ROD1 allele, two specific amplification products will
appear, reflecting both the amplification of the mutant and the
wild type ROD1 allele.
[0205] Identification of the wild type and mutant ROD1 specific
amplification products can occur e.g. by size estimation after gel
or capillary electrophoresis (e.g. for mutant ROD1 alleles
comprising a number of inserted or deleted nucleotides which
results in a size difference between the fragments amplified from
the wild type and the mutant ROD1 allele, such that said fragments
can be visibly separated on a gel); by evaluating the presence or
absence of the two different fragments after gel or capillary
electrophoresis, whereby the diagnostic amplification of the mutant
ROD1 allele can, optionally, be performed separately from the
diagnostic amplification of the wild type ROD1 allele; by direct
sequencing of the amplified fragments; or by fluorescence-based
detection methods.
[0206] Alternatively, to determine the zygosity status of a
specific mutant ROD1 allele, a hybridization-based assay can be
developed to determine the presence of a mutant and/or
corresponding wild type ROD1 specific allele:
[0207] To determine the zygosity status of a specific mutant ROD1
allele, two specific probes recognizing the wild-type ROD1 allele
can be designed in such a way that each probe specifically
recognizes a sequence within the ROD1 wild type allele and that the
mutation region is located in between the sequences recognized by
the probes. These probes may be probes specifically recognizing the
5' and 3' flanking sequences, respectively. The use of one or,
preferably, both of these probes allows simultaneous diagnostic
hybridization of the mutant, as well as of the corresponding wild
type ROD1 allele.
[0208] Alternatively, to determine the zygosity status of a
specific mutant ROD1 allele, two specific probes recognizing the
wild-type ROD1 allele can be designed in such a way that one of
them specifically recognizes a sequence within the ROD1 wild type
allele upstream or downstream of the mutation region, preferably
upstream of the mutation region, and that one of them specifically
recognizes the mutation region. These probes may be probes
specifically recognizing the sequence of the 5' or 3' flanking
region, preferably the 5' flanking region, and the mutation region
of the wild type ROD1 allele, respectively. The use of one or,
preferably, both of these probes, optionally, together with a third
probe which specifically recognizes the sequence of the mutation
region in the mutant ROD1 allele, allow diagnostic hybridization of
the mutant and of the wild type ROD1 gene.
[0209] Alternatively, to determine the zygosity status of a
specific mutant ROD1 allele, a specific probe recognizing the
wild-type ROD1 allele can be designed in such a way that the probe
specifically recognizes the joining region between the 5' or 3'
flanking region, preferably the 5' flanking region, and the
mutation region of the wild type ROD1 allele. This probe,
optionally, together with a second probe that specifically
recognizes the joining region between the 5' or 3' flanking region,
preferably the 5' flanking region, and the mutation region of the
mutant ROD1 allele, allows diagnostic hybridization of the mutant
and of the wild type ROD1 gene.
[0210] Alternatively, the zygosity status of a specific mutant ROD1
allele can be determined by using alternative sets of probes that
specifically recognize mutant and wild type ROD1 alleles.
[0211] If the plant is homozygous for the mutant ROD1 gene or the
corresponding wild type ROD1 gene, the diagnostic hybridization
assays described above will give rise to a single specific
hybridization product, such as one or more hybridizing DNA
(restriction) fragments, typical, preferably typical in length, for
either the mutant or wild type ROD1 allele. If the plant is
heterozygous for the mutant ROD1 allele, two specific hybridization
products will appear, reflecting both the hybridization of the
mutant and the wild type ROD1 allele.
[0212] Identification of the wild type and mutant ROD1 specific
hybridization products can occur e.g. by size estimation after gel
or capillary electrophoresis (e.g. for mutant ROD1 alleles
comprising a number of inserted or deleted nucleotides which
results in a size difference between the hybridizing DNA
(restriction) fragments from the wild type and the mutant ROD1
allele, such that said fragments can be visibly separated on a
gel); by evaluating the presence or absence of the two different
specific hybridization products after gel or capillary
electrophoresis, whereby the diagnostic hybridization of the mutant
ROD1 allele can, optionally, be performed separately from the
diagnostic hybridization of the wild type ROD1 allele; by direct
sequencing of the hybridizing DNA (restriction) fragments; or by
fluorescence-based detection methods.
[0213] Furthermore, detection methods specific for a specific
mutant ROD1 allele that differ from PCR- or hybridization-based
amplification methods can also be developed using the specific
mutant ROD1 allele specific sequence information provided herein.
Such alternative detection methods include linear signal
amplification detection methods based on invasive cleavage of
particular nucleic acid structures, also known as Invader.TM.
technology, (as described e.g. in U.S. Pat. No. 5,985,557 "Invasive
Cleavage of Nucleic Acids", U.S. Pat. No. 6,001,567 "Detection of
Nucleic Acid sequences by Invader Directed Cleavage, incorporated
herein by reference), RT-PCR-based detection methods, such as
Taqman, or other detection methods, such as SNPlex. Briefly, in the
Invader.TM. technology, the target mutation sequence may e.g. be
hybridized with a labeled first nucleic acid oligonucleotide
comprising the nucleotide sequence of the mutation sequence or a
sequence spanning the joining region between the 5' flanking region
and the mutation region and with a second nucleic acid
oligonucleotide comprising the 3' flanking sequence immediately
downstream and adjacent to the mutation sequence, wherein the first
and second oligonucleotide overlap by at least one nucleotide. The
duplex or triplex structure that is produced by this hybridization
allows selective probe cleavage with an enzyme (Cleavase.RTM.)
leaving the target sequence intact. The cleaved labeled probe is
subsequently detected, potentially via an intermediate step
resulting in further signal amplification.
[0214] A "kit", as used herein, refers to a set of reagents for the
purpose of performing the method of the invention, more
particularly, the identification of a specific mutant ROD1 allele
in biological samples or the determination of the zygosity status
of plant material comprising a specific mutant ROD1 allele. More
particularly, a preferred embodiment of the kit of the invention
comprises at least two specific primers, as described above, for
identification of a specific mutant ROD1 allele, or at least two or
three specific primers for the determination of the zygosity
status. Optionally, the kit can further comprise any other reagent
described herein in the PCR identification protocol. Alternatively,
according to another embodiment of this invention, the kit can
comprise at least one specific probe, which specifically hybridizes
with nucleic acid of biological samples to identify the presence of
a specific mutant ROD1 allele therein, as described above, for
identification of a specific mutant ROD1 allele, or at least two or
three specific probes for the determination of the zygosity status.
Optionally, the kit can further comprise any other reagent (such as
but not limited to hybridizing buffer, label) for identification of
a specific mutant ROD1 allele in biological samples, using the
specific probe.
[0215] The kit of the invention can be used, and its components can
be specifically adjusted, for purposes of quality control (e.g.,
purity of seed lots), detection of the presence or absence of a
specific mutant ROD1 allele in plant material or material
comprising or derived from plant material, such as but not limited
to food or feed products.
[0216] The term "primer" as used herein encompasses any nucleic
acid that is capable of priming the synthesis of a nascent nucleic
acid in a template-dependent process, such as PCR. Typically,
primers are oligonucleotides from 10 to 30 nucleotides, but longer
sequences can be employed. Primers may be provided in
double-stranded form, though the single-stranded form is preferred.
Probes can be used as primers, but are designed to bind to the
target DNA or RNA and need not be used in an amplification
process.
[0217] The term "recognizing" as used herein when referring to
specific primers, refers to the fact that the specific primers
specifically hybridize to a nucleic acid sequence in a specific
mutant ROD1 allele under the conditions set forth in the method
(such as the conditions of the PCR identification protocol),
whereby the specificity is determined by the presence of positive
and negative controls.
[0218] The term "hybridizing", as used herein when referring to
specific probes, refers to the fact that the probe binds to a
specific region in the nucleic acid sequence of a specific mutant
ROD1 allele under standard stringency conditions. Standard
stringency conditions as used herein refers to the conditions for
hybridization described herein or to the conventional hybridizing
conditions as described by Sambrook et al., 1989 (Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbour
Laboratory Press, NY) which for instance can comprise the following
steps: 1) immobilizing plant genomic DNA fragments or BAC library
DNA on a filter, 2) prehybridizing the filter for 1 to 2 hours at
65.degree. C. in 6.times.SSC, 5.times.Denhardt's reagent, 0.5% SDS
and 20 .mu.g/ml denaturated carrier DNA, 3) adding the
hybridization probe which has been labeled, 4) incubating for 16 to
24 hours, 5) washing the filter once for 30 min. at 68.degree. C.
in 6.times.SSC, 0.1% SDS, 6) washing the filter three times (two
times for 30 min. in 30 ml and once for 10 min in 500 ml) at
68.degree. C. in 2.times.SSC, 0.1% SDS, and 7) exposing the filter
for 4 to 48 hours to X-ray film at -70.degree. C.
[0219] As used in herein, a "biological sample" is a sample of a
plant, plant material or product comprising plant material. The
term "plant" is intended to encompass plant tissues, at any stage
of maturity, as well as any cells, tissues, or organs taken from or
derived from any such plant, including without limitation, any
seeds, leaves, stems, flowers, roots, single cells, gametes, cell
cultures, tissue cultures or protoplasts. "Plant material", as used
herein refers to material that is obtained or derived from a plant.
Products comprising plant material relate to food, feed or other
products that are produced using plant material or can be
contaminated by plant material. It is understood that, in the
context of the present invention, such biological samples are
tested for the presence of nucleic acids specific for a specific
mutant ROD1 allele, implying the presence of nucleic acids in the
samples. Thus the methods referred to herein for identifying a
specific mutant ROD1 allele in biological samples, relate to the
identification in biological samples of nucleic acids that comprise
the specific mutant ROD1 allele.
[0220] Another embodiment provides a chimeric gene comprising the
following operably linked elements: a plant-expressible promoter; a
DNA region, which when transcribed yields an RNA molecule
inhibitory to at least one ROD1 gene, said ROD1 gene encoding a
protein having at least 90% sequence identity to SEQ ID No. 2 or
SEQ ID No. 4; and optionally a transcription termination and
polyadenylation region functional in plant cells.
[0221] In again another embodiment, a knock-out allele of a ROD1
gene is provided, wherein the knock-out ROD1 allele is a mutated
version of the native ROD1 gene selected from the group consisting
of: a nucleic acid molecule which comprises at least 90% sequence
identity to SEQ ID No. 1 or SEQ ID No. 3; or a nucleic acid
molecule encoding an amino acid sequence comprising at least 90%
sequence identity to SEQ ID No. 2 or SEQ ID No. 4, wherein said
mutant rod1 allele comprises a mutated DNA region consisting of one
or more inserted, deleted or substituted nucleotides compared to a
corresponding wild-type DNA region in the functional ROD1 gene and
wherein said mutant rod1 allele encodes no functional ROD1 protein
or encodes a ROD1 protein with reduced activity.
[0222] The chimeric gene according to the invention can be used to
produce plants, such as Brassica juncea plants, with increased
levels of C18:1 in the seeds, or with decreased levels of C18:2 or
SATS in the seeds, or to produce seed oil with increased levels of
C18:1, or with decreased levels of C18:2 or SATS.
[0223] In a further embodiment, a method is provided for producing
oil, comprising harvesting seeds from the plants according to the
invention, i.e. Brassica juncea plants comprising a knock-out ROD1
gene or an RNA inhibitory to a ROD1 gene, and extracting the oil
from said seeds. In yet a further embodiment, a method is provided
of producing food or feed, such as oil, meal, grain, starch, flour
or protein, or an industrial product, such as biofuel, fiber,
industrial chemicals, a pharmaceutical or a neutraceutical,
comprising obtaining the Brassica juncea plant or a part thereof
according to the invention, and preparing the food, feed or
industrial product from the plant or part thereof.
[0224] Plants according to the invention, such as plants comprising
at least one knock-out ROD1 gene or plants comprising an RNA
molecule inhibitory to at least one ROD1 gene can further be used
to produce seeds, such as seeds with increased levels of C18:1, or
seeds with decreased levels of C18:2 or SATS, or to produce seed
oil with increased levels of C18:1, or with decreased levels of
C18:2 or SATS.
[0225] The plants according to the invention may additionally
contain an endogenous or a transgene, which confers herbicide
resistance, such as the bar or pat gene, which confer resistance to
glufosinate ammonium (Liberty.RTM., Basta.RTM. or Ignite.RTM.); or
any modified EPSPS gene, such as the 2mEPSPS gene from maize, or
glyphosate acetyltransferase, or glyphosate oxidoreductase, which
confer resistance to glyphosate (RoundupReady.RTM.), or
bromoxynitril nitrilase to confer bromoxynitril tolerance, or any
modified AHAS gene, which confers tolerance to sulfonylureas,
imidazolinones, sulfonylaminocarbonyltriazolinones,
triazolopyrimidines or pyrimidyl(oxy/thio)benzoates. Further, the
plants according to the invention may additionally contain an
endogenous or a transgene which confers increased oil content or
improved oil composition, such as a 12:0 ACP thioesteraseincrease
to obtain high laureate, which confers pollination control, such as
such as barnase under control of an anther-specific promoter to
obtain male sterility, or barstar under control of an
anther-specific promoter to confer restoration of male sterility,
or such as the Ogura cytoplasmic male sterility and nuclear
restorer of fertility.
[0226] The plants and seeds according to the invention may be
further treated with a chemical compound, such as a chemical
compound selected from the following lists: Herbicides: Clethodim,
Clopyralid, Diclofop, Ethametsulfuron, Fluazifop, Glufosinate,
Glyphosate, Metazachlor, Quinmerac, Quizalofop, Tepraloxydim,
Trifluralin. Fungicides/PGRs: Azoxystrobin,
N-[9-(dichloromethylene)-1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-yl]-3-
-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide
(Benzovindiflupyr, Benzodiflupyr), Bixafen, Boscalid, Carbendazim,
Carboxin, Chlormequat-chloride, Coniothryrium minitans,
Cyproconazole, Cyprodinil, Difenoconazole, Dimethomorph,
Dimoxystrobin, Epoxiconazole, Famoxadone, Fluazinam, Fludioxonil,
Fluopicolide, Fluopyram, Fluoxastrobin, Fluquinconazole,
Flusilazole, Fluthianil, Flutriafol, Fluxapyroxad, Iprodione,
Isopyrazam, Mefenoxam, Mepiquat-chloride, Metalaxyl, Metconazole,
Metominostrobin, Paclobutrazole, Penflufen, Penthiopyrad,
Picoxystrobin, Prochloraz, Prothioconazole, Pyraclostrobin,
Sedaxane, Tebuconazole, Tetraconazole, Thiophanate-methyl, Thiram,
Triadimenol, Trifloxystrobin, Bacillus firmus, Bacillus firmus
strain I-1582, Bacillus subtilis, Bacillus subtilis strain GB03,
Bacillus subtilis strain QST 713, Bacillus pumulis, Bacillus.
pumulis strain GB34.
[0227] Insecticides: Acetamiprid, Aldicarb, Azadirachtin,
Carbofuran, Chlorantraniliprole (Rynaxypyr), Clothianidin,
Cyantraniliprole (Cyazypyr), (beta-)Cyfluthrin, gamma-Cyhalothrin,
lambda-Cyhalothrin, Cypermethrin, Deltamethrin, Dimethoate,
Dinetofuran, Ethiprole, Flonicamid, Flubendiamide, Fluensulfone,
Fluopyram, Flupyradifurone, tau-Fluvalinate, Imicyafos,
Imidacloprid, Metaflumizone, Methiocarb, Pymetrozine,
Pyrifluquinazon, Spinetoram, Spinosad, Spirotetramate, Sulfoxaflor,
Thiacloprid, Thiamethoxam,
1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-
-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide,
1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-
-{[5-(trifluoromethyl)-1H-tetrazol-1-yl]methyl}-1H-pyrazole-5-carboxamide,
1-{2-fluoro-4-methyl-5-[(2,2,2-trifluorethyl)
sulfinyl]phenyl}-3-(trifluoromethyl)-1H-1,2,4-triazol-5-amine,
(1E)-N-[(6-chloropyridin-3-yl)methyl]-N'-cyano-N-(2,2-difluoroethyl)ethan-
imidamide, Bacillus firmus, Bacillus firmus strain 1-1582, Bacillus
subtilis, Bacillus subtilis strain GB03, Bacillus subtilis strain
QST 713, Metarhizium anisopliae F52. In some embodiments, the plant
cells of the invention, i.e. a plant cell comprising a knock-out
rod1 gene or an RNA inhibitory to a ROD1 gene, as well as plant
cells generated according to the methods of the invention, may be
non-propagating cells.
[0228] The obtained plants according to the invention can be used
in a conventional breeding scheme to produce more plants with the
same characteristics or to introduce the characteristic according
to the invention in other varieties of the same or related plant
species, or in hybrid plants. The obtained plants can further be
used for creating propagating material. Plants according to the
invention can further be used to produce gametes, seeds (including
crushed seeds and seed cakes), seed oil, embryos, either zygotic or
somatic, progeny, or to produce food or feed, such as oil, meal,
grain, starch, flour or protein, or an industrial product, such as
biofuel, fiber, industrial chemicals, a pharmaceutical or a
neutraceutical, or to produce hybrids of plants obtained by methods
of the invention.
[0229] All patents, patent applications, and publications or public
disclosures (including publications on internet) referred to or
cited herein are incorporated by reference in their entirety.
[0230] The sequence listing contained in the file named
"BCS12-2011_ST25.txt", which is 47.5 kilobytes (size as measured in
Microsoft Windows.RTM.), contains 14 sequences SEQ ID NO: 1 through
SEQ ID NO: 14 and was created on 2 Jul. 2012 is filed herewith by
electronic submission and is incorporated by reference herein.
[0231] In the description and examples, reference is made to the
following sequences:
[0232] Sequences
[0233] SEQ ID No. 1: cDNA sequence of ROD1-A1 from Brassica
juncea.
[0234] SEQ ID No. 2: protein sequence of ROD1-A1 from Brassica
juncea.
[0235] SEQ ID No. 3: cDNA sequence of ROD1-B1 from Brassica
juncea.
[0236] SEQ ID No. 4: protein sequence of ROD1-B1 from Brassica
juncea.
[0237] SEQ ID No. 5: cDNA sequence of ROD1-A2 from Brassica
juncea.
[0238] SEQ ID No. 6: protein sequence of ROD1-A2 from Brassica
juncea.
[0239] SEQ ID No. 7: cDNA sequence of ROD1-B2 from Brassica
juncea.
[0240] SEQ ID No. 8: protein sequence of ROD1-B2 from Brassica
juncea.
[0241] SEQ ID No. 9: cDNA sequence of ROD1-A3 from Brassica
juncea.
[0242] SEQ ID No. 10: protein sequence of ROD1-A3 from Brassica
juncea.
[0243] SEQ ID No. 11: cDNA sequence of ROD1-B3 from Brassica
juncea.
[0244] SEQ ID No. 12: protein sequence of ROD1-B3 from Brassica
juncea.
[0245] SEQ ID No. 13: cDNA sequence of ROD1-B4 from Brassica
juncea.
[0246] SEQ ID No. 14: protein sequence of ROD1-B4 from Brassica
juncea.
EXAMPLES
[0247] Unless stated otherwise in the Examples, all recombinant DNA
techniques are carried out according to standard protocols as
described in Sambrook and Russell (2001) Molecular Cloning: A
Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory
Press, NY, in Volumes 1 and 2 of Ausubel et al. (1994) Current
Protocols in Molecular Biology, Current Protocols, USA and in
Volumes I and II of Brown (1998) Molecular Biology LabFax, Second
Edition, Academic Press (UK). Standard materials and methods for
plant molecular work are described in Plant Molecular Biology
Labfax (1993) by R. D. D. Croy, jointly published by BIOS
Scientific Publications Ltd (UK) and Blackwell Scientific
Publications, UK. Standard materials and methods for polymerase
chain reactions can be found in Dieffenbach and Dveksler (1995) PCR
Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
and in McPherson at al. (2000) PCR--Basics: From Background to
Bench, First Edition, Springer Verlag, Germany. Standard procedures
for AFLP analysis are described in Vos et al. (1995, NAR
23:4407-4414) and in published EP patent application EP 534858.
Example 1--Isolation of the DNA Sequences of Brassica juncea ROD1
Genes
[0248] The B. juncea cDNA sequence BjROD1_A1 was obtained by
Sequencher mediated assembly of 80 bp sequencing reads retrieved by
BLAST analysis of in-house B. juncea cv J0005006 sequencing read
databases using a ROD1 sequence from the Brassica napus A genome as
the query.
[0249] The B. juncea cDNA sequences BjROD1_B1, BjROD1_A2,
BjROD1_B2, BjROD1_A3, BjROD1_B3, and BjROD1_B4 were obtained by
assembly of 80 bp sequencing reads retrieved by running the
GeneXpression program with different ROD1 sequences from the
Brassica napus A and C genome as queries using a B. juncea cv
J0005006 sequencing read databases. For the BjROD1 cDNA sequence
assemblies the ROD1 cDNAs from B. napus cv. PPS02-144B were used as
a reference sequence.
[0250] Thus, seven cDNAs were identified, three of which were
annotated to the A genome and four of which were annotated on the B
genome: BjROD1-A1 (SEQ ID No. 1), BjROD1-B1 (SEQ ID No. 3),
BjROD1-A2 (SEQ ID No. 5), BjROD1-B2 (SEQ ID No. 7), BjROD1-A3 (SEQ
ID No. 9), BjROD1-B3 (SEQ ID No. 11), and BjROD1-B4 (SEQ ID No.
13). It is plausible that a fourth ROD1 gene is present on the A
genome (BjROD1-A4), which is homeologous to BjROD1-B4, which has a
low or no expression, and has therefore not been identified in the
cDNA sequence database.
Example 2--Generation and Isolation of Mutant Brassica Juncea rod1
Alleles
[0251] Mutations in the ROD1 genes from Brassica juncea identified
in Example 1 are generated and identified as follows: [0252] Seeds
are preimbibed for two hours on wet filter paper in deionized or
distilled water. Half of the seeds are exposed to 0.8% EMS and half
to 1% EMS (Sigma: M0880) and incubated for 4 hours. [0253] The
mutagenized seeds (M1 seeds) are rinsed 3 times and dried in a fume
hood overnight. M1 plants are grown in soil and selfed to generate
M2 seeds. M2 seeds are harvested for each individual M1 plant.
[0254] M2 plants, derived from different M1 plants, are grown and
DNA samples are prepared from leaf samples of each individual M2
plant. [0255] The DNA samples are screened for the presence of
point mutations in the ROD1 genes causing the introduction of STOP
codons in the protein-encoding regions of the ROD1 genes, amino
acid substitutions, or the disruption of splice sites in the ROD1
mRNA, by direct sequencing by standard sequencing techniques and
analyzing the sequences for the presence of the point mutations
using the NovoSNP software.
[0256] Mutant rod1 alleles have been identified of the BjROD1-A1
gene, the BjROD1-B1, gene, the BjROD1-A2 gene, the BjROD1-B2 gene,
the BjROD1-A3 gene, the BjROD1-B3 gene and of the BjROD1-B4
gene.
Example 3--Activity of BjROD1 Alleles in Yeast
[0257] The activity of the Brassica juncea ROD1-1 and ROD1-2
alleles, as well as mutant alleles thereof, are tested in
yeast.
[0258] Cloning of the ROD1 Alleles in Yeast Expression Vectors
[0259] BjROD1-A1, BjROD1-B1, BjROD1-A2, BjROD1-B2, BjROD1-A3,
BjROD1-B3 and BjROD1-B4 and their mutant alleles are amplified by
KOD DNA polymerase (Toyobo Life Science Department,
http://www.toyobo-global.com), using primers that created 5' BamHI
and 3'EcoRI restriction sites.
[0260] Following BamHI and EcoRI double digestion, each product is
ligated into the p424GPD vector (ATCC, http://www.atcc.org/), in
which the CDNA is expressed under control of the constitutive
Glyceraldehyde-3-P dehydrogenase promoter, and then transformed
into E. coli competent cells (TOP10, Invitrogen). Plasmids with
correct inserts confirmed by sequencing are transformed into yeast
HJ091 cells (cpt1::LEU2 ept1-), and transformants are selected by
synthetic minimal media (SD base) with dropout leucine and
tryptophan (DO-Leu/-Trp) (Clontech, http://www.clontech.com).
[0261] Activity Testing of the ROD1 Alleles in Yeast
[0262] ROD1 activity assay is modified based on Supplementary
Information in Lu et al., 2009 (PNAS, 2009, 106 (44):18837-18842.,
S1 Materials and Methods). Yeast cells are inoculated from
overnight cultures and grown to mid-log phase (OD600=0.5-1.5) at
30.degree. C. in liquid media SD/-Leu/-Trp. To prepare a total
membrane fraction, 100 ml yeast cells are harvested by
centrifugation at 1500 g for 5 min. Each cell pellet is washed once
with sterile water and then resuspended in ice-cold
glucose-Tris-EDTA (GTE) buffer [20% glycerol, 50 mM glucose, 25 mM
Tris-HCl, pH 7.4, 10 mM EDTA]. Cells are then vortexed for 30
seconds.times.8 times with 30 seconds gaps on ice. The resulting
homogenate is centrifuged at 2,500 g at 4.degree. C. for 10 min. to
pellet cell debris. The supernatant is centrifuged at 100,000 g at
4.degree. C. for 1h and the membrane pellet is resuspended in 200
.mu.L GTE buffer. The protein concentration is determined by
Bradford assay.
[0263] The PDCT activities in membrane preparations of HJ091 cells
transformed with p424GPD (control) or p424ROD1 and mutant alleles
are determined as the amount of [14C]dioleoyl-PC produced from
1,2-dioleoyl-rac-glycerol [14C(U)] ([14C-glycerol]diolein). The
substrates of 1.8 nmol (200,000 cpm) [14C-glycerol]diolein
(American Radiolabeled Chemicals, Inc. (http://www.arcinc.com) and
0.1 .mu.mol dioleoyl-PC are dried under nitrogen gas and
resuspended in 50 .mu.L of 4.times. reaction buffer [final
concentrations: 50 mM 3-(N-morpholino)propanesulfonic acid
(MOPS)/NaOH (pH 7.5), 20 mM MgCl2, 0.45% Triton X-100] by 2 minutes
sonication in a bath sonicator. Reactions (200 .mu.L) are started
by adding 50 ng of microsomal proteins suspended in the GTE buffer.
Assays are incubated at 15.degree. C. for 15 min and are terminated
by the addition of 3 mL of chloroform/ethanol (2:1, vol./vol.),
followed by 1.5 mL of 0.9% KCl. Tubes are mixed by vortexing, and
phase separation was facilitated by centrifugation at 2,000 g for 2
min. The aqueous phase is aspirated, and the organic phase is
washed twice with 1.5 mL of 40% (vol./vol.) ethanol. Samples are
analyzed by TLC on Whatman Partisil.RTM. K6 silica gel 60 .ANG.
20.times.20 cm glass plates (Whatman, http://www.whatman.com) in a
solvent system of chloroform/methanol/water (65:25:4, by volume),
followed by phosphorimaging analysis (phosphorimager 445 SI, Lab
Extreme, Inc, http://www.labextreme.com). Corresponding bands are
scraped, and radioactivity is determined by scintillation counting
on a TRI-CARB.RTM. liquid scintillation analyzer (Packard
Instrument Company).
[0264] It is found that BjROD1-A1 and BjROD1-B1 have activity,
whereas no activity of the other BjROD1 genes and mutant BjROD1-A1
and mutant BjROD1-B1 alleles can be detected.
Example 4--Downregulation of BjROD1 in Brassica juncea
[0265] The ROD1 genes are downregulated in Brassica juncea using
hairpin constructs of ROD1.
[0266] Construction of the ROD1 Hairpin Constructs
[0267] Host Escherichia coli strains are TOP10 (with Gateway entry
and expression clones) or DB3.1 (with pHELLSGATE12 destination
vector; Invitrogen). Bacterial cultures are grown at 37.degree. C.
in Luria broth medium with appropriate antibiotics.
[0268] Generation of ROD1 hpRNA Suppression Constructs:
[0269] To specifically knock down the expression of the BjROD1
genes, a hairpin construct is generated which contains at least 20
bp identical to both BjROD1-A1 and BjROD1-B1, or to BjROD1-A1,
BjROD1-B1, BjROD1-A2, BjROD1-B2, BjROD1-A3, BjROD1-B3 and of
BjROD1-B4. Therefore, a fragment of BjROD1-A1 is amplified by PCR
on BjROD1-A1 DNA as template: The PCR reaction (50p1) contains 0.3
.mu.M of each primer, 2 ng/pt template DNA, 0.2 mM of dNTP mix,
0.02 unit/.mu.L of KOD DNA polymerase (Toyobo), 5 .mu.l of
10.times.PCR buffer, and 1.5 mM MgSO4. Programmed cycles are as
follows: 2 min initial denaturing step at 95.degree. C.; 40 cycles
of 20 s denaturation at 95.degree. C., 15 s annealing at 55.degree.
C., 20 s extension at 70.degree. C. PCR products are purified with
QIAquick Gel Extraction Kit (QIAGEN) and ligated into the
pENTR.TM./D-TOPO.RTM. cloning vector (Invitrogen) to generate entry
clones according to the manual's instruction. To generate hairpin
constructs, 100 ng BjROD1 entry clone and 150 ng pHELLSGATE12
destination vector are mixed, and LR recombination reaction is
conducted using Gateway.RTM. LR Clonase.TM. Enzyme following the
manual's instruction (Invitrogen). After transformation into TOP10
competent cells, clones are screened by restriction analysis to
identify plasmids with the expected insert in the correct
orientation, and are validated by sequencing.
[0270] The transformation vectors are obtained by extracting the
hairpin region from the above hairpin constructs and placing this
cassette into a transformation vector under control of the
Cauliflower Mosaic Virus 35S promoter containing bar as selectable
marker.
[0271] Transformation of Brassica juncea with the ROD1 Hairpin
Constructs
[0272] A DNA fragment comprising the hairpin construct and the bar
selectable marker is HPLC purified and used to obtain transformed
Brassica juncea plants by means of direct gene transfer into cells
of Brassica juncea, followed by regeneration of transformed plant
cells into transgenic fertile Brassica juncea plants.
[0273] Single-copy regenerated transformation events are
back-crossed with a Brassica juncea (elite) line. Following 2
rounds of selfing seeds from both homozygous transformation events
and wild type segregants are harvested for subsequent seed oil
analysis.
[0274] Oil Composition in Seeds from Brassica juncea Transformed
with the ROD1 Hairpin Constructs
[0275] The fatty acid composition of the seed oil of individual
progeny Brassica juncea plants for homozygous transformation events
and the corresponding wild type segregants as well as a
non-transformed reference line is determined by extracting the
fatty acyls from the seeds and analyzing their relative levels in
the seed oil by capillary gas-liquid chromatography as described in
WO09/007091.
[0276] It is found that the levels of C18:1 is significantly
increased in seed lipids of the plants comprising the hairpin
construct as compared to wild-type controls or wild-type
segregants. These results show that downregulation of the BjROD1-A1
and BjROD1-B1 alleles, and of the BjROD1-A1, BjROD1-B1, BjROD1-A2,
BjROD1-B2, BjROD1-A3, BjROD1-B3 and of BjROD1-B4 alleles
contributes significantly to the increase of C18:1 levels in the
seed lipid fraction. Further, it is found that the levels of C18:2
and of saturated fatty acids (SATS; C12:0, C14:0, C16:0, C18:0,
C20:0, C22:0 and C24:0) are decreased in seeds of plants comprising
the ROD1 hairpin construct as compared to wild-type controls or
wild-type segregants.
Example 5--Oil Composition in Brassica juncea Comprising ROD1
Knock-Out Alleles
[0277] Brassica juncea plants comprising mutant ROD1-A1 and ROD1-B1
alleles are crossed. Following 2 rounds of selfing seeds from
plants homozygous for ROD1-A1 and ROD1-B1 mutations, for the
ROD1-A1 mutation, for the ROD1-B1 mutation or wild type segregants
(i.e. not comprising any mutant ROD1 allele that would impact the
normal function of a ROD1 protein) are obtained.
[0278] Fatty acid composition is determined as described above in
F1S2 seeds of the Brassica juncea lines with mutant BjROD1-A1,
BjROD1-B1, and combinations thereof. For each combination of
mutants, oil composition is determined in wild-type segregants not
comprising the respective mutations in BjROD1-A1 and BjROD1-B1, in
lines homozygous for either the mutant BjROD1-A1 or for the mutant
BjROD1-B1 allele, and in lines homozygous for both mutants
BjROD1-A1 and BjROD1-B1.
[0279] It is found that the levels of C18:1 are increased in lines
comprising either the mutant BjROD1-A1, or for the mutant BjROD1-B1
allele, or both mutants BjROD1-A1 and BjROD1-B1 as compared to the
wild-type segregant. Further, the levels of C18:2 and of SATS
(SATS; C12:0, C14:0, C16:0, C18:0, C20:0, C22:0 and C24:0) are
decreased in lines comprising either the mutant BjROD1-A1, or the
mutant BjROD1-B1 allele, or both mutants BjROD1-A1 and BjROD1-B1 as
compared to the wild-type segregant.
Example 6--Detection and/or Transfer of Mutant ROD1 Alleles into
(Elite) Brassica juncea Lines
[0280] The mutant ROD1 genes are transferred into (elite) Brassica
juncea breeding lines by the following method: A plant containing a
mutant ROD1 gene (donor plant), is crossed with an (elite) Brassica
juncea line (elite parent/recurrent parent) or variety lacking the
mutant ROD1 gene. The following introgression scheme is used (the
mutant ROD1 allele is abbreviated to rod/while the wild type is
depicted as ROD1):
[0281] BC1 cross: rod1/rod1 (donor plant).times.ROD1/ROD1 (elite
parent)
[0282] F1 plant: ROD1/rod1
[0283] BC2 cross: ROD1/rod1.times.ROD1/ROD1 (recurrent parent)
[0284] BC2 plants: 50% ROD1/rod1 and 50% ROD1/ROD1
[0285] The 50% ROD1/rod1 are selected using molecular markers (e.g.
AFLP, PCR, Invader.TM., TaqMan.RTM., KASP assay, and the like; see
also below) for the mutant ROD1 allele (rod1).
[0286] BC3 cross: ROD1/rod1 (BC1 plant).times.ROD1/ROD1 (recurrent
parent) BC3 plants: 50% ROD1/rod1 and 50% ROD1/ROD1 The 50%
ROD1/rod1 are selected using molecular markers for the mutant ROD1
allele (rod1).
[0287] Backcrossing is repeated until BC4 to BC7.
[0288] BC4-7 plants: 50% ROD1/rod1 and 50% ROD1/ROD1
[0289] The 50% ROD1/rod1 are selected using molecular markers for
the mutant ROD1 allele (rod1). To reduce the number of
backcrossings (e.g. until BC4 instead of BC7), molecular markers
can be used specific for the genetic background of the elite
parent.
[0290] BC4-7 S1 cross: ROD1/rod1.times.ROD1/rod1 BC4-7 S1 plants:
25% ROD1/ROD1 and 50% ROD1/rod1 and 25% rod1/rod1
[0291] Plants containing rod1 are selected using molecular markers
for the mutant ROD1 allele (rod1).
[0292] Individual BC4-7 S1 or BC4-7 S2 plants that are homozygous
for the mutant ROD1 allele (rod1/rod1) are selected using molecular
markers for the mutant and the wild-type ROD1 alleles. These plants
are then used for seed production.
[0293] To select for plants comprising a point mutation in a ROD1
allele, direct sequencing by standard sequencing techniques known
in the art can be used.
[0294] Alternatively, Invader.TM. technology (Third Wave Agbio) can
be used to discriminate plants comprising a specific point mutation
in an ROD1 allele from plants not comprising that specific point
mutation. Discriminating Invader.TM. probes are thus developed to
detect the presence or absence and the zygosity status of mutant
alleles identified in Example 3, based on the single nucleotide
difference between the mutant and wildtype allele. Briefly, probes
specific for the mutant or corresponding wild-type target ROD1 gene
and "invading" probes which can be used in combination with them
are developed. Generally, each probe set consists of one probe
specific for the mutant or the wild type target gene of which the
first nucleotide after the "5' flap" sequence matches with the
nucleotide difference (the so-called "primary probe") and one probe
specific for the nucleotides upstream of the nucleotide difference
(the so-called "Invader.RTM. oligo"). The last nucleotide of the
latter primer may match with the nucleotide difference in the
mutant, but other nucleotides may be used as well for this last
nucleotide as long as the primary probe and the Invader.RTM. oligo
are still able to form a single base overlap when hybridized to the
target DNA to generate the specific invasive structure recognized
by the Cleavase.RTM. enzymes (Third Wave Agbio). The Invader.TM.
assay procedure and interpretation of the data are performed as
prescribed by the manufacturer (Third Wave Agbio). Briefly, 5'
"flap" nucleotide sequences (flap1 for the mutant allele and flap2
for the wild-type allele) are cleaved from the primary probes in
the primary phase of the Invader.TM. assay and are complementary to
sequences in FRET.TM. cassette 1 and 2, respectively, and not
complementary to the target mutant or wild type sequences. If the
primary probes are cleaved in the primary phase and the flap1-probe
and/or flap2-probe hybridise to FRET.TM. cassette 1 and 2,
respectively, in the secondary phase, a signal is generated
indicative of the presence in the sample of the mutant or
corresponding wild-type target ROD1 gene, respectively.
[0295] Alternatively, KASP assays (KBioscience) can be used to
discriminate plants comprising a specific point mutation in an ROD1
allele from plants not comprising that specific point mutation.
Discriminating primers are developed to detect the presence or
absence and the zygosity status of mutant alleles identified in
Example 2.
[0296] Briefly, forward primers specific for the mutant or
corresponding wild-type target ROD1 gene and a reverse primer that
can be used in combination with them are developed. The nucleotide
at the 3' end of the forward primers corresponds to the nucleotide
which differs between the mutant and the corresponding wild-type
allele. The primers can be used in combination with fluorescent
dyes, such as FAM and VIC according to the protocol as described by
the manufacturer (KBioscience).
Sequence CWU 1
1
1411142DNABrassica junceaCDS(238)..(1080) 1acaagtaaag cccaacaaag
acagatgaga aaatagcaaa gacttgcgta aacgtcgctc 60tcaaacctca tctcatactc
atcgttttcg tatgagtttt tgtagcccaa acaatcttcc 120tttctacagt
ttataatata agaaacaata cttccttcgt aatctccgcc tcgtatctct
180tatataactc atctctctaa acctaaaaaa tgttcctctc cgttaaatct aacggtc
237atg tca act aat acc gtc gtc cct ctc cgt cgc aga tct aac gga aat
285Met Ser Thr Asn Thr Val Val Pro Leu Arg Arg Arg Ser Asn Gly Asn
1 5 10 15 cac act aac ggc gag gcc ttt aac gga atg gag aac att gtc
aag aaa 333His Thr Asn Gly Glu Ala Phe Asn Gly Met Glu Asn Ile Val
Lys Lys 20 25 30 acc gac gac tgc tac acc aac ggc aac gga gga gta
gag aga agc aaa 381Thr Asp Asp Cys Tyr Thr Asn Gly Asn Gly Gly Val
Glu Arg Ser Lys 35 40 45 gcc tcg ttt ctg aca tgg acc atg cgt gac
gct gtc tac gta gcg aga 429Ala Ser Phe Leu Thr Trp Thr Met Arg Asp
Ala Val Tyr Val Ala Arg 50 55 60 tac cat tgg ata ccg tgt ttc ttt
gcg gtc gga gtt ctg ttc ttt atg 477Tyr His Trp Ile Pro Cys Phe Phe
Ala Val Gly Val Leu Phe Phe Met 65 70 75 80 ggg gtt gag tac acg ctc
cag atg gtt ccg gcg aag tct gag ccg ttc 525Gly Val Glu Tyr Thr Leu
Gln Met Val Pro Ala Lys Ser Glu Pro Phe 85 90 95 gat att ggg ttt
gtg gcc acg cgc tct ctg aac cgc gtc ttg gcg agt 573Asp Ile Gly Phe
Val Ala Thr Arg Ser Leu Asn Arg Val Leu Ala Ser 100 105 110 tca ccg
gat ctt aac acc ctt tta gcg gct cta aac acg gta ttc gta 621Ser Pro
Asp Leu Asn Thr Leu Leu Ala Ala Leu Asn Thr Val Phe Val 115 120 125
gcg atg caa acg acg tat att gta tgg aca tgg ttg atg gaa gga aga
669Ala Met Gln Thr Thr Tyr Ile Val Trp Thr Trp Leu Met Glu Gly Arg
130 135 140 cca cga gcc act atc tcg gct tgc ttc atg ttt act tgt cgc
ggc att 717Pro Arg Ala Thr Ile Ser Ala Cys Phe Met Phe Thr Cys Arg
Gly Ile 145 150 155 160 ctt ggt tac tct act cag ctc cct cta cca cag
gat ttt tta gga tca 765Leu Gly Tyr Ser Thr Gln Leu Pro Leu Pro Gln
Asp Phe Leu Gly Ser 165 170 175 gga gtt gat ttt ccg gtg gga aac gtc
tca ttc ttc ctc ttc tat tct 813Gly Val Asp Phe Pro Val Gly Asn Val
Ser Phe Phe Leu Phe Tyr Ser 180 185 190 ggc cac gta gcc ggt tca atg
atc gca tcc ttg gac atg agg aga atg 861Gly His Val Ala Gly Ser Met
Ile Ala Ser Leu Asp Met Arg Arg Met 195 200 205 cag agg ttg aga cta
gcg atg ctt ttt gac atc ctc aac ata tta caa 909Gln Arg Leu Arg Leu
Ala Met Leu Phe Asp Ile Leu Asn Ile Leu Gln 210 215 220 tcg atc aga
ctg ctc ggg acg aga gga cac tac acg atc gat ctt gcg 957Ser Ile Arg
Leu Leu Gly Thr Arg Gly His Tyr Thr Ile Asp Leu Ala 225 230 235 240
gtc gga gtt ggc gct ggg att ctc ttt gac tca ttg gcc ggg aag tac
1005Val Gly Val Gly Ala Gly Ile Leu Phe Asp Ser Leu Ala Gly Lys Tyr
245 250 255 gaa gag atg atg agc aag aga cac aat tta gcc aat ggt ttt
agt ttg 1053Glu Glu Met Met Ser Lys Arg His Asn Leu Ala Asn Gly Phe
Ser Leu 260 265 270 att tct aaa gac tcg cta gtc aat taa tcttttgttt
tcattttaaa 1100Ile Ser Lys Asp Ser Leu Val Asn 275 280 tgattagttg
aacttgaaca tatttgattt agttaaagac tt 1142 2280PRTBrassica juncea
2Met Ser Thr Asn Thr Val Val Pro Leu Arg Arg Arg Ser Asn Gly Asn 1
5 10 15 His Thr Asn Gly Glu Ala Phe Asn Gly Met Glu Asn Ile Val Lys
Lys 20 25 30 Thr Asp Asp Cys Tyr Thr Asn Gly Asn Gly Gly Val Glu
Arg Ser Lys 35 40 45 Ala Ser Phe Leu Thr Trp Thr Met Arg Asp Ala
Val Tyr Val Ala Arg 50 55 60 Tyr His Trp Ile Pro Cys Phe Phe Ala
Val Gly Val Leu Phe Phe Met 65 70 75 80 Gly Val Glu Tyr Thr Leu Gln
Met Val Pro Ala Lys Ser Glu Pro Phe 85 90 95 Asp Ile Gly Phe Val
Ala Thr Arg Ser Leu Asn Arg Val Leu Ala Ser 100 105 110 Ser Pro Asp
Leu Asn Thr Leu Leu Ala Ala Leu Asn Thr Val Phe Val 115 120 125 Ala
Met Gln Thr Thr Tyr Ile Val Trp Thr Trp Leu Met Glu Gly Arg 130 135
140 Pro Arg Ala Thr Ile Ser Ala Cys Phe Met Phe Thr Cys Arg Gly Ile
145 150 155 160 Leu Gly Tyr Ser Thr Gln Leu Pro Leu Pro Gln Asp Phe
Leu Gly Ser 165 170 175 Gly Val Asp Phe Pro Val Gly Asn Val Ser Phe
Phe Leu Phe Tyr Ser 180 185 190 Gly His Val Ala Gly Ser Met Ile Ala
Ser Leu Asp Met Arg Arg Met 195 200 205 Gln Arg Leu Arg Leu Ala Met
Leu Phe Asp Ile Leu Asn Ile Leu Gln 210 215 220 Ser Ile Arg Leu Leu
Gly Thr Arg Gly His Tyr Thr Ile Asp Leu Ala 225 230 235 240 Val Gly
Val Gly Ala Gly Ile Leu Phe Asp Ser Leu Ala Gly Lys Tyr 245 250 255
Glu Glu Met Met Ser Lys Arg His Asn Leu Ala Asn Gly Phe Ser Leu 260
265 270 Ile Ser Lys Asp Ser Leu Val Asn 275 280 3936DNABrassica
junceaCDS(65)..(907)misc_feature(540)..(541)n is a, c, g, or t
3gtatctctta tataactcat ctctctaaac atagatatgt tcctctccgt taaatctaac
60ggtc atg tca act aat acc gtc gtc cct ctc cgt cgc aga tct aac gga
109 Met Ser Thr Asn Thr Val Val Pro Leu Arg Arg Arg Ser Asn Gly 1 5
10 15 tat cac agt aac ggc gtg gcc ttt aac gga atg gag aac att gtc
aag 157Tyr His Ser Asn Gly Val Ala Phe Asn Gly Met Glu Asn Ile Val
Lys 20 25 30 aaa aca gac gac tgc tac acc aac ggc aac gga gga gga
ggg aag agc 205Lys Thr Asp Asp Cys Tyr Thr Asn Gly Asn Gly Gly Gly
Gly Lys Ser 35 40 45 aag gcg tcg ttt ctg aca tgg acc atg cgc gac
gct gtc tac gtg gcg 253Lys Ala Ser Phe Leu Thr Trp Thr Met Arg Asp
Ala Val Tyr Val Ala 50 55 60 aga tac cat tgg ata ccg tgt ttc ttt
gcg gtc gga gtt ctg ttc ttt 301Arg Tyr His Trp Ile Pro Cys Phe Phe
Ala Val Gly Val Leu Phe Phe 65 70 75 atg ggc gtt gag tat acg ctc
cag atg gtt ccg gcg aag tct gag ccg 349Met Gly Val Glu Tyr Thr Leu
Gln Met Val Pro Ala Lys Ser Glu Pro 80 85 90 95 ttc gat att ggg ttt
gtg gcc acg cgc tct ctg aac cgc gtc ttg gcg 397Phe Asp Ile Gly Phe
Val Ala Thr Arg Ser Leu Asn Arg Val Leu Ala 100 105 110 agt tca ccg
gat ctt aac acc ctt tta gcg gct cta aac acg gta ttc 445Ser Ser Pro
Asp Leu Asn Thr Leu Leu Ala Ala Leu Asn Thr Val Phe 115 120 125 gta
gcg atg caa acg acg tat att gta tgg aca tgg ttg atg gaa gga 493Val
Ala Met Gln Thr Thr Tyr Ile Val Trp Thr Trp Leu Met Glu Gly 130 135
140 aga cca cga gcc act atc tct gct tgc ttt atg ttt act tgt cgc gnn
541Arg Pro Arg Ala Thr Ile Ser Ala Cys Phe Met Phe Thr Cys Arg Xaa
145 150 155 att ctt ggt tac tct act cag ctc cct ctc cca cag gat ttt
tta gga 589Ile Leu Gly Tyr Ser Thr Gln Leu Pro Leu Pro Gln Asp Phe
Leu Gly 160 165 170 175 tca gga gtt gat ttt cca gtg gga aac gtc tca
ttc ttc ctc ttc tat 637Ser Gly Val Asp Phe Pro Val Gly Asn Val Ser
Phe Phe Leu Phe Tyr 180 185 190 tct ggt cac gtc gcc ggt tca atg atc
gca tcc ttg gac atg agg aga 685Ser Gly His Val Ala Gly Ser Met Ile
Ala Ser Leu Asp Met Arg Arg 195 200 205 atg cgg agg ttg aga cta gcg
atg ctt ttt gac atc ctc aac gta tta 733Met Arg Arg Leu Arg Leu Ala
Met Leu Phe Asp Ile Leu Asn Val Leu 210 215 220 caa tct atc agg ctg
ctc ggg aca aga gga cat tac acg att gat ctt 781Gln Ser Ile Arg Leu
Leu Gly Thr Arg Gly His Tyr Thr Ile Asp Leu 225 230 235 gcg gtc gga
gtt ggc gct ggg att ctc ttt gac tct ttg gcc ggg aag 829Ala Val Gly
Val Gly Ala Gly Ile Leu Phe Asp Ser Leu Ala Gly Lys 240 245 250 255
tac gaa gag atg atg agc aag aga cac aat tta gcc aat ggt ttt agt
877Tyr Glu Glu Met Met Ser Lys Arg His Asn Leu Ala Asn Gly Phe Ser
260 265 270 ttg att tcg aaa gac tcg cta gtc aat taa tcttttgttt
tcattttaaa 927Leu Ile Ser Lys Asp Ser Leu Val Asn 275 280 tgattagtt
9364280PRTBrassica junceamisc_feature(159)..(159)The 'Xaa' at
location 159 stands for Glu, Asp, Gly, Ala, or Val. 4Met Ser Thr
Asn Thr Val Val Pro Leu Arg Arg Arg Ser Asn Gly Tyr 1 5 10 15 His
Ser Asn Gly Val Ala Phe Asn Gly Met Glu Asn Ile Val Lys Lys 20 25
30 Thr Asp Asp Cys Tyr Thr Asn Gly Asn Gly Gly Gly Gly Lys Ser Lys
35 40 45 Ala Ser Phe Leu Thr Trp Thr Met Arg Asp Ala Val Tyr Val
Ala Arg 50 55 60 Tyr His Trp Ile Pro Cys Phe Phe Ala Val Gly Val
Leu Phe Phe Met 65 70 75 80 Gly Val Glu Tyr Thr Leu Gln Met Val Pro
Ala Lys Ser Glu Pro Phe 85 90 95 Asp Ile Gly Phe Val Ala Thr Arg
Ser Leu Asn Arg Val Leu Ala Ser 100 105 110 Ser Pro Asp Leu Asn Thr
Leu Leu Ala Ala Leu Asn Thr Val Phe Val 115 120 125 Ala Met Gln Thr
Thr Tyr Ile Val Trp Thr Trp Leu Met Glu Gly Arg 130 135 140 Pro Arg
Ala Thr Ile Ser Ala Cys Phe Met Phe Thr Cys Arg Xaa Ile 145 150 155
160 Leu Gly Tyr Ser Thr Gln Leu Pro Leu Pro Gln Asp Phe Leu Gly Ser
165 170 175 Gly Val Asp Phe Pro Val Gly Asn Val Ser Phe Phe Leu Phe
Tyr Ser 180 185 190 Gly His Val Ala Gly Ser Met Ile Ala Ser Leu Asp
Met Arg Arg Met 195 200 205 Arg Arg Leu Arg Leu Ala Met Leu Phe Asp
Ile Leu Asn Val Leu Gln 210 215 220 Ser Ile Arg Leu Leu Gly Thr Arg
Gly His Tyr Thr Ile Asp Leu Ala 225 230 235 240 Val Gly Val Gly Ala
Gly Ile Leu Phe Asp Ser Leu Ala Gly Lys Tyr 245 250 255 Glu Glu Met
Met Ser Lys Arg His Asn Leu Ala Asn Gly Phe Ser Leu 260 265 270 Ile
Ser Lys Asp Ser Leu Val Asn 275 280 51059DNABrassica
junceaCDS(242)..(1057) 5aatataaaaa gaacttaaca acatgttggt acaaaattaa
agtaaagccc aacaaagaga 60gaaaacaaag aaaaaaaata ataaggcaaa gactttgcgt
aaacgtagct ctcgaaactc 120aatactcatc gttttcgtat gaatttttgt
agaccaaaca atcttccttc cacagttcac 180aaaataaaaa caatacctcc
ttcgaaatct ctgcctctta tagaactcat ctctgacgct 240t atg tca act gaa
act agc gtc cct ctc cgt cgc aga tct acc tct ctt 289 Met Ser Thr Glu
Thr Ser Val Pro Leu Arg Arg Arg Ser Thr Ser Leu 1 5 10 15 aac gga
cat cac tct aac gac gtc gcc ttt gac gga acc gtc cca tta 337Asn Gly
His His Ser Asn Asp Val Ala Phe Asp Gly Thr Val Pro Leu 20 25 30
atg gag aac aac att gtt aag aaa aca gac gac ggc tac gcc aat gga
385Met Glu Asn Asn Ile Val Lys Lys Thr Asp Asp Gly Tyr Ala Asn Gly
35 40 45 gga gga aag gcg tcg ttt atg aca tgg acg gcg cgt gac gct
atc tac 433Gly Gly Lys Ala Ser Phe Met Thr Trp Thr Ala Arg Asp Ala
Ile Tyr 50 55 60 gtg gcg aga gtc cat tgg ata ccg tgt gtg ttc gcg
gtt gga gtt ctc 481Val Ala Arg Val His Trp Ile Pro Cys Val Phe Ala
Val Gly Val Leu 65 70 75 80 ttc ttc atg ggc gtc gag tat acg ctt caa
atg att ccc gcg agg tct 529Phe Phe Met Gly Val Glu Tyr Thr Leu Gln
Met Ile Pro Ala Arg Ser 85 90 95 gag ccg ttc gat att ggg ttt gtg
gtc acg cgc tct ctg aac cgc gtc 577Glu Pro Phe Asp Ile Gly Phe Val
Val Thr Arg Ser Leu Asn Arg Val 100 105 110 ttg gca aat tca ccg gct
ctt aac acc gtt tta gcc gca cta aac acg 625Leu Ala Asn Ser Pro Ala
Leu Asn Thr Val Leu Ala Ala Leu Asn Thr 115 120 125 gtg ttc gta ggg
atg caa act acg tat att gta tgg aca tgg ttg atg 673Val Phe Val Gly
Met Gln Thr Thr Tyr Ile Val Trp Thr Trp Leu Met 130 135 140 gaa gga
aga cca cgg gcc acc atc tcg gct tgc ttc atg ttt act tgt 721Glu Gly
Arg Pro Arg Ala Thr Ile Ser Ala Cys Phe Met Phe Thr Cys 145 150 155
160 cgc gac tct acc cag ctt cct ctc cct cag gag ttt tta gga tca gga
769Arg Asp Ser Thr Gln Leu Pro Leu Pro Gln Glu Phe Leu Gly Ser Gly
165 170 175 gtc gat ttt ccg gtg gga aac gtc tca ttc ttc ctc ttc tac
tcg ggt 817Val Asp Phe Pro Val Gly Asn Val Ser Phe Phe Leu Phe Tyr
Ser Gly 180 185 190 cac gtc gcc ggt tcc atg ata gca tcc ttg gac atg
agg aga atg cag 865His Val Ala Gly Ser Met Ile Ala Ser Leu Asp Met
Arg Arg Met Gln 195 200 205 agg ttg aga cta gcg atg ctt ttt gac atc
ctc aat gta cta caa tcc 913Arg Leu Arg Leu Ala Met Leu Phe Asp Ile
Leu Asn Val Leu Gln Ser 210 215 220 atc agg ctg ctc ggg acg aga gga
cat tac acc atc gat ctt gcg gtc 961Ile Arg Leu Leu Gly Thr Arg Gly
His Tyr Thr Ile Asp Leu Ala Val 225 230 235 240 gga gtt ggc gct ggg
att ctc ttt gac tcg ttg gcc ggg aag tac gaa 1009Gly Val Gly Ala Gly
Ile Leu Phe Asp Ser Leu Ala Gly Lys Tyr Glu 245 250 255 gag atg atg
agc aaa aga cac aat tta ggc aat ggt ttt agt ttg att 1057Glu Met Met
Ser Lys Arg His Asn Leu Gly Asn Gly Phe Ser Leu Ile 260 265 270 tc
10596272PRTBrassica juncea 6Met Ser Thr Glu Thr Ser Val Pro Leu Arg
Arg Arg Ser Thr Ser Leu 1 5 10 15 Asn Gly His His Ser Asn Asp Val
Ala Phe Asp Gly Thr Val Pro Leu 20 25 30 Met Glu Asn Asn Ile Val
Lys Lys Thr Asp Asp Gly Tyr Ala Asn Gly 35 40 45 Gly Gly Lys Ala
Ser Phe Met Thr Trp Thr Ala Arg Asp Ala Ile Tyr 50 55 60 Val Ala
Arg Val His Trp Ile Pro Cys Val Phe Ala Val Gly Val Leu 65 70 75 80
Phe Phe Met Gly Val Glu Tyr Thr Leu Gln Met Ile Pro Ala Arg Ser 85
90 95 Glu Pro Phe Asp Ile Gly Phe Val Val Thr Arg Ser Leu Asn Arg
Val 100 105 110 Leu Ala Asn Ser Pro Ala Leu Asn Thr Val Leu Ala Ala
Leu Asn Thr 115 120 125 Val Phe Val Gly Met Gln Thr Thr Tyr Ile Val
Trp Thr Trp Leu Met 130 135 140
Glu Gly Arg Pro Arg Ala Thr Ile Ser Ala Cys Phe Met Phe Thr Cys 145
150 155 160 Arg Asp Ser Thr Gln Leu Pro Leu Pro Gln Glu Phe Leu Gly
Ser Gly 165 170 175 Val Asp Phe Pro Val Gly Asn Val Ser Phe Phe Leu
Phe Tyr Ser Gly 180 185 190 His Val Ala Gly Ser Met Ile Ala Ser Leu
Asp Met Arg Arg Met Gln 195 200 205 Arg Leu Arg Leu Ala Met Leu Phe
Asp Ile Leu Asn Val Leu Gln Ser 210 215 220 Ile Arg Leu Leu Gly Thr
Arg Gly His Tyr Thr Ile Asp Leu Ala Val 225 230 235 240 Gly Val Gly
Ala Gly Ile Leu Phe Asp Ser Leu Ala Gly Lys Tyr Glu 245 250 255 Glu
Met Met Ser Lys Arg His Asn Leu Gly Asn Gly Phe Ser Leu Ile 260 265
270 71089DNABrassica junceaCDS(173)..(979) 7agaaaagaat aacgaggcaa
aagacttgcg taaacgtagc tctagaacct catactcatc 60gttttcgtat gaatttttgt
agaccaaaca atcttccttc cacagttcac aaaatataaa 120acaatacctc
cttcgagatc tctgcctctt acataaccca tatctcacgc tt atg tca 178 Met Ser
1 act gaa act ggc gtc cct ctc cgt cgc aga tct aac tct ctt aac gga
226Thr Glu Thr Gly Val Pro Leu Arg Arg Arg Ser Asn Ser Leu Asn Gly
5 10 15 cat cac act aac ggc gtc gcc tct gac gga aca aac gtc cca tta
atg 274His His Thr Asn Gly Val Ala Ser Asp Gly Thr Asn Val Pro Leu
Met 20 25 30 gag aag gcg tcg ttt atg aca tgg acg gcg cgt gac gct
atc tac gtg 322Glu Lys Ala Ser Phe Met Thr Trp Thr Ala Arg Asp Ala
Ile Tyr Val 35 40 45 50 gcg aga gtc cat tgg ata ccg tgt gtg ttc gcg
gtc gga gtt ctg ttc 370Ala Arg Val His Trp Ile Pro Cys Val Phe Ala
Val Gly Val Leu Phe 55 60 65 ttc atg ggc gtc gag tat acg ctt cag
atg att ccc gcg agg tct gag 418Phe Met Gly Val Glu Tyr Thr Leu Gln
Met Ile Pro Ala Arg Ser Glu 70 75 80 ccg ttc gat att ggg ttc gtg
gcc acg cgc tct ctg aat cgc gtc ttg 466Pro Phe Asp Ile Gly Phe Val
Ala Thr Arg Ser Leu Asn Arg Val Leu 85 90 95 gca gat tca ccg gat
ctt aac acc gtt tta gct gca cta aac acg gtt 514Ala Asp Ser Pro Asp
Leu Asn Thr Val Leu Ala Ala Leu Asn Thr Val 100 105 110 ttc gta ggg
atg caa act acg tat att gta tgg aca tgg ttg atg gaa 562Phe Val Gly
Met Gln Thr Thr Tyr Ile Val Trp Thr Trp Leu Met Glu 115 120 125 130
gga aga cca cgg gcc acc atc tcg gct tgc ttc atg ttt act tgt cgc
610Gly Arg Pro Arg Ala Thr Ile Ser Ala Cys Phe Met Phe Thr Cys Arg
135 140 145 ggt att ctt ggt tac tct act cag ctc cct ctc cct cag gag
ttt tta 658Gly Ile Leu Gly Tyr Ser Thr Gln Leu Pro Leu Pro Gln Glu
Phe Leu 150 155 160 gga tca gga gtc gat ttt ccg gtg gga aac gtc tca
ttc ttc ctc ttc 706Gly Ser Gly Val Asp Phe Pro Val Gly Asn Val Ser
Phe Phe Leu Phe 165 170 175 tac tcg ggt cac gtc gcc ggt tcc atg ata
gca tcc ttg gac atg agg 754Tyr Ser Gly His Val Ala Gly Ser Met Ile
Ala Ser Leu Asp Met Arg 180 185 190 aga atg cag agg ttg aga cta gcg
atg ctt ttt gac atc ctc aat gta 802Arg Met Gln Arg Leu Arg Leu Ala
Met Leu Phe Asp Ile Leu Asn Val 195 200 205 210 cta caa tcc atc agg
ctg ctc ggg acg aga gga cat tac acc atc gat 850Leu Gln Ser Ile Arg
Leu Leu Gly Thr Arg Gly His Tyr Thr Ile Asp 215 220 225 ctt gcg gtc
gga gtt ggc gct ggg att ctc ttt gac tcg ttg gcc ggg 898Leu Ala Val
Gly Val Gly Ala Gly Ile Leu Phe Asp Ser Leu Ala Gly 230 235 240 aag
tac gaa gag atg atg agc aaa aga cac aat tta ggc aat ggt ttt 946Lys
Tyr Glu Glu Met Met Ser Lys Arg His Asn Leu Gly Asn Gly Phe 245 250
255 agt ttg att tct aaa gac tcg cta gtc aat taa ttttgtttaa
tttcttttga 999Ser Leu Ile Ser Lys Asp Ser Leu Val Asn 260 265
aatgtttagt tgaacttgaa catattaaat ttaattgatg tccaatgaat taaatttatt
1059ttctttccga tgattctgac tgaaaaggat 10898268PRTBrassica juncea
8Met Ser Thr Glu Thr Gly Val Pro Leu Arg Arg Arg Ser Asn Ser Leu 1
5 10 15 Asn Gly His His Thr Asn Gly Val Ala Ser Asp Gly Thr Asn Val
Pro 20 25 30 Leu Met Glu Lys Ala Ser Phe Met Thr Trp Thr Ala Arg
Asp Ala Ile 35 40 45 Tyr Val Ala Arg Val His Trp Ile Pro Cys Val
Phe Ala Val Gly Val 50 55 60 Leu Phe Phe Met Gly Val Glu Tyr Thr
Leu Gln Met Ile Pro Ala Arg 65 70 75 80 Ser Glu Pro Phe Asp Ile Gly
Phe Val Ala Thr Arg Ser Leu Asn Arg 85 90 95 Val Leu Ala Asp Ser
Pro Asp Leu Asn Thr Val Leu Ala Ala Leu Asn 100 105 110 Thr Val Phe
Val Gly Met Gln Thr Thr Tyr Ile Val Trp Thr Trp Leu 115 120 125 Met
Glu Gly Arg Pro Arg Ala Thr Ile Ser Ala Cys Phe Met Phe Thr 130 135
140 Cys Arg Gly Ile Leu Gly Tyr Ser Thr Gln Leu Pro Leu Pro Gln Glu
145 150 155 160 Phe Leu Gly Ser Gly Val Asp Phe Pro Val Gly Asn Val
Ser Phe Phe 165 170 175 Leu Phe Tyr Ser Gly His Val Ala Gly Ser Met
Ile Ala Ser Leu Asp 180 185 190 Met Arg Arg Met Gln Arg Leu Arg Leu
Ala Met Leu Phe Asp Ile Leu 195 200 205 Asn Val Leu Gln Ser Ile Arg
Leu Leu Gly Thr Arg Gly His Tyr Thr 210 215 220 Ile Asp Leu Ala Val
Gly Val Gly Ala Gly Ile Leu Phe Asp Ser Leu 225 230 235 240 Ala Gly
Lys Tyr Glu Glu Met Met Ser Lys Arg His Asn Leu Gly Asn 245 250 255
Gly Phe Ser Leu Ile Ser Lys Asp Ser Leu Val Asn 260 265
9953DNABrassica junceaCDS(53)..(922) 9acccatctct ctaagcctct
caaaacgttc ttctccgtta aatctaacgg tc atg tca 58 Met Ser 1 act aca
aca atc gtc cct ctc cgt cgc act tct aac tct ctc aat gaa 106Thr Thr
Thr Ile Val Pro Leu Arg Arg Thr Ser Asn Ser Leu Asn Glu 5 10 15 tac
cac act aac gca gtc gcc ttt gac gga atc gtc ggg tca gca agt 154Tyr
His Thr Asn Ala Val Ala Phe Asp Gly Ile Val Gly Ser Ala Ser 20 25
30 act agc caa atg gag gag att gtt acg caa acc gac gac tgc tac gcc
202Thr Ser Gln Met Glu Glu Ile Val Thr Gln Thr Asp Asp Cys Tyr Ala
35 40 45 50 aac ccc aac gga gat gga ggg aga agc aag gtg tcg tta atg
acg tgg 250Asn Pro Asn Gly Asp Gly Gly Arg Ser Lys Val Ser Leu Met
Thr Trp 55 60 65 agg atg tgc aat cct gtc cac gtg gtg aga gtc cat
tgg ata ccg tgt 298Arg Met Cys Asn Pro Val His Val Val Arg Val His
Trp Ile Pro Cys 70 75 80 ttg tta gcg gta gga gtt ctg ttc ttc acg
tgc gta gag gag tac atg 346Leu Leu Ala Val Gly Val Leu Phe Phe Thr
Cys Val Glu Glu Tyr Met 85 90 95 ctc cag atg att ccg gcg agt tct
gag ccg ttc gat att ggt ttt gtg 394Leu Gln Met Ile Pro Ala Ser Ser
Glu Pro Phe Asp Ile Gly Phe Val 100 105 110 gcg acg ggc tct ctg tat
cgc ctc ttg gct tct tca ccg gat ctt aat 442Ala Thr Gly Ser Leu Tyr
Arg Leu Leu Ala Ser Ser Pro Asp Leu Asn 115 120 125 130 acc gtt tta
gct gct ctc aac acg gtg ttt gta ggg atg caa acg acg 490Thr Val Leu
Ala Ala Leu Asn Thr Val Phe Val Gly Met Gln Thr Thr 135 140 145 tat
att gta tgg aca tgg ttg atg gaa gga cga cca cga gcg acc atc 538Tyr
Ile Val Trp Thr Trp Leu Met Glu Gly Arg Pro Arg Ala Thr Ile 150 155
160 tcg gct tgc ttt atg ttt act tgc cgt ggc att ctg ggt tac tct act
586Ser Ala Cys Phe Met Phe Thr Cys Arg Gly Ile Leu Gly Tyr Ser Thr
165 170 175 cag ctc cct ctt cct cag gat ttt cta gga tca ggg gta gat
ttt ccg 634Gln Leu Pro Leu Pro Gln Asp Phe Leu Gly Ser Gly Val Asp
Phe Pro 180 185 190 gta gga aac gtc tcg ttc ttc ctc ttc tac tca ggc
cat gtc gca ggg 682Val Gly Asn Val Ser Phe Phe Leu Phe Tyr Ser Gly
His Val Ala Gly 195 200 205 210 tcg acg ata gca tcc ttg gat atg agg
aga atg aag agg ttg aga ctt 730Ser Thr Ile Ala Ser Leu Asp Met Arg
Arg Met Lys Arg Leu Arg Leu 215 220 225 gcc ttg ctt ttt gac atc ctc
aat gta tta caa tcg atc agg ctt ctc 778Ala Leu Leu Phe Asp Ile Leu
Asn Val Leu Gln Ser Ile Arg Leu Leu 230 235 240 ggg acg aga gga caa
tac acg atc gat ctc gct gtc gga gtt ggc gct 826Gly Thr Arg Gly Gln
Tyr Thr Ile Asp Leu Ala Val Gly Val Gly Ala 245 250 255 ggg gtt ctc
ttt gac tca ctg gct gga aaa tac gaa gag atg atg agc 874Gly Val Leu
Phe Asp Ser Leu Ala Gly Lys Tyr Glu Glu Met Met Ser 260 265 270 aag
aga cac aat gta ggc aat ggt ttt agt tta att tcg act cgc tag 922Lys
Arg His Asn Val Gly Asn Gly Phe Ser Leu Ile Ser Thr Arg 275 280 285
ttattaattt ttgttttttt ctttatgttt t 95310289PRTBrassica juncea 10Met
Ser Thr Thr Thr Ile Val Pro Leu Arg Arg Thr Ser Asn Ser Leu 1 5 10
15 Asn Glu Tyr His Thr Asn Ala Val Ala Phe Asp Gly Ile Val Gly Ser
20 25 30 Ala Ser Thr Ser Gln Met Glu Glu Ile Val Thr Gln Thr Asp
Asp Cys 35 40 45 Tyr Ala Asn Pro Asn Gly Asp Gly Gly Arg Ser Lys
Val Ser Leu Met 50 55 60 Thr Trp Arg Met Cys Asn Pro Val His Val
Val Arg Val His Trp Ile 65 70 75 80 Pro Cys Leu Leu Ala Val Gly Val
Leu Phe Phe Thr Cys Val Glu Glu 85 90 95 Tyr Met Leu Gln Met Ile
Pro Ala Ser Ser Glu Pro Phe Asp Ile Gly 100 105 110 Phe Val Ala Thr
Gly Ser Leu Tyr Arg Leu Leu Ala Ser Ser Pro Asp 115 120 125 Leu Asn
Thr Val Leu Ala Ala Leu Asn Thr Val Phe Val Gly Met Gln 130 135 140
Thr Thr Tyr Ile Val Trp Thr Trp Leu Met Glu Gly Arg Pro Arg Ala 145
150 155 160 Thr Ile Ser Ala Cys Phe Met Phe Thr Cys Arg Gly Ile Leu
Gly Tyr 165 170 175 Ser Thr Gln Leu Pro Leu Pro Gln Asp Phe Leu Gly
Ser Gly Val Asp 180 185 190 Phe Pro Val Gly Asn Val Ser Phe Phe Leu
Phe Tyr Ser Gly His Val 195 200 205 Ala Gly Ser Thr Ile Ala Ser Leu
Asp Met Arg Arg Met Lys Arg Leu 210 215 220 Arg Leu Ala Leu Leu Phe
Asp Ile Leu Asn Val Leu Gln Ser Ile Arg 225 230 235 240 Leu Leu Gly
Thr Arg Gly Gln Tyr Thr Ile Asp Leu Ala Val Gly Val 245 250 255 Gly
Ala Gly Val Leu Phe Asp Ser Leu Ala Gly Lys Tyr Glu Glu Met 260 265
270 Met Ser Lys Arg His Asn Val Gly Asn Gly Phe Ser Leu Ile Ser Thr
275 280 285 Arg 111040DNABrassica junceaCDS(70)..(939) 11ataatatctc
ttatatattc catttctcta agcctctcga aatgttcttc tccgttaaat 60ctaacggcc
atg tca act aca aca atc gtc cct ctc cgt cgg agt tct aac 111 Met Ser
Thr Thr Thr Ile Val Pro Leu Arg Arg Ser Ser Asn 1 5 10 tct ctc aat
gaa tac cac act aac gca gtc gcc ttt gac gga atc gtc 159Ser Leu Asn
Glu Tyr His Thr Asn Ala Val Ala Phe Asp Gly Ile Val 15 20 25 30 ggg
tca aca agt act agc caa atg gag gag att gtt acg caa atg gac 207Gly
Ser Thr Ser Thr Ser Gln Met Glu Glu Ile Val Thr Gln Met Asp 35 40
45 gaa ggc tac gcc aac ccc aac gga gat gga ggg aga agc aag gtg tcg
255Glu Gly Tyr Ala Asn Pro Asn Gly Asp Gly Gly Arg Ser Lys Val Ser
50 55 60 ttc atg acg tgg agg atg tgc agt gct gtc cac gtg gtg aga
gtc cac 303Phe Met Thr Trp Arg Met Cys Ser Ala Val His Val Val Arg
Val His 65 70 75 tgg ata ccg tgt ttg tta gcg gta gga gtt ctg ttc
ttc acg ggg gtg 351Trp Ile Pro Cys Leu Leu Ala Val Gly Val Leu Phe
Phe Thr Gly Val 80 85 90 gag gag tac atg ctc cag atg att ccc ccg
agt tct gag ccg ttc gat 399Glu Glu Tyr Met Leu Gln Met Ile Pro Pro
Ser Ser Glu Pro Phe Asp 95 100 105 110 att ggt ttt gtg gcg acg cgc
tct ctc tat cgc ctc ttg gct tct tca 447Ile Gly Phe Val Ala Thr Arg
Ser Leu Tyr Arg Leu Leu Ala Ser Ser 115 120 125 ccg gat ctc aac acc
gtt tta gcc gct ctc aac acg gtg ttc gta ggg 495Pro Asp Leu Asn Thr
Val Leu Ala Ala Leu Asn Thr Val Phe Val Gly 130 135 140 atg caa acg
acg tat att gta tgg aca tgg ttg atg gaa gga cga cca 543Met Gln Thr
Thr Tyr Ile Val Trp Thr Trp Leu Met Glu Gly Arg Pro 145 150 155 cga
gcg acc atc tcg gct tgc ttt atg ttt aca tgt cgt ggc att ctt 591Arg
Ala Thr Ile Ser Ala Cys Phe Met Phe Thr Cys Arg Gly Ile Leu 160 165
170 ggt tac tct act cag ctc cct ctt cct cag gat ttt cta gga tca ggg
639Gly Tyr Ser Thr Gln Leu Pro Leu Pro Gln Asp Phe Leu Gly Ser Gly
175 180 185 190 gta gac ttt cct gta gga aac gtc tcc ttc ttc ctc ttc
tac tca ggc 687Val Asp Phe Pro Val Gly Asn Val Ser Phe Phe Leu Phe
Tyr Ser Gly 195 200 205 cat gtg gca ggg tcg acg ata gca tcc ttg gac
atg agg aga atg aag 735His Val Ala Gly Ser Thr Ile Ala Ser Leu Asp
Met Arg Arg Met Lys 210 215 220 agg ttg aga cta gcc ttg ctt ttt gac
atc ctc aat gta tta caa tcg 783Arg Leu Arg Leu Ala Leu Leu Phe Asp
Ile Leu Asn Val Leu Gln Ser 225 230 235 atc agg ctt ctc ggg acg aga
gga caa tac acg atc gat ctc gct gtc 831Ile Arg Leu Leu Gly Thr Arg
Gly Gln Tyr Thr Ile Asp Leu Ala Val 240 245 250 gga gtt ggc gct ggg
gtt ctc ttt gac tca ctg gct gga aaa tac gaa 879Gly Val Gly Ala Gly
Val Leu Phe Asp Ser Leu Ala Gly Lys Tyr Glu 255 260 265 270 gag atg
atg agc aag aga cac aat gta ggc aat ggt ttt agt ttg att 927Glu Met
Met Ser Lys Arg His Asn Val Gly Asn Gly Phe Ser Leu Ile 275 280 285
tcg tct cgc tag ttattaattt ttgttttttt tttatgtttt tagcctggac 979Ser
Ser Arg atatttaatt tagttgaaat ctaatgactt aaatttactt tctttcaaaa
tgctctaact 1039g 104012289PRTBrassica juncea 12Met Ser Thr Thr Thr
Ile Val Pro Leu Arg Arg
Ser Ser Asn Ser Leu 1 5 10 15 Asn Glu Tyr His Thr Asn Ala Val Ala
Phe Asp Gly Ile Val Gly Ser 20 25 30 Thr Ser Thr Ser Gln Met Glu
Glu Ile Val Thr Gln Met Asp Glu Gly 35 40 45 Tyr Ala Asn Pro Asn
Gly Asp Gly Gly Arg Ser Lys Val Ser Phe Met 50 55 60 Thr Trp Arg
Met Cys Ser Ala Val His Val Val Arg Val His Trp Ile 65 70 75 80 Pro
Cys Leu Leu Ala Val Gly Val Leu Phe Phe Thr Gly Val Glu Glu 85 90
95 Tyr Met Leu Gln Met Ile Pro Pro Ser Ser Glu Pro Phe Asp Ile Gly
100 105 110 Phe Val Ala Thr Arg Ser Leu Tyr Arg Leu Leu Ala Ser Ser
Pro Asp 115 120 125 Leu Asn Thr Val Leu Ala Ala Leu Asn Thr Val Phe
Val Gly Met Gln 130 135 140 Thr Thr Tyr Ile Val Trp Thr Trp Leu Met
Glu Gly Arg Pro Arg Ala 145 150 155 160 Thr Ile Ser Ala Cys Phe Met
Phe Thr Cys Arg Gly Ile Leu Gly Tyr 165 170 175 Ser Thr Gln Leu Pro
Leu Pro Gln Asp Phe Leu Gly Ser Gly Val Asp 180 185 190 Phe Pro Val
Gly Asn Val Ser Phe Phe Leu Phe Tyr Ser Gly His Val 195 200 205 Ala
Gly Ser Thr Ile Ala Ser Leu Asp Met Arg Arg Met Lys Arg Leu 210 215
220 Arg Leu Ala Leu Leu Phe Asp Ile Leu Asn Val Leu Gln Ser Ile Arg
225 230 235 240 Leu Leu Gly Thr Arg Gly Gln Tyr Thr Ile Asp Leu Ala
Val Gly Val 245 250 255 Gly Ala Gly Val Leu Phe Asp Ser Leu Ala Gly
Lys Tyr Glu Glu Met 260 265 270 Met Ser Lys Arg His Asn Val Gly Asn
Gly Phe Ser Leu Ile Ser Ser 275 280 285 Arg 131014DNABrassica
junceaCDS(23)..(730) 13aaaaaaaaca aggaataata aa atg tct caa atg gac
att tct acg aga act 52 Met Ser Gln Met Asp Ile Ser Thr Arg Thr 1 5
10 gag gaa gga gga tgg aga agc aag cct tcg ttc atg acg tgg aga gcg
100Glu Glu Gly Gly Trp Arg Ser Lys Pro Ser Phe Met Thr Trp Arg Ala
15 20 25 cgc gac gtt gtc tac gtg atg aga cac cat tgg ata ccg tgt
ctg ttc 148Arg Asp Val Val Tyr Val Met Arg His His Trp Ile Pro Cys
Leu Phe 30 35 40 gcg gcc gga ttc ttg ttc gtc gta agc gtg gag tcc
tcg atc aag atg 196Ala Ala Gly Phe Leu Phe Val Val Ser Val Glu Ser
Ser Ile Lys Met 45 50 55 gtt tcc gag agt tct cca ccg ttc gat att
ggg ttt gtg gcc acg gag 244Val Ser Glu Ser Ser Pro Pro Phe Asp Ile
Gly Phe Val Ala Thr Glu 60 65 70 tct ctg cat cat atc ttg gct tct
tca ccg gat ctg aac acc ggt ttg 292Ser Leu His His Ile Leu Ala Ser
Ser Pro Asp Leu Asn Thr Gly Leu 75 80 85 90 gcc gct cta aac tcg gtg
tta gga gtg atg caa gta tcg tat att gca 340Ala Ala Leu Asn Ser Val
Leu Gly Val Met Gln Val Ser Tyr Ile Ala 95 100 105 tgg aca tgg tta
ata gaa gga cgg cca cga gcc acc atc acg gct tta 388Trp Thr Trp Leu
Ile Glu Gly Arg Pro Arg Ala Thr Ile Thr Ala Leu 110 115 120 ttc ctc
ttc act tgt cgc ggc gtt ctc ggt tac tgt acg cag ctc cct 436Phe Leu
Phe Thr Cys Arg Gly Val Leu Gly Tyr Cys Thr Gln Leu Pro 125 130 135
ctt tca aag gag tat cta gga tca gca atc gat ttc ccg cta gga aac
484Leu Ser Lys Glu Tyr Leu Gly Ser Ala Ile Asp Phe Pro Leu Gly Asn
140 145 150 ctc tcg ttc ttc tat ttt ttc tcg ggt cac gtg gca ggc acg
acc atc 532Leu Ser Phe Phe Tyr Phe Phe Ser Gly His Val Ala Gly Thr
Thr Ile 155 160 165 170 gca tct ttg gac atg agg aga atg cag agg ttg
aga ctt gcg atg gtt 580Ala Ser Leu Asp Met Arg Arg Met Gln Arg Leu
Arg Leu Ala Met Val 175 180 185 ttt gac atc ctc aat gta tta cag tcg
atc agg ctg ctt gcg acg aga 628Phe Asp Ile Leu Asn Val Leu Gln Ser
Ile Arg Leu Leu Ala Thr Arg 190 195 200 gga cac tac acg atc gat ctc
gca ggt gga gtt gcc gcc gcg att ctc 676Gly His Tyr Thr Ile Asp Leu
Ala Gly Gly Val Ala Ala Ala Ile Leu 205 210 215 ttt gac tca ttg gcc
ggc aag tac gaa gca aat aca aga aag agg caa 724Phe Asp Ser Leu Ala
Gly Lys Tyr Glu Ala Asn Thr Arg Lys Arg Gln 220 225 230 ttg tag
gaacaggttt cagcttgatt accaaaagac ttcaaagatt tcattcaaca 780Leu 235
tgtttagttg ctgttgaatt aagtctactg tggttcggca attattctcc ccatgagcca
840gtggcttgga cttcttcgac cctaatgttc atggtcagac tgtatatgtt
gtttatttct 900cattttttca ttcaactccg caatttgtga tatgggtttg
gttaacacta gttggttcag 960ttgttttcaa ttggttttac tctgaaagtt
ataaacgttt tgtaatacca gatt 101414235PRTBrassica juncea 14Met Ser
Gln Met Asp Ile Ser Thr Arg Thr Glu Glu Gly Gly Trp Arg 1 5 10 15
Ser Lys Pro Ser Phe Met Thr Trp Arg Ala Arg Asp Val Val Tyr Val 20
25 30 Met Arg His His Trp Ile Pro Cys Leu Phe Ala Ala Gly Phe Leu
Phe 35 40 45 Val Val Ser Val Glu Ser Ser Ile Lys Met Val Ser Glu
Ser Ser Pro 50 55 60 Pro Phe Asp Ile Gly Phe Val Ala Thr Glu Ser
Leu His His Ile Leu 65 70 75 80 Ala Ser Ser Pro Asp Leu Asn Thr Gly
Leu Ala Ala Leu Asn Ser Val 85 90 95 Leu Gly Val Met Gln Val Ser
Tyr Ile Ala Trp Thr Trp Leu Ile Glu 100 105 110 Gly Arg Pro Arg Ala
Thr Ile Thr Ala Leu Phe Leu Phe Thr Cys Arg 115 120 125 Gly Val Leu
Gly Tyr Cys Thr Gln Leu Pro Leu Ser Lys Glu Tyr Leu 130 135 140 Gly
Ser Ala Ile Asp Phe Pro Leu Gly Asn Leu Ser Phe Phe Tyr Phe 145 150
155 160 Phe Ser Gly His Val Ala Gly Thr Thr Ile Ala Ser Leu Asp Met
Arg 165 170 175 Arg Met Gln Arg Leu Arg Leu Ala Met Val Phe Asp Ile
Leu Asn Val 180 185 190 Leu Gln Ser Ile Arg Leu Leu Ala Thr Arg Gly
His Tyr Thr Ile Asp 195 200 205 Leu Ala Gly Gly Val Ala Ala Ala Ile
Leu Phe Asp Ser Leu Ala Gly 210 215 220 Lys Tyr Glu Ala Asn Thr Arg
Lys Arg Gln Leu 225 230 235
* * * * *
References