U.S. patent application number 10/755328 was filed with the patent office on 2004-10-28 for methods and means for obtaining modified phenotypes.
Invention is credited to Graham, Michael Wayne, Wang, Ming-Bo, Waterhouse, Peter Michael.
Application Number | 20040214330 10/755328 |
Document ID | / |
Family ID | 33298159 |
Filed Date | 2004-10-28 |
United States Patent
Application |
20040214330 |
Kind Code |
A1 |
Waterhouse, Peter Michael ;
et al. |
October 28, 2004 |
Methods and means for obtaining modified phenotypes
Abstract
Methods and means are provided for reducing the phenotypic
expression of a nucleic acid of interest in eucaryotic cells,
particularly in plant cells, by introducing chimeric genes encoding
sense and antisense RNA molecules directed towards the target
nucleic acid, which are capable of forming a double stranded RNA
region by base-pairing between the regions with sense and antisense
nucleotide sequence or by introducing the RNA molecules themselves.
Preferably, the RNA molecules comprises simultaneously both sense
and antisense nucleotide sequence.
Inventors: |
Waterhouse, Peter Michael;
(Canberra, AU) ; Wang, Ming-Bo; (Canberra, AU)
; Graham, Michael Wayne; (St. Lucia, AU) |
Correspondence
Address: |
BURNS DOANE SWECKER & MATHIS L L P
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Family ID: |
33298159 |
Appl. No.: |
10/755328 |
Filed: |
January 13, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10755328 |
Jan 13, 2004 |
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09287632 |
Apr 7, 1999 |
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Current U.S.
Class: |
435/455 ;
435/375 |
Current CPC
Class: |
C12N 15/8218 20130101;
C12N 15/8282 20130101; C12N 15/8247 20130101; C12N 15/8283
20130101; A61K 48/00 20130101; C12N 15/8203 20130101; A01K 2217/05
20130101 |
Class at
Publication: |
435/455 ;
435/375 |
International
Class: |
C12N 015/85 |
Claims
1-38. (Canceled)
39. A method to inhibit expression of a target gene in a eukaryotic
cell in vitro comprising introduction of a ribonucleic acid (RNA)
into the cell in an amount sufficient to inhibit expression of the
target gene, introduction of a ribonucleic acid (RNA) into the cell
in an amount sufficient to inhibit expression of the target gene,
wherein the RNA is a double-stranded molecule with a first strand
consisting essentially of a ribonucleotide sequence which
corresponds to a nucleotide sequence of the target gene and a
second strand consisting essentially of a ribonucleotide sequence
which is complementary to the nucleotide sequence of the target
gene, wherein the first and the second ribonucleotide strands are
separate complementary strands that hybridize to each other to form
said double-stranded molecule, and the double-stranded molecule
inhibits expression of the target gene.
40. The method of claim 39 in which the target gene is a cellular
gene.
41. The method of claim 39 in which the target gene is an
endogenous gene.
42. The method of claim 39 in which the target gene is a
transgene.
43. The method of claim 39 in which the target gene is a viral
gene.
44. The method of claim 39 in which the cell is from a plant.
45. The method of claim 39 in which at least one strand of the RNA
is produced by transcription of an expression construct.
Description
FIELD OF THE INVENTION
[0001] The invention relates to methods for reducing the phenotypic
expression of a nucleic acid sequence of interest in eucaryotic
cells, particularly plant cells, by simultaneously providing the
cells with chimeric genes encoding sense and anti sense RNA
molecules comprising nucleotide sequences respectively homologous
and complementary to at least part of the nucleotide sequence of
the nucleic acid of interest. The sense and antisense RNA molecules
may be provided as one RNA molecule, wherein the sense and
antisense RNA may be linked by a spacer nucleotide sequence and are
capable of forming a double stranded RNA molecule. In one aspect of
the invention, the methods are directed towards reducing viral
infection, resulting in extreme virus resistance. In another
embodiment the methods are directed towards reducing the phenotypic
expression of an endogenous gene in a plant cell. The invention
further relates to high throughput screening methods for
identifying the phenotype endowed by the nucleic acid of interest
in plant cells. Also provided are plant cells comprising such RNA
molecules, as well as plants consisting essentially of such plant
cells.
BACKGROUND OF THE INVENTION
[0002] In 1985, Sanford and Johnston proposed the concept of
parasite-derived resistance. They postulated that key gene products
from a parasite expressed in the host in a dysfunctional form, in
excess or at a wrong developmental stage, should disrupt the
function of the parasite with minimal effect on the host (Sanford
& Johnston, 1985). Using the QB bacteriophage as a model, they
proposed that expression, in bacteria, of the bacteriophage coat
protein or modified replicase or an antisense RNA complementary to
the QB genome could all give resistance. They also proposed that
such approaches would be applicable, in plants, to plant viruses
and particularly the use of a modified plant virus replicase. The
expression of the coat protein of the plant virus, tobacco mosaic
virus (TMV), in tobacco was the first practical validation of this
concept for plant virus resistance. This work (Powell-Abel et al.,
1986) showed that the expression of the TMV coat protein, from a
transgene under the control of the cauliflower mosaic virus 35S
promoter, conferred on the plants resistance to TMV. The same group
(Powell et al., 1990) showed that, generally, plants expressing
higher levels of coat protein were more resistant to TMV than
plants expressing low levels. Since this demonstration there have
been very many examples of plants transformed with virus coat
protein genes showing resistance (Table 1). There have also been a
number of reports of plant virus resistance in plants expressing
wild-type replicase (Braun and Hemenway, 1992, Brederode et al.,
1995), truncated replicase (Carr et al. 1992), modified replicase
(Longstaff et al. 1993), or antisense viral RNA (Kawchuck et al.
1991).
[0003] In 1992, Dougherty and colleagues were using different forms
of the coat protein gene of tobacco etch virus (TEV) and discovered
that some plants containing untranslatable "sense" coat protein
genes and antisense coat protein genes showed extreme resistance
(ER) to the virus (Lindbo & Dougherty, 1992 a,b). This
resistance was functional at the whole plant level and at the
single cell level. TEV was unable to replicate in protoplasts
derived from ER plants but replicated to high levels in protoplasts
from non-transgenic tobacco. Dougherty et al. concluded that the
mechanism creating the extreme resistance for the untranslatable
sense construct was not the same as the often reported coat
protein-mediated strategy. They proposed that the mRNA of the
untranslatable sense construct was hybridizing with the minus sense
genome of the virus and interfering with the procession of
replication complexes on the minus strand. They suggested that the
use of viral sequence that could form intramolecular pairing should
be avoided as this would interfere with their ability to hybridize
to the target viral RNA.
1TABLE 1 Plant species that have been genetically engineered for
virus resistance (from Rebecca Grumet, Hort Science 30[3] 1995)
Species Viruses Tobacco AIMV, ArMV, CMV, PVX, PVY, TEV, (Nicotiana
tabacum L.) TGMV, TMV, TRV, TSV, TSWV Other Nicotiana spp. ACMV,
BYMV, CyMV, CyRSV, BCTV, (N. debneyii, N. benthamiana, PEBV, PPV,
PVS, WMV N. clevelandii) Potato PI, RV, PVY (Solanum tuberusom L.)
Tomato AIMV, CMV, TMV, TYLCV (Lycopersicon esculentum L.) Cucumber
CMV (Cucumis sativus L.) Melon (Cucumis melo L.) CMV, ZYMV Alfalfa
(Medicago sativa L.) AIMV Papaya (Carica papaya L.) PRSV Corn (Zea
mays L.) MDMV Rice (Oryza sativa L.) RSV Rapeseed (Brassica napus
L.) TYMV
[0004] The Dougherty group expanded their investigations to plants
containing untranslatable sense potato virus Y (PVY) coat protein
genes. They obtained results similar to those with TEV and showed
that the plants with ER had high transgene copy number, highly
active transcription of the transgenes, and low levels of steady
state mRNA from the PVY transgene (Lindbo et al., 1993, Smith et
al. 1994). The following model for this mechanism of the resistance
was proposed: the high level of transcription of the viral
transgene triggers a cytoplasmic based, post transcriptional
cellular surveillance system that targets specific RNAs for
elimination. As the transgene encodes a transcript comprising viral
sequences the mechanism not only degrades the transgene mRNA but
also the same sequences in the viral genomic RNA. A key point in
this model is the need for a high level of transcription of the
transgene provided by high copy number (3-8; Goodwin et al. 1996).
Alternatively, the RNA threshold required to trigger the mechanism
can be reached by a more modest transcription level aided by the
viral RNA from replication in early infection. This gives rise to a
"recovery phenotype" where the plant is initially infected and
shows symptoms but then produces new growth without virus symptoms
and which are extremely resistant to infection.
[0005] This proposal was substantiated by the findings that gene
silencing of non-viral transgenes could also be due to a post
transcriptional mechanism (Ingelbrecht et al. 1994; de Carvalho
Niebel et al., 1995) operating at an RNA level.
[0006] A link between non-viral gene silencing and this pathogen
derived resistance was provided by inoculating transgenic plants,
in which a GUS transgene was known to be silenced by a post
transcriptional mechanism, with a virus containing GUS sequences
(English et al. 1996). In this situation the plants were extremely
resistant to the viral infection. However, the same plants were
susceptible to the virus if they contained no GUS sequences.
[0007] The degree of viral resistance is not always directly
related to the level of viral transgene transcription (Mueller et
al. 1995; English et al. 1996) suggesting that there may be an
alternative mechanism of inducing the resistance. To accommodate
these discrepancies, an alternative model has been proposed in
which the crucial factor affecting the resistance is not the level
but the quality of the transgene mRNA (English et al. 1996).
According to this model, the transgene can only induce resistance
(or gene silencing) if it is transcribed to produce "aberrant" RNA.
There have been many examples of post-transcriptional gene
silencing and methylation of the transgene (Hobbs et al. 1990;
Ingelbrecht et al. 1994) and methylation of the transgene has also
been found to be associated in some cases of extreme viral
resistance (Smith et al. 1994, English 1996). In the proposed
model, methylation of the transgene leads to the production of
aberrant RNAs which induce the cytoplasmic RNA surveillance system.
Baulcombe and English have suggested that this method of induction
may be the same as that found for the silencing of met2 in
A.immersus. In this system transcription of the met2 RNA was
terminated in the methylated regions of the endogenous gene thus
producing aberrant truncated RNAs. It was suggested that the
methylation was a consequence of ectopic interaction between the
transgene and a homologous region of a corresponding region of the
endogenous gene (Barry et al. 1993). The presence of multiple
transgenes would create an increased likelihood of ectopic pairing
and is therefore consistent with the correlation between high copy
number and extreme viral resistance (Mueller et al., 1995; Goodwin
et al. 1996; Pang et al., 1996).
[0008] This whole area has been reviewed recently (e.g. Baulcombe
(1996) and Stam et al. (1997)) and several models were presented.
All models call for a high degree of sequence specificity because
the resistance is very (strain)-specific and therefore invoke base
pairing interaction with an RNA produced from the transgene. One
explanation for the induction of the virus resistance or gene
silencing with sense transgenes is that the plant's RNA dependent
RNA polymerase generates complementary RNAs using the transgene
mRNA as a template (Schiebel et al. 1993a,b). This hypothetical
complementary RNA (cRNA) has not been detected (Baulcombe 1996) but
it is expected that the cRNAs will be small and heterodisperse RNAs
rather than full complements (Schiebel 1993ab, Baulcombe 1996) and
therefore difficult to detect.
[0009] The possible methods of action of the cRNA in mediating the
virus resistance or gene silencing (as proposed by Baulcombe, 1996)
are:
[0010] 1: The cRNA hybridizes with transgene mRNA or viral RNA and
the duplex becomes a target for dsRNases.,
[0011] 2: The cRNA hybridizes with target RNA to form a duplex
which can arrest translation and consequently have an indirect
effect on stability (Green, 1993);
[0012] 3: The duplex formed between the cRNA and viral RNA causes
hybrid arrest of translation of co-factors required for viral
replication; and
[0013] 4. The hybridization of the cRNA affects intra-molecular
base pairing required for viral replication.
[0014] The current models for virus resistance or gene silencing
thus involve the induction of a cytoplasmic surveillance system by
either high levels of transgene transcription or by the production
of aberrant single stranded mRNA. Once the system is triggered, RNA
dependent RNA polymerase makes cRNA from the transgene mRNA. These
cRNAs hybridize to the target RNA either directly affecting its
translatability or stability, or marking the RNA for
degradation.
[0015] U.S. Pat. No. 5,190,131 and EP 0 467 349 A1 describe methods
and means to regulate or inhibit gene expression in a cell by
incorporating into or associating with the genetic material of the
cell a non-native nucleic acid sequence which is transcribed to
produce an mRNA which is complementary to and capable of binding to
the mRNA produced by the genetic material of that cell.
[0016] EP 0 223 399 A1 describes methods to effect useful somatic
changes in plants by causing the transcription in the plant cells
of negative RNA strands which are substantially complementary to a
target RNA strand. The target RNA strand can be a mRNA transcript
created in gene expression, a viral RNA, or other RNA present in
the plant cells. The negative RNA strand is complementary to at
least a portion of the target RNA strand to inhibit its activity in
vivo.
[0017] EP 0 240 208 describes a method to regulate expression of
genes encoded for in plant cell genomes, achieved by integration of
a gene under the transcriptional control of a promoter which is
functional in the host and in which the transcribed strand of DNA
is complementary to the strand of DNA that is transcribed from the
endogenous gene(s) one wishes to regulate.
[0018] EP 0 647 715 A1 and U.S. Pat. Nos. 5,034,323, 5,231,020 and
5,283,184 describe methods and means for producing plants
exhibiting desired phenotypic traits, by selecting transgenotes
that comprise a DNA segment operably linked to a promoter, wherein
transcription products of the segment are substantially homologous
to corresponding transcripts of endogenous genes, particularly
endogenous flavonoid biosynthetic pathway genes.
[0019] WO 93/23551 describes methods and means for the inhibition
of two or more target genes, which comprise introducing into the
plant a single control gene which has distinct DNA regions
homologous to each of the target genes and a promoter operative in
plants adapted to transcribe from such distinct regions RNA that
inhibits expression of each of the target genes.
[0020] WO92/13070 describes a method for the regulation of nucleic
acid translation, featuring a responsive RNA molecule which encodes
a polypeptide and further includes a regulatory domain, a substrate
region and a ribosome recognition sequence. This responsive RNA
molecule has an inhibitor region in the regulatory domain, which
regulatory domain is complementary to both a substrate region of
the responsive RNA molecule and to an anti-inhibitor region of a
signal nucleic acid such that, in the absence of the signal nucleic
acid, the inhibitor and substrate regions form a base-paired domain
the formation of which reduced the level of translation of one of
the protein-coding regions in the responsive RNA molecule compared
to the level of translation of that one protein-coding region
observed in the presence of the signal nucleic acid.
[0021] Metzlaff et al., 1997 describe a model for the RNA-mediated
RNA degradation and chalcone synthase A silencing in Petunia,
involving cycles of RNA-RNA pairing between complementary sequences
followed by endonucleolytic RNA cleavages to describe how RNA
degradation is likely to be promoted. Fire et al., 1998 describe
specific genetic interference by experimental introduction of
double-stranded RNA in Caenorhabditis elegans. The importance of
these findings for functional genomics is discussed (Wagner and
Sun, 1998).
[0022] Que et al., 1998 describe distinct patterns of pigment
suppression which are produced by allelic sense and antisense
chalcone synthase transgenes in petunia flowers and have also
analyzed flower color patterns in plants heterozygous for sense and
antisense chalcone synthase alleles.
[0023] WO 98/05770 discloses antisense RNA with special secondary
structures which may be used to inhibit gene expression.
[0024] WO 94/18337 discloses transformed plants which have
increased or decreased linolenic acids as well as plants which
express a linoleic acid desaturase.
[0025] U.S. Pat. No. 5,850,026 discloses an endogenous oil from
Brassica seeds that contains, after crushing and extracting,
greater than 86% oleic acid and less than 2.5% .alpha.-linolenic
acid. The oil also contains less than 7% linoleic acid. The
Brassica seeds are produced by plants that contain seed-specific
inhibition of microsomal oleate desaturase and microsomal linoleate
desaturase gene expression, wherein the inhibition can be created
by cosuppression or antisense technology.
[0026] U.S. Pat. No. 5,638,637 discloses vegetable oil from
rapeseeds and rapeseed producing the same, the vegetable oil having
an unusually high oleic acid content of 80% to 90% by weight based
on total fatty acid content.
SUMMARY OF THE INVENTION
[0027] The present invention provides methods for reducing the
phenotypic expression of a nucleic acid of interest, which is
normally capable of being expressed in a eucaryotic cell,
particularly for reducing the phenotypic expression of a gene,
particularly a endogenous gene or a foreign transgene, integrated
in the genome of a eucaryotic cell or for reducing the phenotypic
expression of nucleic acid of interest which is comprised in the
genome of an infecting virus, comprising the step of introducing,
preferably integrating, in the nuclear genome of the eucaryotic
cell, a chimeric DNA comprising a promoter, capable of being
expressed in that eucaryotic cell, and optionally a DNA region
involved in transcription termination and polyadenylation and in
between a DNA region, which when transcribed, yields an RNA
molecule with a nucleotide sequence comprising a sense nucleotide
sequence of at least 10 consecutive nucleotides, particularly at
least about 550 consecutive nucleotides, having between 75 and 100%
sequence identity with at least part of the nucleotide sequence of
the nucleic acid of interest, and an antisense nucleotide sequence
including at least 10 consecutive nucleotides, having between about
75.degree./to about 100% sequence identity with the 10 nucleotide
stretch of the complement of the sense nucleotide sequence, wherein
the RNA is capable of forming an artificial hairpin RNA structure
with a double stranded RNA stem by base-pairing between the regions
with sense and antisense nucleotide sequence such that at least the
10 consecutive nucleotides of the sense sequence base pair with the
10 consecutive nucleotides of the antisense sequence, resulting in,
preferably an artificial hairpin structure. Preferably the chimeric
DNA is stably integrated in the genome of the DNA.
[0028] The invention also provides a method for reducing the
phenotypic expression of a nucleic acid of interest, which is
normally capable of being expressed in a eucaryotic cell comprising
the step of introducing a chimeric RNA molecule with a nucleotide
sequence comprising a sense nucleotide sequence of at least 10
consecutive nucleotides having between 75 and 100% sequence
identity with at least part of the nucleotide sequence of the
nucleic acid of interest; and an antisense nucleotide sequence
including at least 10 consecutive nucleotides, having between about
75% to about 100% sequence identity with 10 nt stretch of the
complement of the sense nucleotide sequence; wherein the RNA is
capable of forming a double stranded RNA region by base-pairing
between the regions with sense and antisense nucleotide sequence
such that at least the 10 consecutive nucleotides of the sense
sequence base pair with the 10 consecutive nucleotides of the
antisense sequence, resulting in a(n artificial) hairpin RNA
structure.
[0029] The invention further provides a method for reducing the
gene expression of a gene of interest in plant cells, comprising
the-step of introducing a first and second chimeric DNA, linked on
one recombinant DNA such that both chimeric DNAs are integrated
together in the nuclear genome of the transgenic plant cells;
wherein the first chimeric DNA comprises a plant-expressible
promoter, a first DNA region capable of being transcribed into a
sense RNA molecule with a nucleotide sequence comprising a sense
nucleotide sequence of at least 10 consecutive nucleotides having
between 75 and 100% sequence identity with at least part of the
nucleotide sequence of the gene of interest and optionally a DNA
region involved in transcription termination and polyadenylation
functioning in plant cells. The second chimeric DNA comprises a
plant-expressible promoter, a second DNA region capable of being
transcribed into an antisense RNA molecule with a nucleotide
sequence comprising an antisense nucleotide sequence including at
least 10 consecutive nucleotides, having between about 75% to about
100% sequence identity with the complement of the at least 10
consecutive nucleotides of the sense nucleotide sequence and
optionally a DNA region involved in transcription termination and
polyadenylation functioning in plant cells. The sense and antisense
RNA molecules are capable of forming a double stranded, duplex RNA
by base-pairing between the regions which are complementary.
[0030] Also provided by the invention is a method for obtaining
virus resistant organisms, particularly plants, comprising the
steps of providing cells of the organism with a first and second
chimeric DNA, wherein the first chimeric DNA comprises a promoter,
a first DNA region capable of being transcribed into a sense RNA
molecule with a nucleotide sequence comprising a sense nucleotide
sequence of at least 10 consecutive nucleotides having between 75
and 100% sequence identity with at least part of the nucleotide
sequence of the genome of a virus, capable of infecting the plant
and a DNA region involved in transcription termination and
polyadenylation functioning in plant cells. The second chimeric DNA
comprises a promoter, a second DNA region capable of being
transcribed into an antisense RNA molecule with a nucleotide
sequence comprising an antisense nucleotide sequence including at
least 10 consecutive nucleotides, having between about 75% to about
100% sequence identity with the complement of at least 10
consecutive nucleotides of the sense nucleotide sequence and a DNA
region involved in transcription termination and polyadenylation
functioning in plant cells. The sense and antisense RNA molecules
are capable of forming a double stranded RNA region by base-pairing
between the regions which are complementary.
[0031] The first and second chimeric DNA are either integrated
separately in the nuclear genome of the transformed plant cell or
they are linked on one recombinant DNA such that both chimeric DNAs
are integrated together in the nuclear genome of the transgenic
plant cells.
[0032] The invention also provides a method for identifying a
phenotype associated with the expression of a nucleic acid of
interest in a eucaryotic cell, comprising the steps of selecting
within the nucleotide sequence of interest, a target sequence of at
least 10 consecutive nucleotides; designing a sense nucleotide
sequence corresponding to the length of the selected target
sequence and which has a sequence identity of at least about 75% to
about 100% with the selected target sequence, designing an
antisense nucleotide sequence which has a sequence identity of at
least about 75% to about 100% with the complement of the sense
nucleotide sequence and comprises a stretch of at least about 10
consecutive nucleotides with 100% sequence identity to the
complement of a part of the sense nucleotide sequence. The method
further comprises the steps of introducing an RNA molecule
comprising both the designed sense and antisense nucleotide
sequences into a suitable eucaryotic host cell comprising the
nucleic acid including the nucleotide sequence with hitherto
unidentified phenotype; and observing the phenotype by a suitable
method.
[0033] The invention also provides a eucaryotic cell, comprising a
nucleic acid of interest which is normally capable of being
phenotypically expressed, further comprising a chimeric DNA
molecule comprising a promoter, capable of being expressed in that
eucaryotic cell, a DNA region, which when transcribed, yields an
RNA molecule with a nucleotide sequence comprising a sense
nucleotide sequence of at least 10 consecutive nucleotides having
between 75 and 100% sequence identity with at least part of the
nucleotide sequence of the nucleic acid of interest and an
antisense nucleotide sequence including at least 10 consecutive
nucleotides, having between about 75% to about 100% sequence
identity with the complement of the at least 10 consecutive
nucleotides of the sense nucleotide sequence wherein the RNA
molecule is capable of forming a double stranded RNA region by
base-pairing between the regions with sense and antisense
nucleotide sequence such that at least said 10 consecutive
nucleotides of the sense sequence base pair with said 10
consecutive nucleotides of the antisense sequence, resulting in a
hairpin RNA structure, preferably an artificial hairpin structure
and a DNA region involved in transcription termination and
polyadenylation, wherein the phenotypic expression of the nucleic
acid of interest is significantly reduced.
[0034] Also provided by the invention is a eucaryotic cell,
comprising a nucleic acid of interest, which is normally capable of
being phenotypically expressed, further comprising a chimeric RNA
molecule with a nucleotide sequence comprising a sense nucleotide
sequence of at least 10 consecutive nucleotides having between 75
and 100% sequence identity with at least part of the nucleotide
sequence of the nucleic acid of interest and an antisense
nucleotide sequence including at least 10 consecutive nucleotides,
having between about 75% to about 100% sequence identity with the
complement of the at least 10 consecutive nucleotides of the sense
nucleotide sequence wherein the RNA is capable of forming a double
stranded RNA region by base-pairing between the regions with sense
and antisense nucleotide sequence such that at least said 10
consecutive nucleotides of the sense sequence basepair with said 10
consecutive nucleotides of the antisense sequence, resulting in an
artificial hairpin RNA structure.
[0035] It is another objective of the invention to provide a
eucaryotic cell, comprising a gene of interest, which is normally
capable of being phenotypically expressed, further comprising a
first and second chimeric DNA, linked on one recombinant DNA such
that both chimeric DNAs are integrated together in the nuclear
genome of that eucaryotic cell wherein the first chimeric DNA
comprises the following operably linked parts a promoter capable of
being expressed in the eucaryotic cell a first DNA region capable
of being transcribed into a sense RNA molecule with a nucleotide
sequence comprising a sense nucleotide sequence of at least 10
consecutive nucleotides having between 75 and 100% sequence
identity with at least part of the nucleotide sequence of the gene
of interest; and a DNA region involved in transcription termination
and polyadenylation; and wherein the second chimeric DNA comprises
the following operably linked parts: a promoter operative in the
eucaryotic cell; a second DNA region capable of being transcribed
into an antisense RNA molecule with a nucleotide sequence
comprising an antisense nucleotide sequence including at least 10
consecutive nucleotides, having between about 75.degree./to about
100% sequence identity with the complement of the at least 10
consecutive nucleotides of the sense nucleotide sequence; a DNA
region involved in transcription termination and polyadenylation ;
wherein the sense and antisense RNA molecules are capable of
forming a double stranded RNA region by base-pairing between the
regions which are complementary.
[0036] It is yet another objective of the invention to provide a
virus resistant plant, comprising a first and second chimeric DNA
integrated in the nuclear genome of its cells, wherein the first
chimeric DNA comprises a plant-expressible promoter, a first DNA
region capable of being transcribed into a sense RNA molecule with
a nucleotide sequence comprising a sense nucleotide sequence of at
least 10 consecutive nucleotides having between 75 and 100%
sequence identity with at least part of the nucleotide sequence of
the genome of a virus, capable of infecting the plant, and a DNA
region involved in transcription termination and polyadenylation
functioning in plant cells. The second chimeric DNA comprises a
plant-expressible promoter, a second DNA region capable of being
transcribed into an antisense RNA molecule with a nucleotide
sequence comprising an antisense nucleotide sequence including at
least 10 consecutive nucleotides, having between about 75% to about
100% sequence identity with the complement of the at least 10
consecutive nucleotides of the sense nucleotide sequence, and a DNA
region involved in transcription termination and polyadenylation
functioning in plant cells. The sense and antisense RNA molecules
are capable of forming a double stranded RNA region by base-pairing
between the regions which are complementary. The first and second
chimeric DNA are integrated either in one locus or in different
loci in the nuclear genome.
[0037] The invention also provides a method for modifying the fatty
acid profile in oil, preferably increasing the oleic acid content,
from a plant, preferably from oilseed rape, the method comprising
the step of introducing a chimeric DNA into the cells of the plant,
comprising the following operably linked parts: a). a
plant-expressible promoter, preferably a seed-specific promoter,
b). a DNA region, particularly with the nucleotide sequence of SEQ
ID No 6, which when transcribed yields an RNA molecule comprising
an RNA region capable of forming an artificial stem-loop structure,
wherein one of the annealing RNA sequences of the stem-loop
structure comprises a nucleotide sequence essentially similar to at
least part of the nucleotide sequence of a .DELTA.12 desaturase
encoding open reading frame, and wherein the second of the
annealing RNA sequences comprises a sequence essentially similar to
at least part of the complement of at least part of the nucleotide
sequence of the .DELTA.12 desaturase encoding open reading frame;
and optionally; c) a DNA region involved in transcription
termination and polyadenylation. Plants with modified fatty acid
profile, particularly with increased oleic acid content, comprising
the mentioned chimeric genes are also provided by the invention.
Also encompassed are oils obtained from such plants or seed.
[0038] With the foregoing and other objects, advantages and
features of the invention that will become hereinafter apparent,
the nature of the invention may be more clearly understood by
reference to the following detailed description of the preferred
embodiments of the invention and to the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] FIG. 1 represents schematically the different sense and
antisense constructs, as well as the so-called CoP (complementary
pair) constructs used for obtaining virus resistance (FIG. 1B) or
for reducing the phenotypic expression of a transgenic Gus gene
(FIG. 1A).
[0040] FIG. 2A represents schematically the so-called panhandle
construct or CoP constructs used for reducing the phenotypic
expression of a .DELTA.12 desaturase gene in Arabidopsis (Nos Pro:
nopaline synthase gene promoter; nptlI neomycin phospho-transferase
coding region; Nos term: nopaline syntase gene terminator; FP1:
truncated seed specific napin promoter; 480 bp: 5' end of the Fad2
gene of Arabidopsis thaliana in sense orientation; 623 bp: spacer;
480 bp: 5' end of the Fad2 gene of Arabidopsis thaliana in
antisense orientation.
[0041] FIG. 2B represents schematically a common cosuppression
construct for for reducing the phenotypic expression of a .DELTA.12
desaturase gene in Arabidopsis thaliana.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
[0042] One of the objectives of the invention is to provide a
eucaryotic cell, particularly a plant cell with an RNA molecule
which comprises a hairpin structure including a determined sense
part and a determined antisense part. Potentially, an RNA molecule
is capable of forming several secondary structures, some of which
may contain the desired hairpin. It is expected that the real
secondary structure of the RNA in the cell, will have the lowest
free energy. In accordance with the invention, the RNA molecule to
be produced in the cell is designed in such a way that at least in
its lowest free energy state, which it can assume within that cell,
it will comprise the desired hairpin.
[0043] As used herein "hairpin RNA" refers to any self-annealing
double stranded RNA molecule. In its simplest representation, a
hairpin RNA consists of a double stranded stem made up by the
annealing RNA strands, connected by a single stranded RNA loop, and
is also referred to as a "pan-handle RNA". However, the term
"hairpin RNA" is also intended to encompass more complicated
secondary RNA structures comprising self-annealing double stranded
RNA sequences, but also internal bulges and loops. The specific
secondary structure adapted will be determined by the free energy
of the RNA molecule, and can be predicted for different situations
using appropriate software such as FOLDRNA (Zuker and Stiegler,
1981).
[0044] As used herein, "sequence identity" with regard to
nucleotide sequences (DNA or RNA), refers to the number of
positions with identical nucleotides divided by the number of
nucleotides in the shorter of the two sequences. The alignment of
the two nucleotide sequences is performed by the Wilbur and Lipmann
algorithm (Wilbur and Lipmann, 1983) using a window-size of 20
nucleotides, a word length of 4 nucleotides, and a gap penalty of
4. Computer-assisted analysis and interpretation of sequence data,
including sequence alignment as described above, can, e.g., be
conveniently performed using the programs of the
Intelligenetics.TM. Suite (Intelligenetics Inc., Calif.). Sequences
are indicated as "essentially similar" when such sequence have a
sequence identity of at least about 75%, particularly at least
about 80%, more particularly at least about 85%, quite particularly
about 90%, especially about 95%, more especially about 100%, quite
especially are identical. It is clear than when RNA sequences are
said to be essentially similar or have a certain degree of sequence
identity with DNA sequences, thymine (T) in the DNA sequence is
considered equal to uracil (U) in the RNA sequence.
[0045] As used herein, the term "plant-expressible promoter" means
a DNA sequence which 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, the subterranean
clover virus promoter No 4 or No 7, or T-DNA gene promoters.
[0046] The term "expression of a gene" refers to the process
wherein a DNA region which is operably linked to appropriate
regulatory regions, particularly to a promoter, is transcribed into
an RNA which is biologically active i.e., which is either capable
of interaction with another nucleic acid or which is capable of
being translated into a polypeptide or protein. A gene is said to
encode an RNA when the end product of the expression of the gene is
biologically active RNA, such as e.g. an antisense RNA, a ribozyme
or a replicative intermediate. A gene is said to encode a protein
when the end product of the expression of the gene is a protein or
polypeptide.
[0047] A nucleic acid of interest is "capable of being expressed",
when said nucleic acid, when introduced in a suitable host cell,
particularly in a plant cell; can be transcribed (or replicated) to
yield an RNA, and/or translated to yield a polypeptide or protein
in that host cell.
[0048] The term "gene" means any DNA fragment comprising a DNA
region (the "transcribed DNA region") that is transcribed into a
RNA molecule (e.g., an mRNA) in a cell operably linked to suitable
regulatory regions, e.g., a plant-expressible promoter. A gene may
thus comprise several operably linked DNA fragments such as a
promoter, a 5' leader sequence, a coding region, and a 3' region
comprising a polyadenylation site. A plant gene endogenous to a
particular plant species (endogenous plant gene) is a gene which is
naturally found in that plant species or which can be introduced in
that plant species by conventional breeding. A chimeric gene is any
gene which is not normally found in a plant species or,
alternatively, any gene in which the promoter is not associated in
nature with part or all of the transcribed DNA region or with at
least one other regulatory region of the gene.
[0049] As used herein, "phenotypic expression of a nucleic acid of
interest" refers to any quantitative trait associated with the
molecular expression of a nucleic acid in a host cell and may thus
include the quantity of RNA molecules transcribed or replicated,
the quantity of post-transcriptionally modified RNA molecules, the
quantity of translated peptides or proteins, the activity of such
peptides or proteins.
[0050] A "phenotypic trait" associated with the phenotypic
expression of a nucleic acid of interest refers to any quantitative
or qualitative trait, including the trait mentioned, as well as the
direct or indirect effect mediated upon the cell, or the organism
containing that cell, by the presence of the RNA molecules, peptide
or protein, or posttranslationally modified peptide or protein. The
mere presence of a nucleic acid in a host cell, is not considered a
phenotypic expression or a phenotypic trait of that nucleic acid,
even though it can be quantitatively or qualitatively traced.
Examples of direct or indirect effects mediated on cells or
organisms are, e.g., agronomically or industrial useful traits,
such as resistance to a pest or disease; higher or modified oil
content etc.
[0051] As used herein, "reduction of phenotypic expression" refers
to the comparison of the phenotypic expression of the nucleic acid
of interest to the eucaryotic cell in the presence of the RNA or
chimeric genes of the invention, to the phenotypic expression of
the nucleic acid of interest in the absence of the RNA or chimeric
genes of the invention. The phenotypic expression in the presence
of the chimeric RNA of the invention should thus be lower than the
phenotypic expression in absence thereof, preferably be only about
25%, particularly only about 10%, more particularly only about 5%
of the phenotypic expression in absence of the chimeric RNA,
especially the phenotypic expression should be completely inhibited
for all practical purposes by the presence of the chimeric RNA or
the chimeric gene encoding such an RNA.
[0052] A reduction of phenotypic expression of a nucleic acid where
the phenotype is a qualitative trait means that in the presence of
the chimeric RNA or gene of the invention, the phenotypic trait
switches to a different discrete state when compared to a situation
in which such RNA or gene is absent. A reduction of phenotypic
expression of a nucleic acid may thus, a.o., be measured as a
reduction in transcription of (part on that nucleic acid, a
reduction in translation of (part of) that nucleic acid or a
reduction in the effect the presence of the transcribed RNA(s) or
translated polypeptide(s) have on the eucaryotic cell or the
organism, and will ultimately lead to altered phenotypic traits. It
is clear that the reduction in phenotypic expression of a nucleic
acid of interest, may be accompanied by or correlated to an
increase in a phenotypic trait.
[0053] As used herein "a nucleic acid of interest" or a "target
nucleic acid" refers to any particular RNA molecule or DNA sequence
which may be present in a eucaryotic cell, particularly a plant
cell.
[0054] As used herein "comprising" is to be interpreted as
specifying the presence of the stated features, integers, steps or
components as referred to, but does not preclude the presence or
addition of one or more features, integers, steps or components, or
groups thereof. Thus, e.g., a nucleic acid or protein comprising a
sequence of nucleotides or amino acids, may comprise more
nucleotides or amino acids than the actually cited ones, i.e., be
embedded in a larger nucleic acid or protein. A chimeric gene
comprising a DNA region which is functionally or structurally
defined, may comprise additional DNA regions etc.
[0055] It has unexpectedly been found by the inventors, that
introduction of a chimeric gene capable of being transcribed into
an RNA molecule with a nucleotide sequence comprising both the
sense and antisense nucleotide sequence of a target gene, or part
thereof, integrated in the nuclear genome of a plant cell, could
efficiently and specifically reduce the phenotypic expression of
that target gene. The reduction in phenotypic expression was more
efficient and more predictable than observed when separate chimeric
genes were introduced in similar cells with the target gene,
encoding either sense or antisense RNA molecules.
[0056] At the same time, it has also been found that simultaneously
introducing separate chimeric genes in one cell encoding RNA
molecules with nucleotide sequences comprising sense and antisense
respectively, resulted in extreme virus resistance, even when the
chimeric genes were transcribed from weaker promoters. It is well
known that gene silencing and virus resistance can be mediated by
similar phenomena.
[0057] In one embodiment of the invention, a method for reducing
the phenotypic expression of a nucleic acid of interest, which is
normally capable of being expressed in a eucaryotic cell,
particularly a plant cell, is provided, comprising the steps of
introducing a chimeric DNA comprising the following operably linked
parts:
[0058] a) a promoter, operative in that cell, particularly a
plant-expressible promoter;
[0059] b) a DNA region capable of being transcribed into an RNA
molecule with a nucleotide sequence comprising
[0060] i. a sense nucleotide sequence of at least 10 nt, preferably
15 nt consecutive nucleotides having between 75 and 100% sequence
identity with at least part of the nucleotide sequence of the
nucleic acid of interest; and
[0061] ii. an antisense nucleotide sequence including at least 10,
preferably 15 nt consecutive nucleotides, having between about 75%
to about 100% sequence identity with the complement of the at least
10, preferably 15 nt consecutive nucleotides of the sense
nucleotide sequence;
[0062] wherein the RNA is capable of forming a double stranded RNA
by base-pairing between the regions with sense and antisense
nucleotide sequence resulting in a hairpin RNA structure; and
[0063] c) a DNA region involved in transcription termination and
polyadenylation functioning in the suitable eucaryotic cells,
particularly functioning in plant cells.
[0064] In a preferred embodiment of the invention, the RNA molecule
transcribed from the chimeric gene, consists essentially of the
hairpin RNA.
[0065] The order of the sense and antisense nucleotide sequence in
the RNA molecule is thought not be critical.
[0066] Thus, in other words, the chimeric DNA has a transcribed DNA
region, which when transcribed, yields an RNA molecule comprising
an RNA region capable of forming an artificial stem-loop structure,
wherein one of the annealing RNA sequences of the stem-loop
structure comprises a sequence, essentially similar to at least
part of the nucleotide sequence of the nucleic acid of interest,
and wherein the second of the annealing RNA sequences comprises a
sequence essentially similar to at least part of the complement of
at least part of the nucleotide sequence of the nucleic acid of
interest.
[0067] The RNA molecule may comprise several artificial hairpin
structures, which may be designed to reduce the phenotypic
expression of different nucleic acids of interest.
[0068] In one preferred embodiment, the nucleic acid of interest,
whose phenotypic expression is targeted to be reduced, is a gene
incorporated in the genome of a eucaryotic cell, particularly a
plant cell. It will be appreciated that the means and methods of
the invention can be used for the reduction of phenotypic
expression of a gene which belongs to the genome of the cell as
naturally occurring, (an endogenous gene), as well as for the
reduction of phenotypic expression of a gene which does not belong
to the genome of the cell as naturally occurring, but has been
introduced in that cell (a transgene). The transgene can be
introduced stably or transiently, and can be integrated into the
nuclear genome of the cell, or be present on a replicating vector,
such as a viral vector.
[0069] In another preferred embodiment, the nucleic acid of
interest, whose phenotypic expression is targeted to be reduced is
a viral nucleic acid, particularly a viral RNA molecule, capable of
infecting a eucaryotic cell, particularly a plant cell. In this
case, the phenotype to be reduced is the replication of the virus,
and ultimately, the disease symptoms caused by the infecting
virus.
[0070] Preferably, the nucleotide sequence of the target nucleic
acid corresponding to the sense nucleotide sequence is part of a
DNA region which is transcribed, particularly a DNA region which is
transcribed and translated (in other words a coding region). It is
particularly preferred that the target sequence corresponds to one
or more consecutive exons, more particularly is located within a
single exon of a coding region.
[0071] The length of the sense nucleotide sequence may vary from
about 10 nucleotides (nt) up to a length equaling the length (in
nucleotides) of the target nucleic acid. Preferably the total
length of the sense nucleotide sequence is at least 10 nt,
preferably 15 nt, particularly at least about 50 nt, more
particularly at least about 100 nt, especially at least about 150
nt, more especially at least about 200 nt, quite especially at
least about 550 nt. It is expected that there is no upper limit to
the total length of the sense nucleotide sequence, other than the
total length of the target nucleic acid. However for practical
reason (such as e.g. stability of the chimeric genes) it is
expected that the length of the sense nucleotide sequence should
not exceed 5000 nt, particularly should not exceed 2500 nt and
could be limited to about 1000 nt.
[0072] It will be appreciated that the longer the total length of
the sense nucleotide sequence is, the less stringent the
requirements for sequence identity between the total sense
nucleotide sequence and the corresponding sequence in the target
gene become. Preferably, the total sense nucleotide sequence should
have a sequence identity of at least about 75% with the
corresponding target sequence, particularly at least about 80%,
more particularly at least about 85%, quite particularly about 90%,
especially about 95%, more especially about 100%, quite especially
be identical to the corresponding part of the target nucleic acid.
However, it is preferred that the sense nucleotide sequence always
includes a sequence of about 10 consecutive nucleotides,
particularly about 20 nt, more particularly about 50 nt, especially
about 100 nt, quite especially about 150 nt with 100% sequence
identity to the corresponding part of the target nucleic acid.
Preferably, for calculating the sequence identity and designing the
corresponding sense sequence, the number of gaps should be
minimized, particularly for the shorter sense sequences.
[0073] The length of the antisense nucleotide sequence is largely
determined by the length of the sense nucleotide sequence, and will
preferably correspond to the length of the latter sequence.
However, it is possible to use an antisense sequence which differs
in length by about 10%. Similarly, the nucleotide sequence of the
antisense region is largely determined by the nucleotide sequence
of the sense region, and preferably is identical to the complement
of the nucleotide sequence of the sense region. Particularly with
longer antisense regions, it is however possible to use antisense
sequences with lower sequence identity to the complement of the
sense nucleotide sequence, preferably with at least about 75%
sequence identity, more preferably with at least about 80%,
particularly with at least about 85%, more particularly with at
least about 90% sequence identity, especially with at least about
95% sequence to the complement of the sense nucleotide sequence.
Nevertheless, it is preferred that the antisense nucleotide
sequences always includes a sequence of about 10, preferably 15
consecutive nucleotides, particularly about 20 nt, more
particularly about 50 nt, especially about 100 nt, quite especially
about 150 nt with 100% sequence identity to the complement of a
corresponding part of the sense nucleotide sequence. It is clear
that the length of the stretch of the consecutive nucleotides with
100% sequence identity to the complement of the sense nucleotide
sequence cannot be longer than the sense nucleotide sequence
itself. Again, preferably the number of gaps should be minimized,
particularly for the shorter antisense sequences. Further, it is
also preferred that the antisense sequence has between about 75% to
100% sequence identity with the complement of the target
sequence.
[0074] The RNA molecule resulting from the transcription of the
chimeric DNA may comprise a spacer nucleotide sequence located
between the sense and antisense nucleotide sequence. In the absence
of such a spacer sequence, the RNA molecule will still be able to
form a double-stranded RNA, particularly if the sense and antisense
nucleotide sequence are larger than about 10 nucleotides and part
of the sense and/or antisense nucleotide sequence will be used to
form the loop allowing the base-pairing between the regions with
sense and antisense nucleotide sequence and formation of a double
stranded RNA. It is expected that there are no length limits or
sequence requirements associated with the spacer region, as long as
these parameters do not interfere with the capability of the RNA
regions with the sense and antisense nucleotide sequence to form a
double stranded RNA. In a preferred embodiment, the spacer region
varies in length from 4 to about 200 bp, but as previously
mentioned, it may be absent.
[0075] In a preferred embodiment, the hairpin RNA formed by the
sense and antisense region and if appropriate the spacer region, is
an artificial hairpin RNA. By "artificial hairpin RNA" or
"artificial stem-loop RNA structure", is meant that such hairpin
RNA is not naturally occurring in nature, because the sense and
antisense regions as defined are not naturally occurring
simultaneously in one RNA molecule, or the sense and antisense
regions are separated by a spacer region which is heterologous with
respect to the target gene, particularly, the nucleotide sequence
of the spacer has a sequence identity of less than 75% with the
nucleotide sequence of the target sequence, at the corresponding
location 5' or 3' of the endpoints of the sense nucleotide
sequence. A hairpin RNA can also be indicated as artificial, if it
is not comprised within the RNA molecule it is normally associated
with. It is conceivable to use in accordance with the invention a
chimeric DNA whose transcription results in a hairpin RNA structure
with a naturally occurring nucleotide sequence (which otherwise
meets the limits as set forth in this specification) provided this
hairpin RNA is devoid of the surrounding RNA sequences (not
involved in the hairpin structure formation).
[0076] Although it is preferred that the RNA molecule comprising
the hairpin RNA does not further comprise an intron sequence, it is
clear that the chimeric DNA genes encoding such RNAs may comprise
in their transcribed region one or more introns.
[0077] In fact, the inventors have unexpectedly found that
inclusion of an intron sequence in the chimeric DNA genes encoding
an RNA molecule comprising the hairpin RNA, preferably in the
spacer region, and preferably in sense orientation, enhances the
efficiency of reduction of expression of the target nucleic acid.
The enhancement in efficiency may be expressed as an increase in
the frequency of plants wherein silencing occurs or as an increase
in the level of reduction of the phenotypic trait. In a
particularly preferred embodiment, the intron is essentially
identical in sequence to the Flaveria trinervia pyruvate
orthophosphate dikinase 2 intron 2, more particularly, it comprises
the sequence of SEQ ID No 7.
[0078] It has been observed that contrary to methods using either
antisense or sense nucleotide sequences alone to reduce the
phenotypic expression of a target nucleic acid (which generally
depend on the dosage of sense or antisense molecule, and thus the
chimeric genes encoding the sense and antisense molecules need to
be under the control of relatively strong promoters) the method of
the current invention does not rely on the presence of such strong
promoter regions to drive the transcriptional production of the RNA
comprising both the sense and antisense region. In other words, a
whole range of promoters, particularly plant expressible promoters,
is available to direct the transcription of the chimeric genes of
the invention. These include, but are not limited to strong
promoters such as CaMV35S promoters (e.g., Harpster et al., 1988).
In the light of the existence of variant forms of the CaMV35S
promoter, as known by the skilled artisan, the object of the
invention can equally be achieved by employing these alternative
CaMV35S promoters and variants. It is also clear that other
plant-expressible promoters, particularly constitutive promoters,
such as the opine synthase promoters of the Agrobacterium Ti- or
Ri-plasmids, particularly a nopaline synthase promoter, or
subterranean clover virus promoters can be used to obtain similar
effects. Also contemplated by the invention are chimeric genes to
reduce the phenotypic expression of a nucleic acid in a cell, which
are under the control of single subunit bacteriophage RNA
polymerase specific promoters, such as a T7 or a T3 specific
promoter, provided that the host cells also comprise the
corresponding RNA polymerase in an active form.
[0079] It is a further object of the invention, to provide methods
for reducing the phenotypic expression of a nucleic acid in
specific cells, particularly specific plant cells by placing the
chimeric genes of the invention under control of tissue-specific or
organ-specific promoters. Such tissue-specific or organ-specific
promoters are well known in the art and include but are not limited
to seed-specific promoters (e.g., WO89/03887), organ-primordia
specific promoters (An et al., 1996), stem-specific promoters
(Keller et al., 1988), leaf specific promoters (Hudspeth et
al.,1989), mesophyl-specific promoters (such as the light-inducible
Rubisco promoters), root-specific promoters (Keller et al., 1989),
tuber-specific promoters (Keil et al., 1989), vascular tissue
specific promoters (Peleman et al., 1989 ), stamen-selective
promoters (WO 89/10396, WO 92/13956), dehiscence zone specific
promoters (WO 97/13865) and the like.
[0080] In another embodiment of the invention, the expression of a
chimeric gene to reduce the phenotypic expression of a target
nucleic acid can be controlled at will by the application of an
appropriate chemical inducer, by operably linking the transcribed
DNA region of the chimeric genes of the invention to a promoter
whose expression is induced by a chemical compound, such as the
promoter of the gene disclosed in European Patent publication
("EP") 0332104, or the promoter of the gene disclosed in WO
90/08826.
[0081] It has been found that a similar reduction in phenotypic
expression of a nucleic acid of interest in a eucaryotic cell,
particularly in a plant cell, can be obtained by providing the
sense and antisense RNA encoding transcribable DNA regions as
separate transgenes, preferably located in one locus, particularly
as one allele.
[0082] Thus, in another embodiment of the invention a method for
reducing the phenotypic expression of a nucleic acid which is
normally capable of being expressed in a eucaryotic cell,
particularly a plant cell, is provided, comprising the steps of
introducing a first and second chimeric DNA, linked on one
recombinant DNA such that both chimeric DNAs are integrated
together in the nuclear genome of the transgenic cells;
[0083] wherein the first chimeric DNA comprises the following
operably linked parts:
[0084] a) a promoter, operative in the cell, particularly a
plant-expressible promoter;
[0085] b) a DNA region capable of being transcribed into a sense
RNA molecule with a nucleotide sequence comprising a sense
nucleotide sequence of at least 10, preferably 15 consecutive
nucleotides having between 75 and 100% sequence identity with at
least part of the nucleotide sequence of the nucleic acid of
interest; and
[0086] c) a DNA region involved in transcription termination and
polyadenylation functioning in the corresponding eucaryotic cell;
and
[0087] wherein the second chimeric DNA comprises the following
operably linked parts:
[0088] a) a promoter, operative in the cell, particularly a
plant-expressible promoter;
[0089] b) a DNA region capable of being transcribed into an
antisense RNA molecule with a nucleotide sequence comprising an
antisense nucleotide sequence including at least 10, preferably 15
consecutive nucleotides, having between about 75% to about 100%
sequence identity with the complement of the at least 10,
preferably 15 consecutive nucleotides of the sense nucleotide
sequence;
[0090] c) a DNA region involved in transcription termination and
polyadenylation functioning in the corresponding eucaryotic
cell;
[0091] wherein the sense and antisense RNA are capable of forming a
double stranded RNA by base-pairing between the regions which are
complementary.
[0092] Preferred embodiments for the different structural and
functional characteristics, such as length and sequence of sense,
antisense and spacer regions, of this method are as described
elsewhere in this specification.
[0093] The RNA molecule, comprising the sense and antisense
nucleotide sequences capable of forming a hairpin structure, which
are produced by the transcription of the chimeric genes, can also
be introduced directly in a plant cell to reduce the phenotypic
expression of the target nucleic acid, particularly to reduce the
phenotypic expression of a targeted endogenous gene, or a targeted
transgene.
[0094] Such RNA molecules could be produced e.g. by
[0095] 1. cloning the DNA region capable of being transcribed into
an RNA molecule with a nucleotide sequence comprising a sense
nucleotide sequence of at least 10 consecutive nucleotides having
between 75 and 100% sequence identity with at least part of the
nucleotide sequence of the nucleic acid of interest and an
antisense nucleotide sequence including at least 10 consecutive
nucleotides, having between about 75% to about 100% sequence
identity with the complement of the at least 10 consecutive
nucleotides of the sense nucleotide sequence, whereby the RNA is
capable of forming a double stranded RNA by base-pairing between
the regions with sense and antisense nucleotide sequence resulting
in a hairpin RNA structure, under control of a promoter suitable
for recognition by a DNA-dependent RNA polymerase in an in vitro
transcription reaction, such as but not limited to a T7-polymerase
specific promoter;
[0096] 2. performing an in vitro transcription reaction by adding
inter alia the suitable DNA -dependent RNA polymerase as well as
the required reagents to generate the RNA molecules; and
[0097] 3. isolating the RNA molecules.
[0098] In vitro transcription methods as well as other methods for
in vitro RNA production are well known in the art and commercial
kits are available. Methods for direct introduction of RNA in plant
cells are also available to the skilled person and include but are
not limited to electroporation, microinjection and the like.
[0099] The chimeric gene(s) for reduction of the phenotypic
expression of a target nucleic acid of interest in a cell, may be
introduced either transiently, or may be stably integrated in the
nuclear genome of the cell. In one embodiment, the chimeric gene is
located on a viral vector, which is capable of replicating in the
eucaryotic cell, particularly the plant cell (see e.g., WO 95/34668
and WO 93/03161).
[0100] The recombinant DNA comprising the chimeric gene to reduce
the phenotypic expression of a nucleic acid of interest in a host
cell, may be accompanied by a chimeric marker gene, particularly
when the stable integration of the transgene in the genome of the
host cell is envisioned. The chimeric marker gene can comprise a
marker DNA that is operably linked at its 5' end to a promoter,
functioning in the host cell of interest, particularly a
plant-expressible promoter, preferably a constitutive promoter,
such as the CaMV 35S promoter, or a light inducible promoter such
as the promoter of the gene encoding the small subunit of Rubisco;
and operably linked at its 3' end to suitable plant transcription
3' end formation and polyadenylation signals. It is expected that
the choice of the marker DNA is not critical, and any suitable
marker DNA can be used. For example, a marker DNA can encode a
protein that provides a distinguishable color to the transformed
plant cell, such as the A1 gene (Meyer et al., 1987), can provide
herbicide resistance to the transformed plant cell, such as the bar
gene, encoding resistance to phosphinothricin (EP 0,242,246), or
can provide antibiotic resistance to the transformed cells, such as
the aac(6') gene, encoding resistance to gentamycin
(WO94/01560).
[0101] A recombinant DNA comprising a chimeric gene to reduce the
phenotypic expression of a gene of interest, can be stably
incorporated in the nuclear genome of a cell of a plant. Gene
transfer can be carried out with a vector that is a disarmed
Ti-plasmid, comprising a chimeric gene of the invention, and
carried by Agrobacterium. This transformation can be carried out
using the procedures described, for example, in EP 0 116 718.
[0102] Alternatively, any type of vector can be used to transform
the plant cell, applying methods such as direct gene transfer (as
described, for example, in EP 0 233 247), pollen-mediated
transformation (as described, for example, in EP 0 270 356,
W085/01856 and U.S. Pat. No. 4,684,611), plant RNA virus-mediated
transformation (as described, for example, in EP 0 067 553 and U.S.
Pat. No. 4,407,956), liposome-mediated transformation (as
described, for example, in U.S. Pat. No. 4,536,475), and the
like.
[0103] Other methods, such as microprojectile bombardment as
described for corn by Fromm et al. (1990) and Gordon-Kamm et al.
(1990), are suitable as well. Cells of monocotyledonous plants,
such as the major cereals, can also be transformed using wounded
and/or enzyme-degraded compact embryogenic tissue capable of
forming compact embryogenic callus, or wounded and/or degraded
immature embryos as described in W092/09696. The resulting
transformed plant cell can then be used to regenerate a transformed
plant in a conventional manner.
[0104] The obtained transformed plant can be used in a conventional
breeding scheme to produce more transformed plants with the same
characteristics or to introduce the chimeric gene for reduction of
the phenotypic expression of a nucleic acid of interest of 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.
[0105] It is a further object of the invention to provide
eucaryotic cells, preferably plant cells and organisms (preferably
plants) comprising the chimeric genes for the reduction of the
phenotypic expression of a target nucleic acid as described in the
invention.
[0106] It is a yet a further object of the invention to provide
plant cells, comprising a nucleic acid of interest, which is
normally capable of being expressed phenotypically, further
comprising an RNA molecule with a nucleotide sequence which
includes:
[0107] i. a sense nucleotide sequence of at least 10 consecutive
nucleotides having between 75 and 100% sequence identity with at
least part of the nucleotide sequence of the nucleic acid of
interest; and
[0108] ii. an antisense nucleotide sequence including at least 10
consecutive nucleotides, having between about 75% to about 100%
sequence identity with the complement of the at least 10
consecutive nucleotides of the sense nucleotide sequence and
capable of forming a double stranded RNA by association with the
sense nucleotide sequence;
[0109] wherein the phenotypic expression of the nucleotide acid of
interest is significantly reduced by the presence of the RNA
molecule, when compared to the phenotypic expression of the nucleic
acid of interest in the absence of the RNA molecule. The RNA
molecule may be encoded by chimeric DNA. Preferred embodiments for
the sense and antisense nucleotide sequence, particularly
concerning length and sequence, are as mentioned elsewhere in this
specification.
[0110] It will be appreciated that the methods and means described
in the specification can also be applied in High Throughput
Screening (HTS) methods, for the identification or confirmation of
phenotypes associated with the expression of a nucleic acid
sequence with hitherto unidentified function in a eucaryotic cell,
particularly in a plant cell.
[0111] Such a method comprises the steps of
[0112] 1. selecting a target sequence within the nucleic acid
sequence of interest with unidentified or non-confirmed
function/phenotype when expressed. Preferably, if the nucleic acid
has putative open reading frames, the target sequence should
comprise at least part of one of these open reading frames. The
length of the target nucleotide sequence may vary from about 10
nucleotides up to a length equaling the length (in nucleotides) of
the nucleic acid of interest with unidentified function.
[0113] 2. designing an RNA molecule comprising sense nucleotide
sequence and antisense nucleotide sequence in accordance with the
invention.
[0114] 3. introducing the RNA molecule comprising both the sense
and antisense nucleotide sequences designed on the basis of the
target sequence, into a suited host cell, particularly a plant
cell, comprising the nucleic acid with the nucleotide sequence with
hitherto unidentified phenotype. The RNA can either be introduced
directly, or can be introduced by means of a chimeric DNA
comprising a promoter operative in the host cell of interest,
particularly a plant-expressible promoter, and a DNA region
functioning as a suitable 3' end formation and polyadenylation
signal (terminator) functioning in the host cell, with in-between a
DNA region which can be transcribed to yield the RNA molecule
comprising the sense and antisense nucleotide sequence. The
chimeric DNA can either be introduced transiently or integrated in
the nuclear genome. The chimeric DNA can also be provided on a
viral vector (see, e.g., WO 95/34668 and WO 93/03161)
[0115] 4. observing the phenotype by a suitable method. Depending
on the phenotype expected, it may be sufficient to observe or
measure the phenotype in a single cell, but it may also be required
to culture the cells to obtain an (organized) multicellular level,
or even to regenerate a transgenic organism, particularly a
transgenic plant.
[0116] In its most straightforward embodiment, the RNA molecule
comprising both the sense and antisense nucleotide sequences to at
least part of a nucleic acid of interest, suitable for the methods
of the invention, can be obtained by cloning two copies of a DNA
region with the selected target sequence in inverted repeat
orientation (preferably separated by a short DNA region which does
not contain a transcription termination signal, and encodes the
spacer sequence) under a suitable promoter. This chimeric DNA is
then either used as template DNA in an in vitro transcription
method to generate the RNA molecule, which is introduced in the
host cell, or the chimeric DNA itself is introduced in the host
cell.
[0117] The methods and means of the invention can thus be used to
reduce phenotypic expression of a nucleic acid in a eucaryotic cell
or organism, particularly a plant cell or plant, for obtaining
shatter resistance (WO 97/13865), for obtaining modified flower
color patterns (EP 522 880, U.S. Pat. No. 5,231,020), for obtaining
nematode resistant plants (WO 92/21757, WO 93/10251, WO 94/17194),
for delaying fruit ripening (WO 91/16440, WO 91/05865, WO 91/16426,
WO 92/17596, WO 93/07275, WO 92/04456, U.S. Pat. No. 5,545,366),
for obtaining male sterility (WO 94/29465, W089/10396, WO
92/18625), for reducing the presence of unwanted (secondary)
metabolites in organisms, such as glucosinolates (WO97/16559) or
chlorophyll content (EP 779 364) in plants, for modifying the
profile of metabolites synthesized in a eucaryotic cell or
organisms by metabolic engineering e.g. by reducing the expression
of particular genes involved in carbohydrate metabolism (WO
92/11375, WO 92/11376, U.S. Pat. No. 5, 365, 016, WO 95/07355) or
lipid biosynthesis (WO 94/18337, U.S. Pat. No. 5, 530, 192), for
delaying senescence (WO 95/07993), for altering lignification in
plants (WO 93/05159, WO 93/05160), for altering the fibre quality
in cotton (U.S. Pat. No. 5, 597, 718), for increasing bruising
resistance in potatoes by reducing polyphenoloxidase (WO 94/03607),
etc.
[0118] The methods of the invention will lead to better results
and/or higher efficiencies when compared to the methods using
conventional sense or antisense nucleotide sequences and it is
believed that other sequence-specific mechanisms regulating the
phenotypic expression of target nucleic acids might be involved
and/or triggered by the presence of the double-stranded RNA
molecules described in this specification.
[0119] A particular application for reduction of the phenotypic
expression of a transgene in a plant cell, inter alia, by antisense
or sense methods, has been described for the restoration of male
fertility, the latter being obtained by introduction of a transgene
comprising a male sterility DNA (WO 94/09143, WO 91/02069). The
nucleic acid of interest is specifically the male sterility DNA.
Again, the processes and products described in this invention can
be applied to these methods in order to arrive at a more efficient
restoration of male fertility.
[0120] The methods and means of the invention, particularly those
involving RNA molecules comprising a hairpin RNA and the encoding
chimeric genes, have proven to be particularly suited for the
modification of the composition of oil content in plants,
particularly in seeds. Particularly preferred plants are crop
plants used for oil production such as but not limited to oilseed
rape (Brassica juncea, napus, rapa, oleracea, campestris), corn,
cotton, groundnut, sunflower, castor beans, flax, coconut, linseed,
soybean. Preferred target genes are the desaturase genes,
particularly .DELTA.12 desaturase encoding genes such as those
encoded by the Fad2 genes, especially the genes whose nucleotide
sequence can be found in the Genbank Database under accession
number AF1 23460 (from Brassica carinata), AF1 2042841 (Brassica
rapa), L26296 (Arabidopsis thaliana) or A65102 (Corylus avellana).
It is clear that it is well within the reach of the person skilled
in the art to obtain genes homologous to the disclosed fad2 genes
from other species e.g. by hybridization and/or PCR techniques.
[0121] Preferred embodiments for the configuration of sense and
antisense nucleotide sequences, particularly concerning length and
sequence are as mentioned elsewhere in this specification. Also,
preferred embodiments for chimeric genes encoding hairpin
containing RNA molecules, particularly concerning promoter elements
are as described elsewhere in the specification. For this
application, it is particularly preferred that the promoters are
seed-specific promoters.
[0122] In a preferred embodiment, the artificial hairpin RNA
comprising. RNA molecule thus comprises part of a .DELTA.12
desaturase encoding ORF in sense orientation and a similar part in
antisense orientation, preferably separated by a spacer sequence.
In a particularly preferred embodiment the artificial hairpin RNA
(or its encoding chimeric gene) comprises the nucleotide sequence
of SEQ ID No 6 or a similarly constructed nucleotide sequence based
on the aforementioned Brassica fad2 genes.
[0123] Preferably the chimeric gene encoding the artificial hairpin
RNA is transcribed under control of a seed-specific promoter,
particularly under control of the FPI promoter as described
elsewhere in this application.
[0124] A reduction of the expression of .DELTA.12 desaturase gene
in oil containing plants leads to increase in oleic acid and a
concomitant decrease in linolenic acid and linoleic acid. A higher
frequency of plants with oil wherein the increase in oleic acid and
concomitant decrease in linolenic and linoleic acid is significant
is found using the means and methods of the invention than in
transgenic plants harboring common cosuppression constructs.
Moreover the absolute levels of increase, respectively decrease are
higher respectively lower than in transgenic plants harboring
common cosuppression constructs.
[0125] Using the means and methods of the invention, it is thus
possible to obtain plants and seeds, comprising an oil of which the
composition after crushing and extracting has an increased oleic
acid content (expressed as a percentage of the total fatty acid
composition), particularly a three fold increase, when compared
with control plants.
[0126] It is expected that using the methods and means of the
invention, transgenic Brassica plant can be obtained, whose seeds
comprise an oil wherein the oleic acid content exceeds 90% of the
total fatty acid contents.
[0127] The methods and means of the invention will be especially
suited for obtaining virus resistance, particularly extreme virus
resistance, in eucaryotic cells or organisms, particularly in plant
cells and plants. A non-limiting list of viruses for plants against
which resistance can be obtained, is represented in Table I.
[0128] The methods and means of the invention further allow the use
of viral genes, hitherto unused for obtaining virus resistant
plants in addition to the commonly used coat protein genes or
replicase genes. Such different viral genes include protease
encoded genes (Pro) genome linked protein (Vpg) encoding genes,
cytoplasmic inclusion body encoding genes (Cl) as target nucleic
acid sequences for obtaining virus resistant plants.
[0129] It is clear that the invention will be especially suited for
the reduction of phenotypic expression of genes belonging to
multigene families.
[0130] It is also clear that the methods and means of the invention
are suited for the reduction of the phenotypic expression of a
nucleic acid in all plant cells of all plants, whether they are
monocotyledonous or dicotyledonous plants, particularly crop plants
such as but not limited to corn, rice, wheat, barley, sugarcane,
cotton, oilseed rape, soybean, vegetables (including chicory,
brassica vegetables, lettuce, tomato), tobacco, potato, and
sugarbeet, but also plants used in horticulture, floriculture or
forestry. The means and methods of the invention will be
particularly suited for plants which have complex genomes, such as
polyploid plants.
[0131] It is expected that the chimeric RNA molecules produced by
transcription of the chimeric genes described herein, can spread
systemically throughout a plant, and thus it is possible to reduce
the phenotypic expression of a nucleic acid in cells of a
non-transgenic scion of a plant grafted onto a transgenic stock
comprising the chimeric genes of the invention (or vice versa) a
method which may be important in horticulture, viticulture or in
fruit production.
[0132] The following non-limiting Examples describe the
construction of chimeric genes for the reduction of the phenotypic
expression of a nucleic acid of interest in a eucaryotic cell and
the use of such genes. Unless stated otherwise in the Examples, all
recombinant DNA techniques are carried out according to standard
protocols as described in Sambrook et al. (1989) Molecular Cloning:
A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current
Protocols in Molecular Biology, Current Protocols, USA. 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.
[0133] Throughout the description and Examples, reference is made
to the following sequences:
[0134] SEQ ID N.degree. 1: sequence of the Potato virus Y fragment
of the Nia gene used for the construction of various sense and
antisense constructs, for obtaining virus resistance.
[0135] SEQ ID N.degree. 2: sequence of the coding region of the
Gusd CoP construct of Example 1.
[0136] SEQ ID N.degree. 3: sequence of a modified 5' untranslated
region (5'UTR) from Johnsongrass mosaic virus
[0137] SEQ ID N.degree. 4: sequence of the Subterranean clover
virus promoter No 4 with S7 enhancer.
[0138] SEQ ID N.degree. 5: sequence of the Subterranean clover
virus double enhancer promoter No 4.
[0139] SEQ ID N.degree. 6: sequence of the CoP construct for the
.DELTA.12 desaturase gene expression inhibition.
[0140] SEQ ID N.degree. 7: sequence of the Flaveria trinervia
pyruvate orthophosphate dikinase intron
[0141] The following free text is included in the sequence
listing:
[0142] <223> fragment of the NIa ORF
[0143] <223> Description of Artificial Sequence:coding region
of the Gusd CoP construct
[0144] <223> deficient Gus coding region
[0145] <223> antisense to the 5' end of the Gus coding
region
[0146] <223> Description of Artificial Sequence:5'UTR of
Johnson mosaic virus
[0147] <223> Description of Artificial Sequence:Subterannean
clover virus S4 promoter with S7 enhancer
[0148] <223> Description of Artificial Sequence: subterranean
clover virus promoter S4 with S4 enhancer
[0149] <223> Description of Artificial Sequence: coding
sequence of the desaturase CoP construct
[0150] <223> corresponding to the 5' end of the
delta12-desaturase (fad2) coding region, in sense orientation
[0151] <223> corresponding to the 5' end of the
delta12-desaturase (fad2) coding region, in anti sense
orientation
[0152] <223> Description of Artificial Sequence: intron 2 of
the Flaveria trinervia puryvate orthophosphate dikinase
[0153] The following examples are presented in order to more fully
illustrate the preferred embodiments of the invention. They should
in no way be construed, however, as limiting the broad scope of the
invention.
EXAMPLE 1
[0154] Experimental Procedures
[0155] Gene Construction
[0156] Standard gene cloning methods (Sambrook et al. 1989) were
used to make the chimeric genes. A schematic representation of the
constructs used is shown in FIGS. 1A and 1B.
[0157] The components for these constructs were:
[0158] Cauliflower mosaic virus 35S promoter from the Cabb-JI
isolate(35S) (Harpster et al., 1988 )
[0159] Octopine synthase terminator (ocs-t) (MacDonald et al.,
1991)
[0160] Subterranean clover virus promoter No 4 (S4) (WO
9606932)
[0161] Subterranean clover virus terminator No 4 (s4t) (WO
9606932)
[0162] Subterranean clover virus double enhancer promoter No 4
(S4S4) (SEQ ID N.degree. 5)
[0163] Subterranean clover virus promoter No 4 with S7 enhancer
(S7S4) (SEQ ID N.degree. 4)
[0164] Maize ubiquitin promoter (Ubi) (Christensen and Quail,
1996)
[0165] Agrobacterium tumour morphology 1 gene terminator (tm1')
(Zheng et al., 1991)
[0166] the Nia gene of an Australian strain of Potato virus Y (Nia)
(SEQ ID N.degree. 1)
[0167] a dysfunctional .beta.-glucuronidase open reading frame
encoding DNA (Gusd)(SEQ ID N.degree. 2 from nucleotide 1 to
nucleotide 1581)
[0168] a modified 5' untranslated region (5'UTR) from Johnsongrass
mosaic virus (JGMV5') (SEQ ID N.degree. 3).
[0169] This contains insertion of a NcoI site at the ATG start
codon followed by three stop codons in frame, and a PstI site (for
insertion of the intron as in constructs 4 and 5 of FIG. 1A). In
vector constructs 2 and 6 of FIG. 1A, the Gusd open reading frame
is inserted in at the NcoI site, removing the stop codons; in all
other constructs of FIG. 1A it is inserted downstream of the PstI
site.
[0170] a castor bean catalase intron (Ohta et al., 1990) as
modified by Wang et al.(1997) ("intron").
[0171] The chimeric genes were constructed by operably assembling
the different parts as schematically indicated in FIG. 1A or FIG.
1B and inserting the obtained chimeric genes in the T-DNA vectors
pART27 and pART7 vectors (Gleave, 1992) between the left T-DNA
border and the chimeric plant expressible neo gene.
[0172] The DNA encoding a dysfunctional -glucuronidase open reading
frame (GUSd) was obtained by deleting from a gus coding region the
sequence between the two EcoRV restriction sites. For the
construction of the-chimeric gene encoding the RNA molecule
comprising both sense and antisense nucleotide sequence to a
.beta.-glucuronidase gene, a sequence was added to the Gusd gene to
be allow base pairing the 5'end over 558 bases resulting in a
sequence as represented in SEQ ID N.degree. 2. This sequence was
cloned between the maize ubiquitin promoter and the tm1' terminator
and inserted in a T-DNA vector.
[0173] T-DNA vectors were constructed which comprised a first and a
second chimeric virus resistance gene, wherein the first chimeric
gene consisted of:
[0174] 1. a CaMV 35S promoter sequence, coupled to
[0175] 2. in sense orientation, the nucleotide sequence from PVY
encoding either
[0176] Vpg protein (see e.g., Genbank Accession Nr ZZ9526 from
nucleotide 1013 to nucleotide 1583), or
[0177] part of the Cl protein (see e.g., Genbank Accession Nr
M95491 from nucleotide 3688 to nucleotide 4215) or
[0178] Protease (Pro) (see e.g., EMBL Accession Nr D00441 from
nucleotide 5714 to nucleotide 7009), followed by
[0179] 3. the S4 terminator from subterranean clover mosaic virus,
as described above.
[0180] The second chimeric gene consists of
[0181] 1. a S4 promoter as described above, coupled to
[0182] 2. in anti-sense orientation, the nucleotide sequence from
PVY encoding either
[0183] Vpg protein, or
[0184] Cl protein or
[0185] Protease, followed by
[0186] 3. the octopine synthase terminator as described above.
[0187] The sense and antisense sequences within one T-DNA vector
were derived from the same PVY coding region.
[0188] Also, T-DNA vectors were constructed for use in altering the
fatty acid composition in oil (see FIG. 2), comprising
[0189] 1. a FP1 promoter (truncated seed specific napin promoter,
containing sequences between -309 and +1, as described in Stalberg
et al; linked to
[0190] 2. the nucleotide sequence of SEQ ID No 6, comprising the
480 bp located 5' in the ORF encoding the .DELTA.12 desaturase from
Arabidopsis thaliana (Fad2) in sense orientation and in antisense
orientation, linked by a 623 bp spacer sequence; followed by
[0191] 3. the terminator from the nopaline synthase gene.
[0192] In addition, T-DNA vectors were constructed to evaluate the
influence of a presence of an intron sequence in the chimeric genes
encoding CoP constructs. To this end, constructs were made
comprising:
[0193] 1. a CamV35S promoter, followed by
[0194] 2. the protease encoding ORF from PVY (see above) in sense
orientation;
[0195] 3. the sequence of SEQ ID No 7 (encoding the Flaveria
trinervia pyruvate orthophospate dikinase intron 2)
[0196] 4. the protease encoding ORF from PVY in antisense
orientation; and
[0197] 5. the octopine synthase gene terminator.
[0198] Plant Transformation
[0199] Nicotiana tabacum (W38) tissue was transformed and
regenerated into whole plants essentially as described by Landsman
et al. (1988). Rice (Oryza sativa) was transformed essentially as
described by Wang et al. (1997).
[0200] Rice Supertransformation
[0201] Mature embryos from a rice plant expressing GUS and
hygromycin phosphotransferase (HPT) activity were excised from
mature seed and placed on callus inducing media for 7 weeks. Calli
were recovered from these cultures, incubated with Agrobacteria
containing various binary vector constructs for 2 days, then placed
on callusing media containing hygromycin, bialaphos and
Timentin.TM.. During the next four weeks hygromycin and bialaphos
resistant calli developed. These callus lines were maintained on
hygromycin and bialaphos containing media for a further 2 months
before being assayed for GUS activity.
[0202] GUS Assay
[0203] Rice calli were tested for GUS activity using the
histochemical stain X-glucuronide or the fluorogenic substrate
4-methyl-umbeliferone glucuronide (MUG) essentially as described by
Jefferson et al. (1987).
EXAMPLE 1
Comparison of Chimeric Genes Comprising only Antisense, only Sense,
or Both Sense and Antisense (Complimentary Pair (CoP)) Sequence for
Reduction in Phenotypic Expression of an Integrated
.beta.-Glucuronidase Gene
[0204] Transgenic rice tissue expressing .beta.-glucuronidase (GUS)
from a single transgene (and hygromycin resistance from a hph gene)
(lines V10-28 and V10-67) was supertransformed using vectors that
contained the bar gene conferring phosphinothricin resistance and
various sense, antisense and CoP constructs (see FIG. 1A) derived
from a crippled GUS (GUSd) gene. The supertransformed tissue was
maintained on hygromycin and bialaphos selection media for 3 weeks
then analyzed for GUS activity. A crippled GUS gene was used so
that expression from this gene would not be superimposed on the
endogenous GUS activity.
[0205] The figures in Table 2 represent the rate of MU production
measured by absorption at 455 nm, with excitation at 365 nm of 1.5
.mu.g of total protein in a reaction volume of 200 .mu.l. The rate
was measured over 30 min at 37.degree. C. The reading for
non-transgenic rice calli was 0.162. The figures in bracket which
follow the description of the introduced construct refer to FIG.
1A.
[0206] The results (Table 2) showed that supertransformation with
the binary vector containing the bar gene without the GUSd gene had
no silencing effect on the endogenous GUS activity.
Supertransformation with GUSd in a sense or antisense orientation,
with or without an intron or an early stop codon, showed some
degree of reduction (in about 25% of the analyzed calli) of the
endogenous GUS activity (see last two rows in Table 2 representing
the percentage of analyzed calli with a MUG assay reading of less
than 2.000). However, supertransformation with a CoP construct gave
in about 75% to 100% of the analyzed calli, reduction of the
endogenous GUS activity. This CoP construct was designed so that
the 3' end of the mRNA produced could form a duplex with the 5' end
of the transcript to give a "pan-handle" structure.
[0207] These data show that a complimentary pair can be made using
one self-annealing transcript, that this design is much more
effective than a conventional sense or antisense construct, and
that the approach can be used to reduce the phenotypic expression
of genes present in a plant cell.
2TABLE 2 MUG assay of Supertransformed Rice Calli Vector Inverted
cassette Sense Sense + Stop Sense + stop + intron Antisense + stop
+ intron repeat (1) (2) (3) (4) (5) CoP (6) V10-28 121,0 97,45
38,43 38,88 0,290 0.565 45,58 6,637 64,16 115,5 0,572 0.316 99,28
71,60 149,2 133,0 37,2 0,351 26,17 0,224 0,955 98,46 53,94 0.210
92,21 0,321 68,32 0,502 105,5 0.701 108,8 5,290 105,6 39,35 56,73
0.733 6,432 0,9460 136,6 1,545 60,36 2,103 90,80 32,44 140,4 10,36
71,12 119,8 98,24 128,8 62,38 111,6 13,17 0.717 93,76 31,28 17,79
14,42 0,424 0.398 5,023 88,06 26,98 0.315 40,27 52,28 115,5 0.270
36,40 30,26 149,7 16,78 53,24 107,5 66,75 67,28 29,97 26,75 145,8
0.217 89,06 105,1 0,534 0.208 0,256 135,1 9,4 68,23 95,04 35,33
5,481 71,5 V10-67 318,8 93,43 0,199 31,82 1,395 0.472 109,5 73,19
0,197 58,08 152,4 0.256 30,35 128,1 0,157 56,32 67,42 0.296 40,04
1,506 128 44,62 12,11 0.452 228 140,6 130,3 0,454 0.668 0.422 23,05
1,275 196,2 17,32 23,34 0.196 241,2 0,272 12,43 73,2 76,10 0.294
118,5 0,209 140,0 20,32 130,1 0.172 11,27 42,05 90,13 107,4 0.841
0.436 110,6 117,5 157,4 0,453 66,12 0.398 19,29 118,9 0,518 87,81
136,9 0.242 121,0 21,44 0,231 0,299 67,92 115,1 155,0 116,1 0,206
50,32 77,1 190,9 43,18 12,47 170,3 106,1 0,773 31,06 0,213 108,9
73,12 0,146 11,15 1.241 29,97 19,22 4,092 50,11 169,6 80,34 76,88
117,8 22,08 159,1 91,6 67,52 7,855 92,32 69,76 27,97 0.822 V10-28
0% 21% 10% 10.5% 22% 75% V10-67 0% 37.5% 33% 29.5% 21% 100%
EXAMPLE 2
Comparison of the Efficiency of using Chimeric Genes Comprising
only Antisense Genes, only Sense Genes, or Both Genes
Simultaneously for Obtaining Virus Resistance in Transgenic
Plants
[0208] Gene constructs were made using the PVY protease encoding
sequence (SEQ ID N.degree. 1) in a sense orientation, an antisense
orientation and in a complimentary pair (CoP) orientation, where
the T-DNA comprised both the sense and antisense chimeric genes
each under control of their own promoter. In all three arrangements
the CaMV35S promoter was used. Five different versions of CoP
constructs were made in which the second promoter was either the
CaMV35S promoter, the S4 promoter, the double S4 promoter, the S7
enhanced S4 promoter, or the vascular specific roIC promoter (see
FIG. 1B).
[0209] These constructs were transformed into tobacco (via
Agrobacterium mediated DNA transfer) and approximately 25
independently transformed plants were recovered per chimeric gene
construct. The transgenic plants were transferred to soil and
maintained in the greenhouse. About 1 month after transplanting to
soil, the plants were inoculated manually with potato virus Y,
using standard application methods. Two and four weeks later the
plants were scored for virus symptoms. The results (Table 3) showed
that after 1 month, 2 on 27 plants comprising only the sense gene,
and 1 on 25 plants comprising only the antisense gene showed no
symptoms.
[0210] In contrast respectively 11 on 24 (35S-Nia/S4-antisenseNia
construct ), 7 on 25 (35S-Nia/RoIC-antisenseNia construct), 10 on
27 (35S-Nia/35S-antisenseNia), 7 on 26 (35S-Nia/S4S4-antisenseNia
construct), and 7 on 25 (35S-Nia/S7S4-antisenseNia construct)
plants which contained both the sense and antisense genes, showed
no symptoms. Plants that showed no symptoms were considered to be
showing extreme resistance to PVY. They continued to show no
symptoms for a further 2 months of monitoring (indicated as Extreme
Resistant (ER) in Table 3). Some other plants, particularly those
containing CoP constructs, showed a delay and restriction of
symptoms. They showed no symptoms 2 weeks after inoculation but
showed some minor lesions in some plants after 4 weeks. These
plants were clearly much less effected by PVY than non-transgenic
or susceptible tobaccos and were scored as resistant (indicated as
ER* in Table 3).
3TABLE 3 Resistance to PVY infection of transgenic tobacco plants
comprising either the sense chimeric PVY protease construct, the
antisense chimeric PVY protease construct, or both (different CoP
constructs). Extreme Total Resistant "Resistant" number of plants
plants transgenic Sense gene Antisense gene (ER) (ER*) plants
35S-Nia 2 2 27 35S-AntisenseNia 1 0 25 35S-Nia 35S-AntisenseNia 10
2 27 35S-Nia S4-AntisenseNia 11 2 24 35S-Nia RolC-AntisenseNia 7 3
25 35S-Nia S4S4-AntisenseNia 7 7 26 35S-Nia S7S4-AntisenseNia 7 4
25
[0211] The data show that using CoP constructs results in a much
higher frequency of transgenic plants with extreme resistance and
resistance than by using either sense or antisense constructs
alone.
[0212] Next, the copy number of the transgenes in the virus
resistant transgenic plants was determined. Therefore, DNA was
extracted from all the transgenic plants showing extreme resistance
or resistance. DNA was also extracted from five susceptible plants
for each construct. The DNA was examined for gene copy number using
Southern analysis. The data (Table 4) showed that the genomes of
some of the CoP plants showing extreme resistance, particularly the
35S-Nia/S4-AntisenseNia plants, only contained a single copy of the
gene construct.
4TABLE 4 Copy number of transgenes comprising sense chimeric PVY
protease construct, the antisense chimeric PVY protease construct,
or both (different CoP constructs) in extreme resistant, resistant
and susceptible plants. Extreme Sense Resistant "Resistant"
Susceptible gene Antisense gene plants (ER) plants (ER*) plants
35S-Nia 6 1 1/1/1/1/1 35S-AntisenseNia 4 -- 1/8/0/2/1 35S-Nia
35S-AntisenseNia 3/1/2/3/6/3/ 1/1 1/2/2/6/1 2/4/2/3 35S-Nia
S4-AntisenseNia 2/4/1/3/2/4/ 2/6 5/8/2/3 6/1/1/3/1 35S-Nia
RolC-AntisenseNia 6/7/6/7/7/7/6 2/1 2/2/2/1/2 35S-Nia
S4S4-AntisenseNia 1/2/4/5/2/2/2 1/1/2/1/1/1/1 1/1/7/1/1 35S-Nia
S7S4-AntisenseNia 2/4/12/5/2/ 3/2/1/2 1/1/3/1/1 2/7
EXAMPLE 3
Inheritance of Extreme Resistance in Plants from Example 2
[0213] Plants from Example 2 were allowed to self-fertilize and
their seeds were collected. Seeds originating from plants showing
extreme resistance and low transgene copy number for CoP constructs
35S-Nia/S4-AntisenseNia and 35S-Nia/35S-AntisenseNia, and seeds
from the sense and the antisense plants showing extreme resistance,
were germinated and grown in the glasshouse. Plants were also grown
from seed collected from two susceptible CoP lines, two susceptible
sense gene only lines and two susceptible antisense gene only
lines. Twenty plants from each line were selected for overall
uniformity of size and development stage, put into individual pots,
allowed to recover for one week, then inoculated with PVY. The
plants were scored for virus symptoms 2,4, and 7 weeks after
inoculation. The results (Table 5) showed that all eight plant
lines of 35S-Nia/S4-antisenseNia and 35S-Nia/35S-antisenseNia
containing one or two gene copies showed an about 3:1 segregation
ratio of extreme resistance : susceptible. The progeny of the
single antisense gene only line that had given extreme resistance
at T.sub.0, and the progeny of the extremely resistant sense plant
containing one gene copy, gave abnormal segregation ratios (2:18;
ER:susceptible). The progeny of the one sense plant that gave
extreme resistance and contained 6 gene copies gave a .about.3:1
ratio (ER: susceptible). All the progeny of the susceptible T.sub.0
plants showed complete susceptibility to PVY.
[0214] These data show extreme resistance from CoP constructs gives
stable expression of the resistance which is inherited in a
Mendelian way. This also indicates that, in these lines the PVY CoP
gene loci are .about.100% effective at conferring extreme
resistance whereas the transgene loci in the antisense line and one
of the two sense lines are only partially effective at conferring
extreme resistance.
5 TABLE 5 35S Sense 35S Sense Nia and S4 Nia and S4 35S Antisense
Antisense 35S Sense Antisense Nia Nia Nia Nia Copy T1 Copy T1 Copy
T1 Copy T1 N.degree. ER:S N.degree. ER:S N.degree. ER:S N.degree.
ER:S ER plant 1 1 15:5 1 16:4 6 17:3 4 2:18 2 1 12:8 2 15:5 1 2:18
3 1 14:6 2 16:4 4 1 15:5 2 16:4 Susceptible Plant 1 8 0:20 1 0:20 1
0:20 1 0:20 2 2 0:20 1 0:20 1 0:20 8 0:20
EXAMPLE 4
Extreme Virus Resistant Transgenic Tobacco with Different
Components (Sense Gene and Antisense Gene) in Different Loci within
the Transgenic Plant
[0215] PVY susceptible plants containing the sense transgene (which
contained single transgene copies; see Table 6) were crossed with
PVY susceptible plants containing the antisense transgene (which
had also been analyzed for copy number by Southern analysis; see
Table 6). Twenty of the resulting progeny per cross were propagated
in the glasshouse, then inoculated with PVY and scored for virus
infection as described in Example 2. The progeny from the crosses
(between single genes/loci containing plants) would be expected to
be in the following ratio: 1/4 sense gene alone, 1/4 antisense gene
alone, 1/4 comprising both sense and antisense genes, and 1/4
comprising no genes at all. The results (Table 4) show that, with
one exception, a proportion of the progeny from all the successful
crosses showed extreme resistance, whereas none of the progeny from
selfed sense or selfed antisense plants showed extreme resistance.
The one cross that gave no extremely resistant progeny was derived
from the parent plant Antisense 2 (As2) which contained 8 copies of
the antisense gene. All twenty progeny plants from crosses Sense 1
(S1 )(male).times.Antisense 1 (As1) (female) and Sense 3 (S3)
(female).times.Antisense 4 (As4) (male) were examined by Southern
analysis. The results showed that in both crosses, the plants that
showed extreme resistance (or in one case resistance) contained
both the sense and antisense genes, whereas plants with no
transgenes (nulls), or sense or antisense genes alone, were all
susceptible to PVY. To further confirm this absolute correlation
between the presence of a complimentary pair (sense with antisense
genes) within a plant and extreme resistance, all progeny plants
showing extreme resistance were analyzed by Southern blots. The
results showed that every extremely resistant or resistant plant
contained both sense and antisense genes.
[0216] These data show that a complimentary pair gives resistance
or extreme resistance even when the genes encoding the sense and
antisense genes are not co-located in the genome. The
"complimentary pair phenomenon" is not simply due to increased
transgene dosage as it would be expected that 1/4 of the selfed
progeny would be homozygous and thus have double the gene dosage,
yet they were susceptible.
6TABLE 6 PVY resistance of the progeny plants resulting from
crosses between susceptible transgenic tobacco plants comprising
the 35S-senseNia gene (S-lines) and susceptible transgenic tobacco
plants comprising the 35S-antisenseNia gene (As-lines). Extreme
"Resistant" Male parent Female parent Resistant plants (ER) plants
(ER*) S1 As1 8 S1 As4 6 5 S2 As4 1 3 S3 As4 3 3 S4 As1 7 1 S3 As5 1
2 S4 As2 0 S4 As4 2 0 S5 As4 9 4 S5 As5 2 3 As4 As4 0 As5 As5 0 S2
S2 0 S4 S4 0
[0217] Extreme resistant plants showed no symptoms of PVY infection
after 7 weeks. Resistant plants showed very minor lesions 7 weeks
after PVY infection. S1, S2, S3, S4 and S5 are PVY susceptible
transgenic tobacco plants comprising the 35S-senseNia gene
construct which all have one copy of the transgene integrated.
[0218] As1, As2, As4 and As5 are PVY susceptible transgenic tobacco
plants comprising the 35S-antisenseNia gene construct which have
respectively 1, 8, 2 and 1 copies of the transgene integrated.
EXAMPLE 5
Evaluation of the use of Different Viral Genes as Target Nucleic
Acid Sequences in Obtaining Extreme Virus Resistant Genes
[0219] The T-DNA vectors comprising first and second chimeric virus
resistance genes based on sequences derived from the coding region
for protease, Vpg or Cl proteins from PVY as described in this
application, were used to obtain transformed tobacco plants, which
were subsequently challenged with PVY. The results are summarized
in the following table:
7 TABLE 7 Number of immune plants/ Number of independent transgenic
plants Construct Replica 1 Replica 2 35S-Pro sense/S4-Pro antisense
11/24 7/25 35S-Vpg sense/S4-Vpg antisense 8/20 6/18 35S-Cl
sense/S4-Cl antisense 2/23 1/20
EXAMPLE 6
Intron Enhanced Silencing
[0220] The T-DNA vectors comprising the chimeric genes encoding the
CoP constructs wherein an intron (Flaveria trinervia pyruvate
orthophosphate dikinase intron 2) has been inserted in either the
sense orientation or the antisense orientation, between the sense
and antisense sequences corresponding to the protease encoding ORF
from PVY (as described elsewhere in this application) were used to
obtain transformed tobacco plants, which were subsequently
challenged with PVY. The results are summarized in the following
table:
8TABLE 8 Number of immune plants/Number of Construct independent
transgenic plants 35S-Pro(sense)-intron(sense)- 22/24
Pro(antisense)-Ocs-t 35S-Pro(sense)-intron(antisense)- 21/24
Pro(antisense)-Ocs-t
EXAMPLE 7
Modifying Oil Profile using CoP Constructs in Arabidopsis
[0221] T-DNA vectors for modifying the fatty acid composition in
oil, extracted from crushed seeds as described elsewhere in this
application were used to introduce the chimeric gene encoding the
CoP construct for reducing the expression (see FIG. 2A; SEQ ID No
6) the .DELTA.12 desaturase gene (Fad2) in Arabidopsis
thaliana.
[0222] For comparison of the efficiency, transgenic Arabidopsis
plants were generated wherein the Fad2 gene expression was reduced
by a plain cosuppression construct, comprising the FPI
seed-specific promoter coupled to the complete ORF from the
.DELTA.12 desaturase gene (Fad2) in Arabidopsis thaliana and the
nopaline synthase promoter (see FIG. 2B).
[0223] As control plants, transgenic Arabidopsis transformed by
unrelated T-DNA constructs were used.
[0224] Seeds were harvested, crushed and extracted and the
percentage of the major fatty acids in the oil was determined by
methods available in the art. The results, which are the mean of
two readings, are summarized in Table 9.
9TABLE 9 Peak Names Sample C18:1/ Name Myristic Palmitic
Palmitoleic Stearic Oleic Linoleic Linolenic 20:0 20:1 22:0 22:1
24:0 (C18:2 + C18:3) Hairpin 1.1 0.00 6.06 0.52 3.21 56.65 7.50
6.82 1.46 16.02 0.00 1.76 0.00 3.95 Hairpin 1.2 0.12 6.86 0.39 3.40
51.28 10.00 8.73 1.64 15.60 0.00 1.97 0.00 2.74 Hairpin 1.3 0.11
8.47 0.50 3.49 21.64 28.99 18.51 2.02 14.19 0.00 2.09 0.00 0.46
Hairpin 1.4 0.00 6.14 0.50 3.37 51.70 9.77 8.02 1.73 16.04 0.00
2.05 0.67 2.91 Hairpin 2.1 0.06 5.19 0.43 3.33 54.84 5.52 7.76 1.77
18.50 0.34 1.83 0.45 4.13 Hairpin 2.2 0.04 7.67 0.46 3.75 19.60
28.29 18.64 2.55 15.96 0.19 2.28 0.56 0.42 Hairpin 3.1 0.00 7.99
0.53 3.62 19.52 28.41 19.24 2.32 15.14 0.00 2.23 0.99 0.41 Hairpin
3.2 0.09 7.00 0.54 3.69 49.02 11.03 9.64 1.71 14.94 0.00 1.72 0.62
2.37 Hairpin 3.3 0.00 5.68 0.49 3.98 46.19 12.82 9.71 2.10 16.70
0.00 1.94 0.39 2.05 Hairpin 3.4 0.17 7.19 0.77 3.69 45.90 11.86
10.65 1.84 15.39 0.00 1.90 0.65 2.04 Hairpin 3.5 0.00 6.45 0.48
3.26 51.76 8.13 10.04 1.51 16.08 0.00 1.92 0.36 2.85 Hairpin 3.6
0.08 7.51 0.23 3.59 19.97 29.13 20.12 2.15 14.54 0.29 2.02 0.36
0.41 Hairpin 3.7 0.14 7.20 0.78 2.90 26.37 24.81 17.18 1.92 15.50
0.36 2.30 0.53 0.63 Hairpin 3.8 0.11 6.34 0.46 3.23 38.58 15.25
13.54 1.89 16.91 0.00 2.36 1.34 1.34 Hairpin 3.9 0.00 6.47 0.49
3.32 47.59 11.44 9.63 1.68 15.96 0.00 1.88 1.55 2.26 Hairpin 3.10
0.00 6.77 0.56 3.48 53.30 7.57 9.34 1.55 15.65 0.00 1.79 0.00 3.15
Hairpin 3.11 0.00 7.05 0.59 3.61 53.62 8.87 8.36 1.55 14.35 0.00
1.99 0.00 3.11 Hairpin 3.12 0.05 8.32 0.36 3.85 18.48 29.24 19.94
2.48 14.75 0.00 2.28 0.26 0.38 Hairpin 4.1 0.09 6.97 0.59 3.61
53.64 8.40 8.44 1.60 15.00 0.00 1.66 0.00 3.19 Hairpin 4.2 0.07
6.81 0.22 3.27 55.06 9.16 8.71 1.26 13.63 0.19 1.33 0.30 3.08
Hairpin 4.3 0.04 6.81 0.50 3.47 46.21 10.67 11.52 1.81 16.50 0.00
1.88 0.58 2.08 Hairpin 5.1 0.00 8.30 0.23 3.71 17.72 28.92 20.63
2.38 14.77 0.00 2.41 0.92 0.36 Hairpin 5.2 0.19 7.15 1.55 3.56
44.58 11.44 11.59 1.77 15.67 0.00 1.84 0.65 1.94 Hairpin 5.3 0.10
6.49 0.40 3.72 54.19 7.01 7.89 1.74 15.91 0.00 1.92 0.62 3.64
Hairpin 5.5 0.12 6.58 0.51 3.84 54.48 6.16 7.23 1.77 16.50 0.42
1.90 0.48 4.07 Hairpin 5.6 0.00 6.67 0.50 3.66 46.32 11.56 10.48
1.83 15.99 0.00 2.15 0.84 2.10 Hairpin 5.7 0.00 5.50 0.51 3.58
57.33 4.75 5.91 1.75 18.03 0.00 1.88 0.76 5.38 Hairpin 5.8 0.16
6.55 1.53 3.54 48.52 9.91 8.97 1.78 16.39 0.00 1.84 0.81 2.57
Hairpin 6.1 0.10 6.35 0.57 3.48 59.00 4.77 6.26 1.48 15.95 0.00
1.80 0.25 5.35 Hairpin 6.2 0.10 7.98 0.37 4.06 20.96 29.01 18.69
2.38 13.63 0.20 2.03 0.60 0.44 Hairpin 6.5 0.08 6.21 0.63 3.61
60.05 5.07 5.27 1.55 15.20 0.00 1.69 0.66 5.81 Columbia pBin 19
0.08 8.81 0.47 3.51 17.07 30.31 20.94 1.78 14.56 0.00 2.17 0.28
0.33 control Cosuppresion 1.1 0.08 8.16 0.62 3.71 26.16 23.77 18.15
2.06 14.65 0.17 1.89 0.57 0.62 Cosuppresion 1.2 0.00 8.49 0.53 3.65
17.90 29.93 20.36 2.34 14.25 0.00 2.33 0.23 0.36 Cosuppresion 1.3
0.07 6.65 0.40 3.42 38.34 15.25 14.16 1.91 17.19 0.31 1.94 0.35
1.30 Cosuppresion 1.4 0.00 8.22 0.57 3.82 18.27 28.82 19.63 2.56
14.83 0.00 2.46 0.83 0.38 Cosuppresion 1.5 0.00 7.51 0.52 3.84
34.59 17.90 14.64 2.18 16.27 0.00 2.02 0.54 1.06 Cosuppresion 1.6
0.07 7.44 0.47 3.16 23.97 27.32 17.29 2.03 15.52 0.18 2.22 0.33
0.54 Cosuppresion 2.1 0.07 7.46 0.43 3.00 23.91 27.21 17.79 1.84
15.27 0.30 2.14 0.58 0.53 Cosuppresion 2.2 0.00 8.19 0.55 4.22
18.59 28.31 18.80 2.77 15.51 0.00 2.46 0.55 0.39 Cosuppresion 2.3
0.00 8.71 0.47 3.48 19.21 30.06 19.49 2.03 13.78 0.00 2.15 0.63
0.39 Cosuppresion 3.1 0.06 7.57 0.50 3.83 32.24 20.00 15.66 2.06
15.65 0.34 1.85 0.23 0.90 Cosuppresion 4.1 0.00 7.29 0.43 3.55
30.26 21.17 17.06 2.01 16.08 0.00 1.92 0.25 0.79 Cosuppresion 4.2
0.08 8.02 0.53 3.62 33.04 20.04 15.68 1.80 14.72 0.00 1.88 0.58
0.92 Cosuppresion 4.3 0.07 8.35 0.54 3.85 30.02 21.72 16.78 2.01
14.25 0.00 1.92 0.49 0.78 Cosuppresion 4.4 0.06 6.98 0.53 3.62
43.38 13.24 12.77 1.74 15.37 0.30 1.67 0.33 1.67 Cosuppresion 4.5
0.13 7.84 0.52 3.76 33.76 18.16 16.21 1.89 14.96 0.35 1.85 0.57
0.98 Cosuppresion 4.6 0.11 8.18 0.32 3.58 19.72 29.19 20.26 2.04
13.92 0.29 1.84 0.55 0.40 Cosuppresion 4.7 0.11 7.88 0.39 3.75
27.40 22.85 17.44 2.08 15.29 0.00 2.04 0.76 0.68 Cosuppresion 4.8
0.13 7.56 0.41 3.46 32.27 20.50 15.45 1.90 15.47 0.00 2.02 0.83
0.90 Cosuppresion 4.9 0.09 7.46 0.29 3.75 36.11 16.96 15.74 1.92
15.38 0.31 1.74 0.25 1.10 Cosuppresion 5.1 0.10 7.68 0.34 3.88
36.00 16.77 15.38 1.90 15.44 0.32 1.82 0.36 1.12 Cosuppresion 5.2
0.08 7.56 0.25 3.58 26.10 25.11 17.79 1.96 15.03 0.30 1.72 0.54
0.61 Cosuppresion 5.3 0.08 7.38 0.20 3.56 42.24 13.33 13.32 1.76
15.19 0.16 1.61 1.18 1.59 Cosuppresion 6.1 0.08 8.04 0.50 3.68
31.37 20.29 17.17 1.84 14.31 0.00 1.76 0.95 0.84 Cosuppresion 6.2
0.00 8.50 0.51 3.91 18.59 29.33 19.66 2.46 14.75 0.00 2.28 0.00
0.38 Control c24 pGNAP- 0.07 8.30 0.10 4.78 19.68 25.91 20.56 2.97
15.29 0.31 1.79 0.24 0.42 p450
[0225] Analysis of the results indicates that transgenic plants
harboring a CoP construct (indicated as "hairpin x.x" in the table)
have a higher frequency of plants with oil wherein the increase in
oleic acid and concomitant decrease in linolenic and linoleic acid
is significant than in transgenic plants harboring cosuppression
constructs. Moreover the absolute levels of increase, respectively
decrease are higher respectively lower than in transgenic plants
harboring cosuppression constructs.
EXAMPLE 7
Modifying Oil Profile using CoP Constructs in Brassica
[0226] The T-DNA vector harboring the chimeric gene encoding the
CoP construct described in Example 6 is introduced in Brassica
oilseed rape. Seeds harvested from the transgenic Brassica sp. are
crashed and oil extracted and the composition of the fatty acids in
the oil is analyzed.
[0227] Oil from transgenic Brassica sp. harboring the CoP construct
have significantly increased oleic acid content and decreased
linoleic and linolenic acid content. A T-DNA vector harboring a
chimeric gene encoding a CoP construct similar to the one described
in Example 6, but wherein the sequence of the sense and antisense
region corresponding to the .DELTA.12 desaturase encoding ORF is
based on a homologous ORF from Brassica spp. is constructed and
introduced in Brassica oilseed rape.
[0228] The sequence of Brassica spp ORFs homologous to .DELTA.12
desaturase encoding ORF from Arabidopsis are available from Genbank
database under Accession nrs AF042841 and AF124360.
[0229] Seeds harvested from the transgenic Brassica sp. are crashed
and oil extracted and the composition of the fatty acids in the oil
is analyzed. Oil from transgenic Brassica sp. harbouring the CoP
construct have significantly increased oleic acid content and
decreased linoleic and linolenic acid content.
EXAMPLE 8
Suppression of an Endogenius Rust Resistance Gene in Flax
[0230] A CoP construct for suppression of the endogenous rust
resistance gene was made consisting of
[0231] 1. a CaMV35S promoter; operably linked to
[0232] 2. part of an endogenous rust resistance gene (n) from flax
(about 1500 bp long) in the sense orientation; ligated to
[0233] 3. a similar part of the endogenous rust resistance gene
from flax (about 1450 bp long) in antisense orientation so that a
perfect inverted repeat without spacer sequence is generated
wherein each repeat is about 1450 bp long; followed by
[0234] 4. a nos terminator.
[0235] Plain antisense constructs were made using a similar
fragment as described sub 3 above inserted between a CaMV35S
promoter and a nos terminator.
[0236] Flax plants containing the n gene (which gives resistance to
a strain of flax rust) were transformed by these CoP and antisense
constructs. If suppression occurs, the plants become susceptible to
the rust strain. If the construct has no effect, the transformed
plants remain resistant to the rust strain.
[0237] Results
10 ngc-b sense/antisense 3 suppressed out of 7 ngc-b antisense 0
suppressed out of 12
[0238] While the invention has been described and illustrated
herein by references to various specific material, procedures and
examples, it is understood that the invention is not restricted to
the particular material, combinations of material, and procedures
selected for that purpose. Numerous variations of such details can
be implied and will be appreciated by those skilled in the art.
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Sequence CWU 1
1
7 1 854 DNA Potato virus Y fragment of the NIa ORF 1 aagctttgaa
gattgatttg atgccacata acccactcaa aatttgtgac aaaacaaatg 60
gcattgccaa atttcctgag agagagttcg agctaaggca gactgggcca gctgtagaag
120 tcgacgtgaa ggacatacca gcacaggagg tggaacatga agctaaatcg
ctcatgagag 180 gcttgagaga cttcaaccca attgcccaaa cagtttgtag
gctgaaagta tctgttgaat 240 atgggacatc agagatgtac ggttttggat
ttggagcgta cataatagcg aaccaccatt 300 tgttcaggag ttataatggt
tccatggagg tacgatccat gcacggtaca ttcagggtaa 360 agaatctaca
cagtttgagc gttctgccaa ttaaaggtag ggacatcatc ctcattaaaa 420
tgccaaaaga tttccctgtc tttccacaga aattgcattt ccgagctcct acacagaacg
480 aaagaatttg tttagttgga accaactttc aggagaagta tgcatcgtcg
atcatcacag 540 aagcaagcac tacttacaat ataccaggca gcacattctg
gaagcattgg attgaaacag 600 ataatggaca ctgtggacta ccagtggtga
gcactgccga tggatgtcta gtcggaattc 660 acagtttggc aaacaatgca
cacaccacga actactactc agccttcgat gaagattttg 720 aaagcaagta
cctccgaacc aatgagcaca atgaatgggt caagtcttgg atttataatc 780
cagacacagt gttgtggggc ccgttgaaac ttaaagacag cactcctaaa gggttattta
840 aaacaacaaa gctt 854 2 2186 DNA Artificial Sequence Description
of Artificial Sequencecoding region of the Gusd CoP construct 2
atggtacgtc ctgtagaaac cccaacccgt gaaatcaaaa aactcgacgg cctgtgggca
60 ttcagtctgg atcgcgaaaa ctgtggaatt gatcagcgtt ggtgggaaag
cgcgttacaa 120 gaaagccggg caattgctgt gccaggcagt tttaacgatc
agttcgccga tgcagatatt 180 cgtaattatg cgggcaacgt ctggtatcag
cgcgaagtct ttataccgaa aggttgggca 240 ggccagcgta tcgtgctgcg
tttcgatgcg gtcactcatt acggcaaagt gtgggtcaat 300 aatcaggaag
tgatggagca tcagggcggc tatacgccat ttgaagccga tgtcacgccg 360
tatgttattg ccgggaaaag tgtacgtatc accgtttgtg tgaacaacga actgaactgg
420 cagactatcc cgccgggaat ggtgattacc gacgaaaacg gcaagaaaaa
gcagtcttac 480 ttccatgatt tctttaacta tgccggaatc catcgcagcg
taatgctcta caccacgccg 540 aacacctggg tggacgatat ctacccgctt
cgcgtcggca tccggtcagt ggcagtgaag 600 ggcgaacagt tcctgattaa
ccacaaaccg ttctacttta ctggctttgg tcgtcatgaa 660 gatgcggact
tgcgtggcaa aggattcgat aacgtgctga tggtgcacga ccacgcatta 720
atggactgga ttggggccaa ctcctaccgt acctcgcatt acccttacgc tgaagagatg
780 ctcgactggg cagatgaaca tggcatcgtg gtgattgatg aaactgctgc
tgtcggcttt 840 aacctctctt taggcattgg tttcgaagcg ggcaacaagc
cgaaagaact gtacagcgaa 900 gaggcagtca acggggaaac tcagcaagcg
cacttacagg cgattaaaga gctgatagcg 960 cgtgacaaaa accacccaag
cgtggtgatg tggagtattg ccaacgaacc ggatacccgt 1020 ccgcaaggtg
cacgggaata tttcgcgcca ctggcggaag caacgcgtaa actcgacccg 1080
acgcgtccga tcacctgcgt caatgtaatg ttctgcgacg ctcacaccga taccatcagc
1140 gatctctttg atgtgctgtg cctgaaccgt tattacggat ggtatgtcca
aagcggcgat 1200 ttggaaacgg cagagaaggt actggaaaaa gaacttctgg
cctggcagga gaaactgcat 1260 cagccgatta tcatcaccga atacggcgtg
gatacgttag ccgggctgca ctcaatgtac 1320 accgacatgt ggagtgaaga
gtatcagtgt gcatggctgg atatgtatca ccgcgtcttt 1380 gatcgcgtca
gcgccgtcgt cggtgaacag gtatggaatt tcgccgattt tgcgacctcg 1440
caaggcatat tgcgcgttgg cggtaacaag aaagggatct tcactcgcga ccgcaaaccg
1500 aagtcggcgg cttttctgct gcaaaaacgc tggactggca tgaacttcgg
tgaaaaaccg 1560 cagcagggag gcaaacaatg aaacagacgc gtggttacag
tcttgcgcga catgcgtcac 1620 cacggtgata tcgtccaccc aggtgttcgg
cgtggtgtag agcatacgct gcgatggatt 1680 ccggcatagt taaagaaatc
atggaagtaa gactgctttt tcttgccgtt ttcgtcggta 1740 atcaccattc
ccggcgggat agtctgccag ttcagttcgt tgttcacaca aacggtgata 1800
cgtacacttt tcccggcaat aacatacggc gtgacatcgg cttcaaatgg cgtatagccg
1860 ccctgatgct ccatcacttc ctgattattg acccacactt tgccgtaatg
agtgaccgca 1920 tcgaaacgca gcacgatacg ctggcctgcc caacctttcg
gtataaagac ttcgcgctga 1980 taccagacgt tgcccgcata attacgaata
tctgcatcgg cgaactgatc gttaaaactg 2040 cctggcacag caattgcccg
gctttcttgt aacgcgcttt cccaccaacg ctgatcaatt 2100 ccacagtttt
cgcgatccag actgaatgcc cacaggccgt cgagtttttt gatttcacgg 2160
gttggggttt ctacaggacg taccat 2186 3 208 DNA Artificial Sequence
Description of Artificial Sequence5'UTR of Johnson mosaic virus 3
cgccccgggc ccaacacaac acaacagaac ctacgtcaat tgattttatc aatcgcaaag
60 ccttacaaag atcttcgcag tcgttcatca acagattcac cgaaccattc
ttgttagctc 120 tcgcacagag ataagcagga aaccatggca ggtgagtgga
acacagtttg atagtaagag 180 aaaccagagg aagactgcag gtacccgc 208 4 1150
DNA Artificial Sequence Description of Artificial
SequenceSubterannean clover virus S4 promoter with S7 enhancer 4
aatctgcagc ggccgcttaa tagtaattat gattaattat gagataagag ttgttattat
60 gcttatgagg aataaagaat gattaatatt gtttaatttt attccgcgaa
gcggtgtgtt 120 atgtttttgt tggagacatc acgtgactct cacgtgatgt
ctccgcgaca ggctggcacg 180 gggcttagta ttaccccgtg ccggatcaga
gacatttgac taaatattga cttggaataa 240 tagcccttgg attagatgac
acgtggacgc tcaggatctg tgatgctagt gaagcgctta 300 agctgaacga
atctgacgga agagcggaca tacgcacatg gattatggcc cacatgtcta 360
aagtgtatct ctttacagct atattgatgt gacgtaagat gctttacttc gcttcgaagt
420 aaagtaggaa attgctcgct aagttattct tttctgaaag aaattattta
attctaatta 480 aattaaatga gtcgctataa atagtgtcga tgctgcctca
catcgtattc ttcttcgcat 540 cgtctgttct ggttttaagc gggatccagg
cctcgagata tcggtacctt gttattatca 600 ataaaagaat ttttattgtt
attgtgttat ttggtaattt atgcttataa gtaattctat 660 gattaattgt
gaattattaa gactaatgag gataataatt gaatttgatt aaattaactc 720
tgcgaagcta tatgtctttc acgtgagagt cacgtgatgt ctccgcgaca ggctggcacg
780 gggcttagta ttaccccgtg ccgggatcag agacatttga ctaaatgttg
acttggaata 840 atagcccttg gattagatga cacgtggacg ctcaggatct
gtgatgctag tgaagcgctt 900 aagctgaacg aatctgacgg aagagcggac
aaacgcacat ggactatggc ccactgcttt 960 attaaagaag tgaatgacag
ctgtctttgc ttcaagacga agtaaagaat agtggaaaac 1020 gcgtaaagaa
taagcgtact cagtacgctt cgtggcttta tataaatagt gcttcgtctt 1080
attcttcgtt gtatcatcaa cgaagaagtt aagctttgtt ctgcgtttta atgatcgatg
1140 gccagtcgac 1150 5 1052 DNA Artificial Sequence Description of
Artificial Sequence subterranean clover virus promoter S4 with S4
enhancer 5 ggatccaggc ctcgagatat cggtaccttg ttattatcaa taaaagaatt
tttattgtta 60 ttgtgttatt tggtaattta tgcttataag taattctatg
attaattgtg aattattaag 120 actaatgagg ataataattg aatttgatta
aattaactct gcgaagctat atgtctttca 180 cgtgagagtc acgtgatgtc
tccgcgacag gctggcacgg ggcttagtat taccccgtgc 240 cgggatcaga
gacatttgac taaatgttga cttggaataa tagcccttgg attagatgac 300
acgtggacgc tcaggatctg tgatgctagt gaagcgctta agctgaacga atctgacgga
360 agagcggaca aacgcacatg gactatggcc cactgcttta ttaaagaagt
gaatgacagc 420 tgtctttgct tcaagacgaa gtaaagaata gtggaaaacg
cgtggatcca ggcctcgaga 480 tatcggtacc ttgttattat caataaaaga
atttttattg ttattgtgtt atttggtaat 540 ttatgcttat aagtaattct
atgattaatt gtgaattatt aagactaatg aggataataa 600 ttgaatttga
ttaaattaac tctgcgaagc tatatgtctt tcacgtgaga gtcacgtgat 660
gtctccgcga caggctggca cggggcttag tattaccccg tgccgggatc agagacattt
720 gactaaatgt tgacttggaa taatagccct tggattagat gacacgtgga
cgctcaggat 780 ctgtgatgct agtgaagcgc ttaagctgaa cgaatctgac
ggaagagcgg acaaacgcac 840 atggactatg gcccactgct ttattaaaga
agtgaatgac agctgtcttt gcttcaagac 900 gaagtaaaga atagtggaaa
acgcgtaaag aataagcgta ctcagtacgc ttcgtggctt 960 tatataaata
gtgcttcgtc ttattcttcg ttgtatcatc aacgaagaag ttaagctttg 1020
ttctgcgttt taatgatcga tggccagtcg ac 1052 6 1583 DNA Artificial
Sequence Description of Artificial Sequence coding sequence of the
desaturase CoP construct 6 atcattatag cctcatgctt ctactacgtc
gccaccaatt acttctctct cctccctcag 60 cctctctctt acttggcttg
gccactctat tgggcctgtc aaggctgtgt cctaactggt 120 atctgggtca
tagcccacga atgcggtcac cacgcattca gcgactacca atggctggat 180
gacacagttg gtcttatctt ccattccttc ctcctcgtcc cttacttctc ctggaagtat
240 agtcatcgcc gtcaccattc caacactgga tccctcgaaa gagatgaagt
atttgtccca 300 aagcagaaat cagcaatcaa gtggtacggg aaatacctca
acaaccctct tggacgcatc 360 atgatgttaa ccgtccagtt tgtcctcggg
tggcccttgt acttagcctt taacgtctct 420 ggcagaccgt atgacgggtt
cgcttgccat ttcttcccca acgctcccat ctacaatgac 480 cgagaacgcc
tccagatata cctctctgat gcgggtattc tagccgtctg ttttggtctt 540
taccgttacg ctgctgcaca agggatggcc tcgatgatct gcctctacgg agtaccgctt
600 ctgatagtga atgcgttcct cgtcttgatc acttacttgc agcacactca
tccctcgttg 660 cctcactacg attcatcaga gtgggactgg ctcaggggag
ctttggctac cgtagacaga 720 gactacggaa tcttgaacaa ggtgttccac
aacattacag acacacacgt ggctcatcac 780 ctgttctcga caatgccgca
ttataacgca atggaagcta caaaggcgat aaagccaatt 840 ctgggagact
attaccagtt cgatggaaca ccgtggtatg tagcgatgta tagggaggca 900
aaggagtgta tctatgtaga accggacagg gaaggtgaca agaaaggtgt gtactggtac
960 aacaataagt tatgagcatg atggtgaaga aattgtcgac ctttctcttg
tctgtttgtc 1020 ttttgttaaa gaagctatgc ttcgttttaa taatcttatt
gtccattttg ttgtgttatg 1080 acattttggc tgctcattat gttcagtaac
atctaccctc gcaacccctt ctttaccgtt 1140 cgcttgggca gtatgccaga
cggtctctgc aatttccgat tcatgttccc ggtgggctcc 1200 tgtttgacct
gccaattgta gtactacgca ggttctccca acaactccat aaagggcatg 1260
gtgaactaac gactaaagac gaaaccctgt ttatgaagta gagaaagctc cctaggtcac
1320 aaccttacca ctgccgctac tgatatgaag gtcctcttca ttccctgctc
ctccttcctt 1380 accttctatt ctggttgaca cagtaggtcg gtaaccatca
gcgacttacg caccactggc 1440 gtaagcaccc gatactgggt ctatggtcaa
tcctgtgtcg gaactgtccg ggttatctca 1500 ccggttcggt tcattctctc
tccgactccc tcctctctct tcattaacca ccgctgcatc 1560 atcttcgtac
tccgatatta cta 1583 7 786 DNA Artificial Sequence Description of
Artificial Sequence intron 2 of the Flaveria trinervia puryvate
orthophosphate dikinase 7 aagcttggta aggaaataat tattttcttt
tttcctttta gtataaaata gttaagtgat 60 gttaattagt atgattataa
taatatagtt gttataattg tgaaaaaata atttataaat 120 atattgttta
cataaacaac atagtaatgt aaaaaaatat gacaagtgat gtgtaagacg 180
aagaagataa aagttgagag taagtatatt atttttaatg aatttgatcg aacatgtaag
240 atgatatact agcattaata tttgttttaa tcataatagt aattctagct
ggtttgatga 300 attaaatatc aatgataaaa tactatagta aaaataagaa
taaataaatt aaaataatat 360 ttttttatga ttaatagttt attatataat
taaatatcta taccattact aaatatttta 420 gtttaaaagt taataaatat
tttgttagaa attccaatct gcttgtaatt tatcaataaa 480 caaaatatta
aataacaagc taaagtaaca aataatatca aactaataga aacagtaatc 540
taatgtaaca aaacataatc taatgctaat ataacaaagc gcaagatcta tcattttata
600 tagtattatt ttcaatcaac attcttatta atttctaaat aatacttgta
gttttattaa 660 cttctaaatg gattgactat taattaaatg aattagtcga
acatgaataa acaaggtaac 720 atgatagatc atgtcattgt gttatcattg
atcttacatt tggattgatt acagttggga 780 aagctt 786
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