U.S. patent application number 12/669659 was filed with the patent office on 2010-11-11 for modification of lignin biosynthesis via sense suppression.
This patent application is currently assigned to DAIRY AUSTRALIA LIMITED. Invention is credited to Megan Elizabeth Griffith, Robyn Louise Heath, Ulrik Peter John, Angela Jane Lidgett, Damian Paul Lynch, Russell Leigh McInnes, Aidyn Mouradov, German Spangenberg.
Application Number | 20100287660 12/669659 |
Document ID | / |
Family ID | 40262645 |
Filed Date | 2010-11-11 |
United States Patent
Application |
20100287660 |
Kind Code |
A1 |
Spangenberg; German ; et
al. |
November 11, 2010 |
Modification of Lignin Biosynthesis Via Sense Suppression
Abstract
The present invention relates to the modification of lignin
biosynthesis in plants, to enzymes involved in the lignin
biosynthetic pathway and nucleic acids encoding such enzymes and,
more particularly, to methods of modifying lignin biosynthesis via
sense suppression and to related nucleic acids and constructs.
Inventors: |
Spangenberg; German;
(Bundoora, AU) ; Lidgett; Angela Jane; (Kew,
AU) ; Heath; Robyn Louise; (Clifton Hill, AU)
; McInnes; Russell Leigh; (Bundoora, AU) ; Lynch;
Damian Paul; (Northcote, AU) ; John; Ulrik Peter;
(Westgarth, AU) ; Mouradov; Aidyn; (Mill Park,
AU) ; Griffith; Megan Elizabeth; (Templestowe,
AU) |
Correspondence
Address: |
Larson & Anderson, LLC
P.O. BOX 4928
DILLON
CO
80435
US
|
Assignee: |
DAIRY AUSTRALIA LIMITED
Southbank, Victoria
AU
MOLECULAR PLANT BREEDING NOMINEES LTD.
Bundoora, Victoria
AU
|
Family ID: |
40262645 |
Appl. No.: |
12/669659 |
Filed: |
July 17, 2008 |
PCT Filed: |
July 17, 2008 |
PCT NO: |
PCT/AU08/01034 |
371 Date: |
July 23, 2010 |
Current U.S.
Class: |
800/278 ;
435/320.1; 435/419; 536/24.5; 800/298 |
Current CPC
Class: |
C12N 15/8255 20130101;
C07H 21/00 20130101; C12N 9/0008 20130101; C12N 9/1007 20130101;
C12N 9/0077 20130101; C12N 15/8251 20130101; C12N 9/93 20130101;
C12N 9/0006 20130101; C12N 9/0073 20130101; C12N 9/88 20130101 |
Class at
Publication: |
800/278 ;
536/24.5; 435/320.1; 435/419; 800/298 |
International
Class: |
A01H 1/00 20060101
A01H001/00; C07H 21/04 20060101 C07H021/04; C12N 15/63 20060101
C12N015/63; C12N 5/10 20060101 C12N005/10; A01H 5/00 20060101
A01H005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 19, 2007 |
AU |
2007203378 |
Claims
1-26. (canceled)
27. A substantially purified or isolated nucleic acid comprising a
fragment or variant of a gene encoding a lignin biosynthetic
enzyme, said nucleic acid being capable of modifying lignin
biosynthesis in a plant via sense suppression; wherein said
fragment or variant comprises a frame shift mutation relative to
the gene upon which the fragment or variant is based, resulting in
a loss or substantial reduction in enzymatic activity in the
encoded polypeptide; and wherein said frame shift mutation is a
mutation that inserts or deletes a number of nucleotides that is
not evenly divisible by three within a short distance of the ATG
start codon of the gene upon which the fragment or variant is
based.
28. The nucleic acid according to claim 27, wherein said lignin
biosynthetic enzyme is selected from the group consisting of 4CL,
CCR, CAD, C3H, C4H, CCoAOMT, COMT, F5H and PAL.
29. The nucleic acid according to claim 27, from a forage, turf or
bioenergy grass species.
30. The nucleic acid according to claim 29, from a Lolium, Festuca,
Cynodon, Bracharia, Paspalum, Panicum, Miscanthus, Pennisetum, or
Phalaris species.
31. The nucleic acid according to claim 27, wherein said gene
encodes 4CL and comprises a nucleotide sequence selected from the
group consisting of the Seq, ID Nos: 1, 3, 5, 21, 25, 29, 33, 37,
and 235-243; and wherein said nucleic acid is capable of modifying
lignin biosynthesis in a plant via sense suppression of a gene
encoding 4CL in said plant.
32. The nucleic acid according to claim 27 wherein said gene
encodes CCR and comprises a nucleotide sequence selected from the
group consisting of sequences shown in Sequence ID No: 7, 117, 121
and 244 to 251 and wherein said nucleic acid is capable of
modifying lignin biosynthesis in a plant via sense suppression of a
gene encoding CCR in said plant.
33. The nucleic acid according to claim 27, wherein said gene
encodes CAD and comprises a nucleotide sequence selected from the
group consisting of the sequences shown in Sequence ID Nos: 9, 11,
14, 16 53, 57, 252 to 269 and 361; and wherein said nucleic acid is
capable of modifying lignin biosynthesis in a plant via sense
suppression of a gene encoding CAD in said plant.
34. The nucleic acid according to claim 27, wherein said gene
encodes C4H and comprises a nucleotide sequence selected from the
group consisting of Sequence ID Nos: 41, 45, 49, and 270-273; and
wherein said nucleic acid is capable of modifying lignin
biosynthesis in a plant via sense suppression of a gene encoding
C4H in said plant.
35. The nucleic acid according to claim 27, wherein said gene
encodes CCoAOMT and comprises a nucleotide sequence selected from
the group consisting of Sequence ID Nos: 89, 93, 274 to 294 and
362; and wherein said nucleic acid is capable of modifying lignin
biosynthesis in a plant via sense suppression of a gene encoding
CCoAOMT in said plant.
36. The nucleic acid according to claim 27, wherein said gene
encodes COMT and comprises a nucleotide sequence selected from the
group consisting of Sequence ID Nos: 133, 137, 141, 145, 295 to
342, 360, 362 and 362; and wherein said nucleic acid is capable of
modifying lignin biosynthesis in a plant via sense suppression of a
gene encoding COMT in said plant.
37. The nucleic acid according to claim 27, wherein said gene
encodes F5H and comprises a nucleotide sequence selected from the
group consisting of Sequence ID Nos: 173, 177, and 343- to 346; and
wherein said nucleic acid is capable of modifying lignin
biosynthesis in a plant via sense suppression of a gene encoding
F5H in said plant.
38. The nucleic acid according to claim 27, wherein said gene
encodes PAL and comprises a nucleotide sequence selected from the
group consisting of Sequence ID Nos: 181, 185, 189, 193, 197, 201,
205, 209, 213, 217, 220, 224 and 347 to 358; and wherein said
nucleic acid is capable of modifying lignin biosynthesis in a plant
via sense suppression of a gene encoding PAL in said plant.
39. The nucleic acid according to claim 27, wherein said gene
encodes C3H and comprises a nucleotide sequence shown in SEQ ID No:
359; and wherein said nucleic acid is capable of modifying lignin
biosynthesis in a plant via sense suppression of a gene encoding
C3H in said plant.
40. The nucleic acid according to claim 27, comprising a nucleotide
sequence selected from the group consisting of SEQ ID Nos: 23, 27,
31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91, 95,
99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147,
151, 155, 159, 163, 167, 171, 175, 179, 183, 187, 191, 195, 199,
203, 207, 211, 215, 218, 222, 225, 229 and 233.
41. The nucleic acid according to claim 27 encoding a polypeptide
comprising a sequence selected from the group consisting of SEQ ID
Nos: 24, 28, 32, 36, 40, 44, 48, 56, 60, 64, 68. 72, 76, 80, 84,
88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140,
144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192,
196, 200, 204, 208, 212, 216, 219, 223, 226, 230 and 234.
42. A genetic construct or vector comprising the nucleic acid
according to claim 27.
43. A transformed plant, plant cell, plant seed or other plant part
or transformed plant biomass comprising the nucleic acid according
to claim 27.
44. A method of modifying lignin biosynthesis in a plant, said
method including introducing into said plant in a sense orientation
an effective amount of a nucleic acid according to claim 27, such
that expression of the corresponding gene is suppressed.
45. The method according to claim 44, wherein said plant is
selected from the group consisting of Lolium, Festuca, Cynodon,
Bracharia, Paspalum, Panicum, Miscanthus, Pennisetum, Phalaris, and
other forage and turf grasses, corn, grains, oat, sugarcane, wheat,
barley, Arabidopsis, tobacco, legumes, Alfalfa, oak, Eucalyptus,
maple, Populus, canola, soybean, chickpea and Pinus.
46. The method according to claim 44, wherein said nucleic acid is
part of a genetic construct or vector.
Description
[0001] The present invention relates to the modification of lignin
biosynthesis in plants, to enzymes involved in the lignin
biosynthetic pathway and nucleic acids encoding such enzymes and,
more particularly, to methods of modifying lignin biosynthesis via
sense suppression and to related nucleic acids and constructs.
[0002] The present invention also relates to a regulatory element
and, more particularly, to a promoter capable of causing expression
of an exogenous gene in plant cells, such as a gene encoding an
enzyme involved in the lignin biosynthetic pathway in plants.
[0003] The invention also relates to vectors including the nucleic
acids and regulatory elements of the invention, plant cells,
plants, seeds and other plant parts transformed with the regulatory
elements, nucleic acids and vectors, and methods of using the
nucleic acids, regulatory elements and vectors.
[0004] Lignins are complex phenolic polymers that strengthen plant
cell walls against mechanical and chemical degradation. The process
of lignification typically occurs during secondary thickening of
the walls of cells with structural, conductive or defensive roles.
Three monolignol precursors, sinapyl, coniferyl and p-coumaryl
alcohol combine by dehydrogenative polymerisation to produce
respectively the syringyl(S), guaiacyl(G) and hydroxyl(H) subunits
of the lignin polymer, which can also become linked to cell-wall
polysaccharides through the action of peroxidases and other
oxidative isozymes. In grasses, biosynthesis of the monolignol
precursors is a multistep process beginning with the aromatic
amino-acids phenylalanine and tyrosine. It is the final two
reduction/dehydrogenation steps of the pathway, catalysed by
Cinnamoyl CoA Reductase (CCR) and Cinnamyl Alcohol Dehydrogenase
(CAD) that are considered to be specific to lignin biosynthesis.
The proportions of monolignols incorporated into the lignin polymer
vary depending on plant species, tissue, developmental stage and
sub-cellular location.
[0005] Caffeic acid O-methyl transferase (OMT), 4 coumarate
CoA-ligase (4CL), cinnamoyl-CoA reductase (CCR) and cinnamyl
alcohol dehydrogenase (CAD) are key enzymes involved in lignin
biosynthesis.
[0006] Worldwide permanent pasture is estimated to cover 70% of
agriculturally cultivated area. Ryegrasses (Lolium spp.) together
with the closely related fescues (Festuca spp.) are of significant
value in temperate grasslands. The commercially most important
ryegrasses are Italian or annual ryegrass (L. multiflorum Lam.) and
perennial ryegrass (L. perenne L.). They are the key forage species
in countries where livestock production is an intensive enterprise,
such as the Netherlands, United Kingdom and New Zealand. The
commercially most important fescues are tall fescue (F. anundinacea
Schreb.), meadow fescue (F. pratensis) and red fescue (F.
rubra).
[0007] Perennial ryegrass (Lolium perenne L.) is the major grass
species sown in temperate dairy pastures in Australia, and the key
pasture grass in temperate climates throughout the world. A marked
decline of the feeding value of grasses is observed in temperate
pastures of Australia during late spring and early summer, where
the nutritive value of perennial ryegrass based pasture is often
insufficient to meet the metabolic demands of lactating dairy
cattle. Perennial ryegrass is also an important turf grass.
[0008] Grass and legume in vitro dry matter digestibility has been
negatively correlated with lignin content. In addition, natural
mutants of lignin biosynthetic enzymes in maize, sorghum and pearl
millet that have higher rumen digestibility have been characterised
as having lower lignin content and altered S/G subunit ratio. Thus,
lignification of plant cell walls is the major factor identified as
responsible for lowering digestibility of forage tissues as they
mature.
[0009] It would be desirable to have methods of altering lignin
biosynthesis in plants, including grass species such as ryegrasses
and fescues, by reducing the activity of key biosynthetic enzymes
in order to reduce lignin content and/or alter lignin composition
for enhancing dry matter digesitibility and improving herbage
quality. However, for some applications it may be desirable to
enhance lignin biosynthesis to increase lignin content and/or alter
lignin composition, for example to increase mechanical strength of
wood, to increase mechanical strength of turf grasses, to reduce
plant height and reduce lodging or improve disease resistance.
[0010] While nucleic acid sequences encoding some of the enzymes
involved in the lignin biosynthetic pathway have been isolated for
certain species of plants, there remains a need for materials
useful in the modification of lignin biosynthesis in plants,
particularly grass species such as ryegrasses and fescues.
[0011] Other phenotypic traits which may be improved by transgenic
manipulation of plants include disease resistance, mineral content,
nutrient quality and drought tolerance.
[0012] However, transgenic manipulation of phenotypic traits in
plants requires the availability of regulatory elements capable of
causing the expression of exogenous genes in plant cells.
[0013] It is an object of the present invention to overcome, or at
least alleviate, one or more of the difficulties or deficiencies
associated with the prior art.
[0014] In one aspect, the present invention provides substantially
purified or isolated nucleic acids or nucleic acid fragments
encoding the following enzymes from a ryegrass (Lolium) or fescue
(Festuca) species: 4 coumarate CoA-ligase (4CL), cinnamoyl-CoA
reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD).
[0015] The ryegrass (Lolium) or fescue (Festuca) species may be of
any suitable type, including Italian or annual ryegrass, perennial
ryegrass, tall fescue, meadow fescue and red fescue. Preferably the
ryegrass or fescue species is a Lolium species such as Lolium
perenne or Lolium arundinaceum which is otherwise known as Festuca
arundinacea.
[0016] By `nucleic acid` is meant a chain of nucleotides capable of
carrying genetic information. The term generally refers to genes or
functionally active fragments or variants thereof and or other
sequences in the genome of the organism that influence its
phenotype. The term `nucleic acid` includes DNA (such as cDNA or
genomic DNA) and RNA (such as mRNA or microRNA) that is single- or
double-stranded, optionally containing synthetic, non-natural or
altered nucleotide bases, synthetic nucleic acids and combinations
thereof.
[0017] The nucleic acid or nucleic acid fragment may be of any
suitable type and includes DNA (such as cDNA or genomic DNA) and
RNA (such as mRNA) that is single- or double-stranded, optionally
containing synthetic, non-natural or altered nucleotide bases, and
combinations thereof.
[0018] By `substantially purified` is meant that the nucleic acid
or promoter is free of the genes, which, in the naturally-occurring
genome of the organism from which the nucleic acid or promoter of
the invention is derived, flank the nucleic acid or promoter. The
term therefore includes, for example, a nucleic acid or promoter
which is incorporated into a vector; into an autonomously
replicating plasmid or virus; or into the genomic DNA of a
prokaryote or eukaryote; or which exists as a separate molecule
(eg. a cDNA or a genomic or cDNA fragment produced by PCR or
restriction endonuclease digestion) independent of other sequences.
It also includes a nucleic acid or promoter which is part of a
hybrid gene. Preferably, the substantially purified nucleic acid or
promoter is at least approximately 90% pure, more preferably at
least approximately 95% pure, even more preferably at least
approximately 98% pure.
[0019] The term "isolated" means that the material is removed from
its original environment (eg. the natural environment if it is
naturally occurring). For example, a naturally occurring nucleic
acid present in a living plant is not isolated, but the same
nucleic acid separated from some or all of the coexisting materials
in the natural system, is isolated. Such nucleic acids could be
part of a vector and/or such nucleic acids could be part of a
composition, and still be isolated in that such a vector or
composition is not part of its natural environment.
[0020] In a preferred embodiment of this aspect of the invention,
the substantially purified or isolated nucleic acid or nucleic acid
fragment encoding 4CL includes a nucleotide sequence selected from
the group consisting of (a) sequences shown in FIGS. 2, 3 and 4
hereto (Sequence ID Nos: 1, 3 and 5; respectively) (b) complements
of the sequences shown in FIGS. 2, 3 and 4 hereto (Sequence ID Nos:
1, 3 and 5, respectively); (c) sequences antisense to the sequences
recited in (a) and (b); and (d) functionally active fragments and
variants of the sequences recited in (a), (b) and (c).
[0021] In a further preferred embodiment of this aspect of the
invention, the substantially purified or isolated nucleic acid or
nucleic acid fragment encoding CCR includes a nucleotide sequence
selected from the group consisting of (a) the sequence shown in
FIG. 10 hereto (Sequence ID No: 7); (b) the complement of the
sequence shown in FIG. 10 hereto (Sequence ID No: 7); (c) sequences
antisense to the sequences recited in (a) and (b); and (d)
functionally active fragments and variants of the sequences recited
in (a), (b) and (c).
[0022] In a still further preferred embodiment of this aspect of
the invention, the substantially purified or isolated nucleic acid
or nucleic acid fragment encoding CAD includes a nucleotide
sequence selected from the group consisting of (a) the sequences
shown in FIGS. 13, 14, 26 and 27 hereto (Sequence ID Nos: 9, 11, 14
and 16, respectively); (c) sequences antisense to the sequences
recited in (a) and (b); and (d) functionally active fragments and
variants of the sequences recited in (a), (b) and (c).
[0023] By "functionally active" is meant that the fragment or
variant (such as an analogue, derivative or mutant) is capable of
participating in or modifying lignin biosynthesis in a plant. Such
variants include naturally occurring allelic variants and
non-naturally occurring variants. Additions, deletions,
substitutions and derivatizations of one or more of the nucleotides
are contemplated so long as the modifications do not result in loss
of functional activity of the fragment or variant. Preferably the
functionally active fragment or variant has at least approximately
80% identity to the relevant part of the above mentioned sequence
to which the fragment or variant corresponds, more preferably at
least approximately 90% identity, even more preferably at least
approximately 95% identity, most preferably at least approximately
98% identity. Such functionally active variants and fragments
include, for example, those having conservative nucleic acid
changes. By `conservative nucleic acid changes` is meant nucleic
acid substitutions that result in conservation of the amino acid in
the encoded protein, due to the degeneracy of the genetic code.
Such functionally active variants and fragments also include, for
example, those having nucleic acid changes which result in
conservative amino acid substitutions of one or more residues in
the corresponding amino acid sequence. By `conservative amino acid
substitutions` is meant the substitution of an amino acid by
another one of the same class, the classes being as follows:
[0024] Nonpolar: Ala, Val, Leu, Ile, Pro, Met Phe, Trp
[0025] Uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln
[0026] Acidic: Asp, Glu
[0027] Basic: Lys, Arg, His
[0028] Other conservative amino acid substitutions may also be made
as follows:
[0029] Aromatic: Phe, Tyr, His
[0030] Proton Donor: Asn, Gln, Lys, Arg, His, Trp
[0031] Proton Acceptor: Glu, Asp, Thr, Ser, Tyr, Asn, Gln
[0032] Preferably the fragment has a size of at least 20
nucleotides, more preferably at least 50 nucleotides, more
preferably at least 100 nucleotides, more preferably at least 200
nucleotides, more preferably at least 500 nucleotides.
[0033] In a still further preferred embodiment of this aspect of
the invention the functionally active fragment or variant may be
capable of modifying lignin biosynthesis in a plant via sense
suppression.
[0034] Accordingly, the present invention provides a substantially
purified or isolated nucleic acid including a fragment or variant
of a gene encoding a lignin biosynthetic enzyme, said nucleic acid
being capable of modifying lignin biosynthesis in a plant via sense
suppression.
[0035] By "sense suppression" is meant that when the functionally
active fragment or variant is introduced into the plant in sense
orientation, it causes an identifiable decrease in expression of
the corresponding gene in the transformed plant relative to an
untransformed control plant.
[0036] By "sense" orientation is meant that the nucleic acid is in
the same orientation or has the same polarity as a messenger RNA
copy that is translated or translatable into protein.
[0037] Fragments and variants for sense suppression include those
with additions, deletions, substitutions or derivatizations of one
or more nucleotides in the nucleic acid or nucleic acid fragment
according to the present invention.
[0038] Fragments and variants for sense suppression preferably
include those with short deletions of, for example 1 to
approximately 500, 1 to approximately 300 or 1 to approximately 100
nucleotides, preferably consecutive nucleotides. In a preferred
embodiment, the short deletion may be located at or near, for
example within approximately 200, 100, 50 or 20 bases of, the 3' or
5' end of the gene upon which the fragment or variant is based.
[0039] In a preferred embodiment of this aspect of the invention,
the functionally active fragment or variant capable of modifying
lignin biosynthesis via sense suppression may be a functionally
active fragment or variant of a nucleic acid or nucleic acid
fragment encoding 4CL, CCR or CAD, for example as herein before
described, or as described in International patent applications WO
02/26994 or WO 03/40306; or a functionally active fragment or
variant of a nucleic acid or nucleic acid fragment encoding
cinnamate-4-hydroxase (C4H), caffeoyl-CoA3-O-methyltransferase
(CCoAOMT or CCoAMT), caffeic acid O-methyltransferase (OMT or
COMT), ferulate-5-hydroxylase (F5H) or phenylalanine ammonia lyase
(PAL), for example as described in International patent application
WO 02/26994 or WO 03/40306; or a functionally active fragment or
variant of cinnamate-3-hydroxylase (C3H), for example as described
in International patent application WO 2008/064289.
[0040] Preferably the functionally active fragment or variant
encodes a 4CL, CCR CAD, C3H, C4H, CCoAOMT, COMT, F5H or PAL
polypeptide without enzymatic activity or with substantially
reduced enzymatic activity.
[0041] By "substantially reduced enzymatic activity" is meant
enzymatic activity which is significantly lower, for example at
least approximately 25%, 50% or 75% lower, than the enzymatic
activity in a wild type plant.
[0042] Preferably the functionally active fragment or variant
includes a frame-shift mutation relative to the corresponding gene
upon which the fragment or variant is based. This may result in a
loss of or substantial reduction in enzymatic activity in the
encoded polypeptide.
[0043] By a "frame-shift mutation" is meant a mutation that inserts
or deletes a number of nucleotides that is not evenly divisible by
three from a nucleic acid sequence. Due to the triplet nature of
gene expression by codons, the insertion or deletion may disrupt
the reading frame, or the grouping of the nucleotides into codons,
resulting in a different translation from the original. The earlier
in the sequence the deletion or insertion occurs, the greater is
the proportion of the protein that is altered.
[0044] A frame-shift mutation may cause the reading of codons to be
different, so most codons after the mutation (with a few exceptions
due to redundancy or coincidental similarity) will code for
different amino acids than the corresponding codon in the wild type
sequence, leading to a substantially altered polypeptide sequence.
Furthermore, the stop codon "UAA, UGA, or UAG" may not be read, or
a stop codon may be created at an earlier site. The protein being
created may be abnormally short, abnormally long, and/or contain
the wrong amino acids. It is unlikely to be functional.
[0045] Deletions or additions occurring at or near the 5' end may
preferably be within a short distance, for example within
approximately 20, 50, 100 or 200 bases of the ATG start codon,
preferably within a short distance downstream of the ATG start
codon, for example within approximately 20, 50, 100 or 200 bases
downstream of the ATG start codon.
[0046] By "downstream" is meant in the 5'.fwdarw.3' direction along
the nucleic acid. Preferably, such deletions or additions occurring
at or near the 5' end may result in a frame-shift mutation, so that
the resulting polypeptide has little or no enzymatic activity.
[0047] In a particularly preferred embodiment, the deletion or
addition at or near the 5' end may be a deletion or addition of
one, two, four, five, seven or eight bases, prefer-ably consecutive
bases, within a short distance downstream of the ATG start codon,
so as to result in a frame-shift mutation, and a resulting
polypeptide with little or no enzymatic activity. More preferably
the frame-shift mutation is a deletion of one base.
[0048] Deletions occurring at or near the 3' end may preferably
start at the 3' end or within a short distance, for example
approximately 20, 50, 100 or 200 bases, of the 3' end, and extend
in a 5' direction. Preferably, such deletions have a size of
between approximately 50 to 500 nucleotides, more preferably
approximately 100 to 300 nucleotides.
[0049] In a particularly preferred embodiment of this aspect of the
invention, the functionally active fragment or variant capable of
modifying lignin biosynthesis via sense suppression may be a
functionally active fragment or variant of a nucleic acid or
nucleic acid fragment encoding CCR, 4CL or CAD, C3H, C4H, CCoAOMT,
COMT, F5H or PAL.
[0050] Accordingly, in a preferred embodiment the present invention
provides a fragment or variant of a nucleic acid selected from the
group consisting of nucleic acids with the sequences shown in FIG.
10 hereto (Sequence ID No: 7), and in FIGS. 38, 40, 41, 43 and 44
of WO 02/26994 (Sequence ID Nos: 244 to 251, respectively) and in
SEQ ID Nos: 147 and 148 of WO 03/40306 (Seq ID Nos. 117 and 121 of
this application); wherein said fragment or variant is capable of
modifying lignin biosynthesis in a plant via sense suppression of a
gene encoding CCR in said plant.
[0051] In a further preferred embodiment the present invention
provides a fragment or variant of a nucleic acid selected from the
group consisting of the nucleic acids with sequences shown in FIGS.
13, 14, 26 and 27 hereto (Sequence ID Nos: 9, 11, 14 and 16,
respectively), and in FIGS. 9, 11, 13, 15, 16, 18, 19, 21 and 22 of
WO 02/26994 (Sequence ID Nos: 252 to 269, respectively) and in SEQ
ID No: 7 of WO 2008/064289 (Seq ID No. 361 of this application) and
in SEQ ID Nos: 35 and 145 of WO 03/40306 (Seq ID Nos 53 and 57 of
this application; wherein said fragment or variant is capable of
modifying lignin biosynthesis in a plant via sense suppression of a
gene encoding CAD in said plant.
[0052] In a further preferred embodiment, the present invention
provides a fragment or variant of a nucleic acid selected from the
group consisting of the nucleic acids with sequences shown in FIGS.
2, 3 and 4 hereto (Sequence ID Nos: 1, 3 and 5, respectively) and
in FIGS. 68, 70, 71 and 73 of WO 02/26994 (Sequence ID Nos:
235-243, respectively) and in SEQ ID Nos: 29, 31, 27, 142 and 143
of WO 03/40306 (Seq ID Nos 21, 25, 33 and 37 of this application);
wherein said fragment or variant is capable of modifying lignin
biosynthesis in a plant via sense suppression of a gene encoding
4CL in said plant.
[0053] In a further preferred embodiment, the present invention
provides a fragment or variant of a nucleic acid selected from the
group consisting of the nucleic acids with sequences shown in FIGS.
32, 34, 36 and 76 of WO 02/26994 (Sequence ID Nos: 270-273,
respectively) and in SEQ ID No: 6 of WO 2008/064289 (Seq ID No. 49
of this application) and in SEQ ID Nos: 33 and 144 of WO 03/40306
(Seq ID Nos. 41 and 45 of this application); wherein said fragment
or variant is capable of modifying lignin biosynthesis in a plant
via sense suppression of a gene encoding C4H in said plant.
[0054] In a further preferred embodiment, the present invention
provides a fragment or variant of a nucleic acid selected from the
group consisting of the nucleic acids with sequences shown in FIGS.
1, 3, 4, 6, 7, 82 and 87 of WO 02/26994 (Sequence ID Nos: 274 to
294, respectively) and in SEQ ID No: 8 of WO 2008/064289 (Seq ID
No. 362 of this application) and in SEQ ID Nos: 37 and 146 of WO
03/40306 (Seq ID Nos. 89 and 93 of this application); wherein said
fragment or variant is capable of modifying lignin biosynthesis in
a plant via sense suppression of a gene encoding CCoAOMT in said
plant.
[0055] In a further preferred embodiment, the present invention
provides a fragment or variant of a nucleic acid selected from the
group consisting of the nucleic acids with sequences shown in FIGS.
24, 26, 27, 29, 30, 93 and 99 of WO 02/26994 (Sequence ID Nos: 295
to 342, respectively) and SEQ ID Nos: 2, 8 and 9 of WO 2008/064289
(Seq ID Nos. 360, 362 and 363 of this application) and in SEQ ID
Nos: 149, 42, 150 and 43 of WO 03/40306 (Seq ID Nos. 133, 137, 141
and 145 of this application); wherein said fragment or variant is
capable of modifying lignin biosynthesis in a plant via sense
suppression of a gene encoding COMT in said plant.
[0056] In a further preferred embodiment, the present invention
provides a fragment or variant of a nucleic acid selected from the
group consisting of the nucleic acids with sequences shown in FIGS.
59 and 61 of WO 02/26994 (Sequence ID Nos: 343 to 346,
respectively) and in SEQ ID Nos: 45 and 151 of WO 03/40306 (Seq ID
Nos. 173 and 177 of this application); wherein said fragment or
variant is capable of modifying lignin biosynthesis in a plant via
sense suppression of a gene encoding F5H in said plant.
[0057] In a further preferred embodiment, the present invention
provides a fragment or variant of a nucleic acid selected from the
group consisting of the nucleic acids with sequences shown in FIGS.
62, 64, 65 and 67 of WO 02/26994 (Sequence ID Nos: 347 to 358,
respectively) and in SEQ ID Nos: 152, 153, 50, 54, 48, 53, 156, 49,
51, 154, 52 and 155 of WO 03/40306 (Seq ID Nos. 181, 185, 189, 193,
197, 201, 205, 209, 213, 217, 220 and 224 of this application);
wherein said fragment or variant is capable of modifying lignin
biosynthesis in a plant via sense suppression of a gene encoding
PAL in said plant.
[0058] In a further preferred embodiment, the present invention
provides a fragment or variant of a nucleic acid selected from the
group consisting of the nucleic acids with sequences shown in SEQ
ID No: 1 of WO 2008/064289 (Seq ID No. 359 of this application)
wherein said fragment or variant is capable of modifying lignin
biosynthesis in a plant via sense suppression of a gene encoding
C3H in said plant.
[0059] Preferably, the fragment or variant includes a short
deletion at or near the 3' or 5' end of a sequence as hereinbefore
described.
[0060] Preferably, the fragment or variant includes a frame-shift
mutation relative to a sequence, as hereinbefore described.
[0061] In a particularly preferred embodiment, the fragment or
variant comprises sequence selected from the group of frame shift
DNA sequences shown in Tables 1 and 2 or encodes a polypeptide
comprising a sequence selected from the group of frame shift
protein sequences shown in Tables 1 and 2.
TABLE-US-00001 TABLE 1 DNA SEQ ID Seq SEQ ID NO. ID No of NO. DNA
PROT Species Gene name Abbrev NO. NT 4CL 29 91 Fescue 4 Coumarate
CoA ligase 2 4CL-2 21 1934 31 93 Fescue 4 Coumarate CoA ligase 3
4CL-3 25 2073 27 89 Lolium 4 Coumarate CoA ligase 1 4CL-1 29 1855
142 90 Lolium 4 Coumarate CoA ligase 2 4CL-2 33 2039 143 178 Lolium
4 Coumarate CoA ligase 3 4CL-3 37 2006 C4H 33 95 Fescue cinnamate
4-hydroxylase C4H 41 1775 144 179 Lolium cinnamate 4-hydroxylase
C4H 45 1789 Fescue cinnamate 4-hydroxylase LaC4H 49 1518 CAD3 35 97
Fescue cinnamyl alcohol CAD 53 1313 dehydrogenase 145 180 Lolium
cinnamyl alcohol CAD 57 1358 dehydrogenase CAD Fescue cinnamyl
alcohol LaCAD1a 61 1501 dehydrogenase Fescue cinnamyl alcohol
LaCAD1b 65 1339 dehydrogenase Fescue cinnamyl alcohol LaCAD2a 69
1322 dehydrogenase Fescue cinnamyl alcohol LaCAD2b 73 1526
dehydrogenase Lolium cinnamyl alcohol LpCAD1 77 1325 dehydrogenase
Lolium cinnamyl alcohol LpCAD2 81 1378 dehydrogenase Lolium
cinnamyl alcohol LpCAD3 85 1382 dehydrogenase CCoAOMT 37 99 Fescue
Caffeoyl CoA O- CCoAOMT 89 1063 methyltransferase 146 98 Lolium
Caffeoyl CoA O- CCoAOMT 93 1051 methyltransferase Lolium Caffeoyl
CoA O- LpCCoAOMT1 97 1126 methyltransferase Lolium Caffeoyl CoA O-
LpCCoAOMT2 101 1164 methyltransferase Lolium Caffeoyl CoA O-
LpCCoAOMT3 105 1088 methyltransferase Lolium Caffeoyl CoA O-
LpCCoAOMT4 109 1241 methyltransferase Lolium Caffeoyl CoA O-
LpCCoAOMT5 113 1151 methyltransferase CCR 148 101 Fescue cinnamoyl
CoA reductase CCR 117 1236 147 181 Lolium cinnamoyl CoA reductase
CCR 121 1332 Lolium cinnamoyl CoA reductase LpCCR1 125 1395 Lolium
cinnamoyl CoA reductase LpCCR2 129 1207 COMT 149 182 Fescue caffeic
acid O- COMT 133 1428 methyltransferase 42 104 Fescue caffeic acid
O- COMT-1 137 1452 methyltransferase 1 150 103 Lolium caffeic acid
O- COMT-1 141 1455 methyltransferase 1 43 105 Lolium caffeic acid
O- COMT-3 145 1440 methyltransferase 3 Fescue caffeic acid O-
LaCOMT1c 149 1438 methyltransferase 1 Fescue caffeic acid O-
LaCOMT3 153 1430 methyltransferase 1 Lolium caffeic acid O- LpOMT1
157 1542 methyltransferase 3 Lolium caffeic acid O- LpOMT2 161 1496
methyltransferase 3 Lolium caffeic acid O- LpOMT3 165 1505
methyltransferase 3 Lolium caffeic acid O- LpOMT4 169 1366
methyltransferase 3 F5H 45 107 Fescue Ferulate 5-hydroxylase F5H
173 2051 151 183 Lolium Ferulate 5-hydroxylase F5H 177 2101 PAL 152
108 Lolium Phenylalanine ammonia PAL 181 2460 lyase 153 184 Fescue
Phenylalanine ammonia PAL 185 2595 lyase 50 112 Fescue Peroxidase
PER 189 1205 54 116 Fescue Peroxidase PER 193 1266 48 110 Fescue
Peroxidase PER 197 1301 53 115 Lolium Peroxidase PER 201 1059 156
185 Peroxidase PER 205 1204 49 111 Lolium Peroxidase PER 209 1236
51 113 Lolium Peroxidase PER 213 1382 154 Peroxidase PER 217 1382
52 114 Lolium Peroxidase PER 220 1261 155 Peroxidase PER 224 1260
Frame shift Frame PROT DNA shift SEQ No SEQ protein SEQ ID ID of
ORF ORF ID SEQ ID NO. DNA NO. AA start end NO. NO. Important Info
4CL 29 22 559 72 1751 23 24 From WO 03/40306 31 26 557 137 1810 27
28 From WO 03/40306 27 30 539 3 1622 31 32 From WO 03/40306 142 34
559 85 1764 35 36 From WO 03/40306 Corrected SEQ 28, no change in
protein seq 143 38 557 126 1799 39 40 From WO 03/40306 Corrected
SEQ 30 and 92 C4H 33 42 505 80 1597 43 44 From WO 03/40306 144 46
501 61 1566 47 47 From WO 03/40306 Corrected SEQ 32 and 94 50 506 1
1518 51 52 From US patent WO 2008/064289 also known as
PCT/US2007/085344 CAD3 35 54 361 86 1171 55 56 From WO 03/40306 145
58 361 67 1152 59 60 From WO 03/40306 FL of SEQ 34 and 96 CAD 62
361 40 1125 63 64 AF188292 66 361 40 1125 67 68 AF188293; Also in
US patent WO 2008/064289 also known as PCT/US2007/085344 70 361 91
1176 71 72 AF188294 74 361 91 1176 75 76 AF188295 78 407 22 1245 79
80 82 370 102 1214 83 84 86 361 81 1166 87 88 CCoAOMT 37 90 265 75
872 91 92 From WO 03/40306 146 94 265 55 852 95 96 From WO 03/40306
Corrected SEQ 36, no change in protein seq 98 261 132 917 99 100
102 261 135 920 103 104 106 243 171 902 107 108 110 243 170 901 111
112 114 265 137 934 115 116 CCR 148 118 342 90 1118 119 120 From WO
03/40306 Corrected SEQ 39, no change in protein seq 147 122 363 148
1239 123 124 From WO 03/40306 Corrected SEQ 38 and 100 126 362 150
1238 127 128 130 344 1 1035 131 132 From NCBI Accession # AF278698
COMT 149 134 360 27 1109 135 136 From WO 03/40306 Corrected SEQ 40
and 102. Also in US patent WO 2008/064289 also known as
PCT/US2007/085344 42 138 360 64 1146 139 140 From WO 03/40306 150
142 360 66 1148 143 144 From WO 03/40306 Corrected SEQ 41, no
change in protein seq 43 146 361 85 1170 147 148 From WO 03/40306
150 360 62 1144 151 152 NCBI accession no AF153825 154 360 78 1160
155 156 NCBI accession no AF153826 158 360 139 1221 159 160 162 351
135 1187 163 164 166 361 156 1238 167 168 170 367 107 1209 171 172
F5H 45 174 542 93 1721 175 176 From WO 03/40306 151 178 543 87 1718
179 180 From WO 03/40306 Corrected SEQ 44 and 106 PAL 152 182 711
111 2246 183 184 From WO 03/40306 Corrected SEQ 46, no change in
protein seq 153 186 713 143 2284 187 188 From WO 03/40306 Corrected
SEQ 47 and 109 50 190 326 87 1067 191 192 From WO 03/40306 54 194
311 80 1015 195 196 From WO 03/40306 48 198 323 22 993 199 200 From
WO 03/40306 53 202 293 1 882 203 204 From WO 03/40306 156 206 324
46 1017 207 208 From WO 03/40306 FL of SEQ 53 and 115, now two
sequences with difference in 5' region with SEQ ID NO 162 49 210
344 4 1038 211 212 From WO 03/40306 51 214 358 59 1135 215 216 From
WO 03/40306 154 218 219 From WO 03/40306 Corrected SEQ 51, no
change in protein seq 52 221 344 18 1052 222 223 From WO 03/40306
155 225 226 From WO 03/40306 Corrected SEQ 52, no change in protein
seq
TABLE-US-00002 TABLE 2 Frame Frame shift shift DNA PROT No DNA
protein SEQ No of SEQ of ORF SEQ SEQ ID Grass Species Gene name
Abbrev ID NO. NT ID NO. AA start ORF end ID NO. NO. Important Info
Bermuda Cynodon cinnamate 3- C3H 227 1539 228 512 1 1539 229 230
From US patent WO grass dactylon hydroxylase 2008/064289 also known
as PCT/US2007/085344 Cynodon caffeic acid O- COMT 231 789 232 262 1
789 233 234 From US patent WO dactylon methyltransferase
2008/064289 also known as PCT/US2007/085344
[0062] In a second aspect of the present invention there is
provided a genetic construct or a vector including a nucleic acid
or nucleic acid fragment according to the present invention.
[0063] In a preferred embodiment of this aspect of the invention,
the vector may include a regulatory element such as a promoter, a
nucleic acid or nucleic acid fragment, according to the present
invention and a terminator; said regulatory element, nucleic acid
or nucleic acid fragment and terminator being operatively
linked.
[0064] By `genetic construct` is meant a recombinant nucleic acid
molecule.
[0065] By a `vector` is meant a genetic construct used to transfer
genetic material to a target cell.
[0066] By `operatively linked` is meant that the nucleic acid(s)
and a regulatory sequence, such as a promoter, are linked in such a
way as to permit expression of said nucleic acid under appropriate
conditions, for example when appropriate molecules such as
transcriptional activator proteins are bound to the regulatory
sequence. Preferably an operatively linked promoter is upstream of
the associated nucleic acid.
[0067] The vector may be of any suitable type and may be viral or
non-viral. The vector may be an expression vector. Such vectors
include chromosomal, non-chromosomal and synthetic nucleic acid
sequences, eg. derivatives of plant viruses; bacterial plasmids;
derivatives of the Ti plasmid from Agrobacterium tumefaciens;
derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage
DNA; yeast artificial chromosomes; bacterial artificial
chromosomes; binary bacterial artificial chromosomes; vectors
derived from combinations of plasmids and phage DNA. However, any
other vector may be used as long as it is replicable or integrative
or viable in the plant cell.
[0068] The regulatory element and terminator may be of any suitable
type and may be endogenous to the target plant cell or may be
exogenous, provided that they are functional in the target plant
cell.
[0069] Preferably the regulatory element is a promoter. A variety
of promoters which may be employed in the vectors of the present
invention are well known to those skilled in the art. Factors
influencing the choice of promoter include the desired tissue
specificity of the vector, and whether constitutive or inducible
expression is desired and the nature of the plant cell to be
transformed (eg. monocotyledon or dicotyledon). Particularly
suitable promoters include the Cauliflower Mosaic Virus 35S (CaMV
35S) promoter, the maize Ubiquitin promoter, the rice Actin
promoter, and ryegrass endogenous OMT, 4CL, CCR or CAD
promoters.
[0070] A variety of terminators which may be employed in the
vectors of the present invention are also well known to those
skilled in the art. The terminator may be from the same gene as the
promoter sequence or a different gene. Particularly suitable
terminators are polyadenylation signals, such as the CaMV 35S polyA
and other terminators from the nopaline synthase (nos) and the
octopine synthase (ocs) genes.
[0071] The vector, in addition to the regulatory element, the
nucleic acid or nucleic acid fragment of the present invention and
the terminator, may include further elements necessary for
expression of the nucleic acid or nucleic acid fragment, in
different combinations, for example vector backbone, origin of
replication (ori), multiple cloning sites, spacer sequences,
enhancers, introns (such as the maize Ubiquitin Ubi intron),
antibiotic resistance genes and other selectable marker genes [such
as the neomycin phosphotransferase (npt2) gene, the hygromycin
phosphotransferase (hph) gene, the phosphinothricin
acetyltransferase (bar or pat) gene], and reporter genes (such as
beta-glucuronidase (GUS) gene (gusA)]. The vector may also contain
a ribosome binding site for translation initiation. The vector may
also include appropriate sequences for amplifying expression.
[0072] As an alternative to use of a selectable marker gene to
provide a phenotypic trait for selection of transformed host cells,
the presence of the vector in transformed cells may be determined
by other techniques well known in the art, such as PCR (polymerase
chain reaction), Southern blot hybridisation analysis,
histochemical GUS assays, northern and Western blot hybridisation
analyses.
[0073] Those skilled in the art will appreciate that the various
components of the vector are operatively linked, so as to result in
expression of said nucleic acid or nucleic acid fragment.
Techniques for operatively linking the components of the vector of
the present invention are well known to those skilled in the art.
Such techniques include the use of linkers, such as synthetic
linkers, for example including one or more restriction enzyme
sites.
[0074] The vectors of the present invention may be incorporated
into a variety of plants, including monocotyledons (such as grasses
from the genera Lolium, Festuca, Cynodon, Bracharia, Paspalum,
Panicum, Miscanthus, Pennisetum, Phalaris, and other forage, turf
and bioenergy grasses, corn, oat, sugarcane, wheat and barley),
dicotyledons (such as Arabidopsis, tobacco, legumes, Alfalfa, oak,
Eucalyptus, maple, Populus, canola, soybean and chickpea) and
gymnosperms (such as Pinus). In a preferred embodiment, the vectors
are used to transform monocotyledons, preferably grass species such
as Lolium, Festuca, Cynodon, Bracharia, Paspalum, Panicum,
Miscanthus, Pennisetum, Phalaris, and other forage, turf and
bioenergy grasses, more preferably a Lolium species such as Lolium
perenne or Lolium arundinaceum, including cultivars for forage and
turf applications.
[0075] Techniques for incorporating the vectors of the present
invention into plant cells (for example by transduction,
transfection or transformation) are well known to those skilled in
the art. Such techniques include Agrobacterium mediated
introduction, electroporation to tissues, cells and protoplasts,
protoplast fusion, injection into reproductive organs, injection
into immature embryos and high velocity projectile introduction to
cells, tissues, calli, immature and mature embryos. The choice of
technique will depend largely on the type of plant to be
transformed.
[0076] Cells incorporating the vector of the present invention may
be selected, as described above, and then cultured in an
appropriate medium to regenerate transformed plants, using
techniques well known in the art. The culture conditions, such as
temperature, pH and the like, will be apparent to the person
skilled in the art. The resulting plants may be reproduced, either
sexually or asexually, using methods well known in the art, to
produce successive generations of transformed plants.
[0077] In a further aspect of the present invention there is
provided a transformed plant cell, plant, plant seed or other plant
part, or plant biomass, including digestible biomass such as hay,
including, eg transformed with, a nucleic acid, genetic construct
or vector of the present invention. Preferably the transgenic plant
cell, plant, plant seed or other plant part is produced by a method
according to the present invention.
[0078] The present invention also provides a transgenic plant,
plant seed or other plant part, or plant biomass, derived from a
plant cell of the present invention and including a nucleic acid,
genetic construct or vector of the present invention.
[0079] The present invention also provides a transgenic plant,
plant seed or other plant, part or plant biomass, derived from a
plant of the present invention and including a nucleic acid,
genetic construct or vector of the present invention.
[0080] The nucleic acid, genetic construct or vector of the present
invention may be stably integrated into the genome of the plant,
plant seed, other plant part or plant biomass.
[0081] The plant cell, plant, plant seed or other plant part may be
from any suitable species, including monocotyledons, dicotyledons
and gymnosperms. In a preferred embodiment the plant cell, plant,
plant seed or other plant part may be from a monocotyledon,
preferably a grass species, such as Lolium, Festuca, Cynodon,
Bracharia, Paspalum, Panicum, Miscanthus, Pennisetum, Phalaris, and
other forage, turf and bioenergy grasses, more preferably a Lolium
species such as Lolium perenne or Lolium arundinaceum.
[0082] In a further aspect of the present invention there is
provided a method of modifying lignin biosynthesis in a plant, said
method including introducing into said plant an effective amount of
a nucleic acid or nucleic acid fragment, genetic construct and/or a
vector according to the present invention.
[0083] By "an effective amount" is meant an amount sufficient to
result in an identifiable phenotypic trait in said plant, or a
plant, plant seed or other plant part, or plant biomass derived
therefrom. Such amounts can be readily determined by an
appropriately skilled person, taking into account the type of
plant, the route of administration and other relevant factors. Such
a person will readily be able to determine a suitable amount and
method of administration. See, for example, Maniatis et al,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, the entire disclosure of which is
incorporated herein by reference.
[0084] Using the methods and materials of the present invention,
plant lignin biosynthesis may be increased, decreased or otherwise
modified relative to an untransformed control plant. It may be
increased or otherwise modified, for example, by incorporating
additional copies of a sense nucleic acid or nucleic acid fragment
of the present invention. It may be decreased, for example, by
incorporating an antisense nucleic acid or nucleic acid fragment of
the present invention or by incorporating a functionally active
fragment or variant which is capable of modifying lignin
biosynthesis in a plant via sense suppression. In addition, the
number of copies of genes encoding for different enzymes in the
lignin biosynthetic pathway may be manipulated to modify the
relative amount of each monolignol synthesized, thereby leading to
the formation of lignin having altered composition.
[0085] Accordingly, in a preferred embodiment of this aspect of the
invention there is provided a method of modifying lignin
biosynthesis in a plant, said method including introducing into
said plant in sense orientation an effective amount of a nucleic
acid, genetic construct or vector according to the present
invention, such that expression of the corresponding gene is
suppressed.
[0086] Preferred functionally active fragments and variants for
sense suppression include those hereinbefore described.
[0087] In a further aspect of the present invention there is
provided use of a nucleic acid, genetic construct or vector
according to the present invention for sense suppression of lignin
biosynthesis in a plant.
[0088] In a still further aspect of the present invention there is
provided use of a nucleic acid or nucleic acid fragment according
to the present invention, and/or nucleotide sequence information
thereof, and/or single nucleotide polymorphisms thereof, as a
molecular genetic marker.
[0089] More particularly, nucleic acids or nucleic acid fragments
according to the present invention, and/or nucleotide sequence
information thereof, and/or single nucleotide polymorphisms
thereof, may be used as a molecular genetic marker for qualitative
trait loci (QTL) tagging, mapping, DNA fingerprinting and in marker
assisted selection, and may be used as candidate genes or perfect
markers, particularly in ryegrasses and fescues. Even more
particularly, nucleic acids or nucleic acid fragments according to
the present invention, and/or nucleotide sequence information
thereof, may be used as molecular genetic markers in forage and
turf grass improvement, eg. tagging QTLs for dry matter
digestibility, herbage quality, mechanical stress tolerance,
disease resistance, insect pest resistance, plant stature and leaf
and stem colour.
[0090] In a still further aspect of the present invention there is
provided a substantially purified or isolated polypeptide from a
ryegrass (Lolium) or fescue (Fustuca) species, selected from the
group consisting of the enzymes 4CL, CCR and CAD.
[0091] The ryegrass (Lolium) or fescue (Festuca) species may be of
any suitable type, including Italian or annual ryegrass, perennial
ryegrass, tall fescue, meadow fescue and red fescue. Preferably the
species is a ryegrass, more preferably perennial ryegrass L.
perenne).
[0092] In a preferred embodiment of this aspect of the invention,
the substantially purified or isolated enzyme 4CL includes an amino
acid sequence selected from the group consisting of sequences shown
in FIGS. 2, 3 and 4 hereto (Sequence ID Nos: 2, 4 and 6,
respectively); and functionally active fragments and variants
thereof.
[0093] In a further preferred embodiment of this aspect of the
invention, the substantially purified or isolated enzyme CCR
includes an amino acid sequence selected from the group consisting
of the sequence shown in FIG. 10 hereto (Sequence ID No: 8); and
functionally active fragments and variants thereof.
[0094] In a still further preferred embodiment of this aspect of
the invention, the substantially purified or isolated enzyme CAD
includes an amino acid sequence selected from the group consisting
of the sequence shown in FIGS. 13, 14, 26 and 27 hereto (Sequence
ID Nos: 10, 12, 15 and 17, respectively); and functionally active
fragments and variants thereof.
[0095] By "functionally active" in this context is meant that the
fragment or variant has one or more of the biological properties of
the enzymes 4CL, CCR and CAD, respectively. Additions, deletions,
substitutions and derivatizations of one or more of the amino acids
are contemplated so long as the modifications do not result in loss
of functional activity of the fragment or variant. Preferably the
fragment or variant has at least approximately 60% identity to the
relevant part of the above mentioned sequence, more preferably at
least approximately 80% identity, most preferably at least
approximately 90% identity. Such functionally active variants and
fragments include, for example, those having conservative amino
acid substitutions of one or more residues in the corresponding
amino acid sequence. Preferably the fragment has a size of at least
10 amino acids, more preferably at least 15 amino acids, most
preferably at least 20 amino acids.
[0096] In a further embodiment of this aspect of the invention,
there is provided a polypeptide recombinantly produced from a
nucleic acid or nucleic acid fragment according to the present
invention. Techniques for recombinantly producing polypeptides are
well known to those skilled in the art.
[0097] In a still further aspect of the present invention there is
provided a lignin or modified lignin substantially or partially
purified or isolated from a plant, plant seed or other plant part
of the present invention.
[0098] Such lignins may be modified from naturally occurring
lignins in terms of the length, the degree of polymerisation
(number of units), degree of branching and/or nature of linkages
between units.
[0099] In a still further aspect, the present invention provides an
isolated regulatory element capable of causing expression of an
exogenous gene in plant cells. Preferably the regulatory element is
isolated from a nucleic acid or nucleic acid fragment encoding OMT,
4CL, CCR or CAD.
[0100] The regulatory element may be a nucleic acid molecule,
including DNA (such as cDNA or genomic DNA) and RNA (such as mRNA)
that is single- or double-stranded, optionally containing
synthetic, non-natural or altered nucleotide bases, and
combinations thereof.
[0101] Preferably the regulatory element includes a promoter, more
preferably an O-methyltransferase promoter, even more preferably an
O-methyltransferase promoter from a ryegrass (Lolium) or fescue
(Festuca) species, more preferably a ryegrass, most preferably
perennial ryegrass (Lolium perenne).
[0102] In a particularly preferred embodiment of this aspect of the
invention, the regulatory element includes a promoter from the
caffeic acid O-methyltransferase gene corresponding to the cDNA
homologue LpOMT1 from perennial ryegrass.
[0103] Preferably the regulatory element includes a nucleotide
sequence including the first approximately 4630 nucleotides of the
sequence shown in FIG. 18 hereto (Sequence ID No: 13); or a
functionally active fragment or variant thereof.
[0104] By "functionally active" in this context is meant that the
fragment or variant (such as an analogue, derivative or mutant) is
capable of causing expression of a transgene in plant cells. Such
variants include naturally occurring allelic variants and
non-naturally occurring variants. Additions, deletions,
substitutions and derivatizations of one or more of the nucleotides
are contemplated so long as the modifications do not result in loss
of functional activity of the regulatory element. Preferably the
functionally active fragment or variant has at least approximately
80% identity to the relevant part of the above sequence, more
preferably at least approximately 90% identity, most preferably at
least approximately 95% identity. Preferably the fragment has a
size of at least 100 nucleotides, more preferably at least 150
nucleotides, most preferably at least 200 nucleotides.
[0105] In a particularly preferred embodiment of this aspect of the
invention, the regulatory element includes a nucleotide sequence
selected from the group consisting of:
[0106] Nucleotides -4581 to -1
[0107] Nucleotides -4285 to -1
[0108] Nucleotides -4020 to -1
[0109] Nucleotides -2754 to -1
[0110] Nucleotides -1810 to -1
[0111] Nucleotides -831 to -1
[0112] Nucleotides -560 to -1
[0113] Nucleotides -525 to -1
[0114] Nucleotides -274 to -1
[0115] Nucleotides -21 to -1
[0116] of FIG. 18 hereto (Sequence ID No: 13);
[0117] or a functionally active fragment or variant thereof.
[0118] In another preferred embodiment the regulatory element
includes a 4 coumarate-CoA ligase promoter, even more preferably a
4 coumarate-CoA ligase promoter from a ryegrass (Lolium) or fescue
(Festuca) species, more preferably a ryegrass, most preferably
perennial ryegrass (Lolium perenne).
[0119] In a particularly preferred embodiment of this aspect of the
invention, the regulatory element includes a promoter from the 4
coumarate-CoA ligase gene corresponding to the cDNA homologue
Lp4CL2 from perennial ryegrass.
[0120] Preferably the regulatory element includes a nucleotide
sequence including the first approximately 2206 nucleotides of the
sequence shown in FIG. 38 hereto (Sequence ID No: 17); or a
functionally active fragment or variant thereof.
[0121] By "functionally active" in this context is meant that the
fragment or variant (such as an analogue, derivative or mutant) is
capable of causing expression of a transgene in plant cells. Such
variants include naturally occurring allelic variants and
non-naturally occurring variants. Additions, deletions,
substitutions and derivatizations of one or more of the nucleotides
are contemplated so long as the modifications do not result in loss
of functional activity of the regulatory element. Preferably the
functionally active fragment or variant has at least approximately
80% identity to the relevant part of the above sequence, more
preferably at least approximately 90% identity, most preferably at
least approximately 95% identity. Preferably the fragment has a
size of at least 100 nucleotides, more preferably at least 150
nucleotides, most preferably at least 200 nucleotides.
[0122] In a particularly preferred embodiment of this aspect of the
invention, the regulatory element includes a nucleotide sequence
selected from the group consisting of:
[0123] Nucleotides -2206 to -1
[0124] Nucleotides -1546 to -1
[0125] Nucleotides -1186 to -1
[0126] Nucleotides -406 to -1
[0127] Nucleotides -166 to -1
[0128] of FIG. 38 hereto (Sequence ID No: 17);
[0129] or a functionally active fragment or variant thereof.
[0130] In another preferred embodiment the regulatory element
includes a cinnamoyl-CoA reductase promoter, even more preferably a
cinnamoyl-CoA reductase promoter from a ryegrass (Lolium) or fescue
(Festuca) species, more preferably a ryegrass, most preferably
perennial ryegrass (Lolium perenne).
[0131] In a particularly preferred embodiment of this aspect of the
invention, the regulatory element includes a promoter from the
cinnamoyl-CoA reductase gene corresponding to the LpCCR1 cDNA from
perennial ryegrass.
[0132] Preferably the regulatory element includes a nucleotide
sequence including the first approximately 6735 nucleotides of the
sequence shown in FIG. 39 hereto (Sequence ID No: 18); or a
functionally active fragment or variant thereof.
[0133] By "functionally active" in this context is meant that the
fragment or variant (such as an analogue, derivative or mutant) is
capable of causing expression of a transgene in plant cells. Such
variants include naturally occurring allelic variants and
non-naturally occurring variants. Additions, deletions,
substitutions and derivatizations of one or more of the nucleotides
are contemplated so long as the modifications do not result in loss
of functional activity of the regulatory element. Preferably the
functionally active fragment or variant has at least approximately
80% identity to the relevant part of the above sequence, more
preferably at least approximately 90% identity, most preferably at
least approximately 95% identity. Preferably the fragment has a
size of at least 100 nucleotides, more preferably at least 150
nucleotides, most preferably at least 200 nucleotides.
[0134] In a particularly preferred embodiment of this aspect of the
invention, the regulatory element includes a nucleotide sequence
selected from the group consisting of:
[0135] Nucleotides -6735 to -1
[0136] Nucleotides -5955 to -1
[0137] Nucleotides -5415 to -1
[0138] Nucleotides -4455 to -1
[0139] Nucleotides -4035 to -1
[0140] Nucleotides -3195 to -1
[0141] Nucleotides -2595 to -1
[0142] Nucleotides -1755 to -1
[0143] Nucleotides -1275 to -1
[0144] Nucleotides -495 to -1
[0145] Nucleotides -255 to -1
[0146] Nucleotides -75 to -1
[0147] of FIG. 39 hereto (Sequence ID No: 18);
[0148] or a functionally active fragment or variant thereof.
[0149] By an "exogenous gene" is meant a gene not natively linked
to said regulatory element. In certain embodiments of the present
invention the exogenous gene is also not natively found in the
relevant plant or plant cell.
[0150] The exogenous gene may be of any suitable type. The
exogenous gene may be a nucleic acid such as DNA (e.g. cDNA or
genomic DNA) or RNA (e.g. mRNA), and combinations thereof. The
exogenous gene may correspond to a target gene, for example a gene
capable of influencing disease resistance, herbage digestibility,
nutrient quality, mineral content or drought tolerance or be a
fragment or variant (such as an analogue, derivative or mutant)
thereof which is capable of modifying expression of said target
gene. Such variants include nucleic acid sequences which are
antisense to said target gene or an analogue, derivative, mutant or
fragment thereof. The transgene may code for a protein or RNA
sequence depending the target condition and whether down or
up-regulation of gene expression is required. Preferably, the
target gene is selected from exogenous coding sequences coding for
mRNA for a protein, this protein may be of bacterial origin (such
as enzymes involved in cell wall modification and cell wall
metabolism, cytokinin biosynthesis), or eukaryotic origin (such as
pharmaceutically active polypeptides) or of plant origin (such as
enzymes involved in the synthesis of phenolic compounds, cell wall
metabolism, sugar metabolism, lignin biosynthesis). Preferably, the
target gene is selected from the group comprising
O-methyltransferase, 4 coumarate CoA-ligase, cinnamoyl CoA
reductase, cinnamyl alcohol dehydrogenase, cinnamate 4 hydroxylase,
phenolase, laccase, peroxidase, coniferol glucosyl transferase,
coniferin beta-glucosidase, phenylalanine ammonia lyase, ferulate
5-hydroxylase, chitinase, glucanase, isopentenyltransferase,
xylanase.
[0151] The plant cells, in which the regulatory element of the
present invention is capable of causing expression of an exogenous
gene, may be of any suitable type. The plant cells may be from
monocotyledons (such as grasses from the genera Lolium, Festuca,
Cynodon, Bracharia, Paspalum, Panicum, Miscanthus, Pennisetum,
Phalaris, and other forage and turf grasses, corn, grains, oat,
sugarcane, wheat and barley), dicotyledons (such as Arabidopsis,
tobacco, legumes, Alfalfa, oak, Eucalyptus, maple, Populus, canola,
soybean and chickpea) and gymnosperms (such as Pinus). Preferably
the plant cells are from a monocotyledon, more preferably a grass
species such as Lolium, Festuca, Cynodon, Bracharia, Paspalum,
Panicum, Miscanthus, Pennisetum, Phalaris, and other forage, turf
and bioenergy grasses, more preferably a Lolium species such as
Lolium perenne or Lolium arundinaceum.
[0152] The regulatory element according to the present invention
may be used to express exogenous genes to which it is operatively
linked in the production of transgenic plants.
[0153] Accordingly, in a further aspect of the present invention
there is provided a vector including a regulatory element according
to the present invention.
[0154] In a preferred embodiment of this aspect of the invention,
the vector may include a regulatory element according to the
present invention, an exogenous gene as hereinbefore described, and
a terminator; said regulatory element, exogenous gene and
terminator being operatively linked, such that said regulatory
element is capable of causing expression of said exogenous gene in
plant cells. Preferably, said regulatory element is upstream of
said exogenous gene and said terminator is downstream of said
exogenous gene.
[0155] The vector may be of any suitable type and may be viral or
non-viral. The vector may be an expression vector. Such vectors
include chromosomal, non-chromosomal and synthetic nucleic acid
sequences, eg. derivatives of plant viruses; bacterial plasmids;
derivatives of the Ti plasmid from Agrobacterium tumefaciens;
derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage
DNA; yeast artificial chromosomes; bacterial artificial
chromosomes; binary bacterial artificial chromosomes; vectors
derived from combinations of plasmids and phage DNA. However, any
other vector may be used as long as it is replicable on integrative
or viable in the plant cell.
[0156] The terminator may be of any suitable type and includes for
example polyadenylation signals, such as the Cauliflower Mosaic
Virus 35S polyA (CaMV 35S polyA) and other terminators from the
nopaline synthase (nos) and the octopine synthase (ocs) genes.
[0157] The vector, in addition to the regulatory element, the
exogenous nucleic acid and the terminator, may include further
elements necessary for expression of the nucleic acid, in different
combinations, for example vector backbone, origin of replication
(ori), multiple cloning sites, spacer sequences, enhancers, introns
(such as the maize Ubiquitin Ubi intron), antibiotic resistance
genes and other selectable marker genes [such as the neomycin
phosphotransferase (npt2) gene, the hygromycin phosphotransferase
(hph) gene, the phosphinothricin acetyltransferase (bar or pat)
gene], and reporter genes (such as beta-glucuronidase (GUS) gene
(gusA)]. The vector may also contain a ribosome binding site for
translation initiation. The vector may also include appropriate
sequences for amplifying expression.
[0158] The regulatory element of the present invention may also be
used with other full promoters or partial promoter elements.
[0159] As an alternative to use of a selectable marker gene to
provide a phenotypic trait for selection of transformed host cells,
the presence of the vector in transformed cells may be determined
by other techniques well known in the art, such as PCR (polymerase
chain reaction), Southern blot hybridisation analysis,
histochemical GUS assays, northern and Western blot hybridisation
analyses.
[0160] Those skilled in the art will appreciate that the various
components of the vector are operatively linked, so as to result in
expression of said transgene. Techniques for operatively linking
the components of the vector of the present invention are well
known to those skilled in the art. Such techniques include the use
of linkers, such as synthetic linkers, for example including one or
more restriction sites.
[0161] The vectors of the present invention may be incorporated
into a variety of plants, including monocotyledons, dicotyledons
and gymnosperms. In a preferred embodiment the vectors are used to
transform monocotyledons, preferably grass species such as
ryegrasses (Lolium species) and fescues (Festuca species), more
preferably perennial ryegrass (Lolium perenne) including cultivars
for forage and turf applications.
[0162] Techniques for incorporating the vectors of the present
invention into plant cells (for example by transduction,
transfection or transformation) are well known to those skilled in
the art. Such techniques include Agrobacterium mediated
introduction, electroporation to tissues, cells and protoplasts,
protoplast fusion, injection into reproductive organs, injection
into immature embryos and high velocity projectile introduction to
cells, tissues, calli, immature and mature embryos. The choice of
technique will depend largely on the type of plant to be
transformed.
[0163] Cells incorporating the vector of the present invention may
be selected, as described above, and then cultured in an
appropriate medium to regenerate transformed plants, using
techniques well known in the art. The culture conditions, such as
temperature, pH and the like, will be apparent to the person
skilled in the art. The resulting plants may be reproduced, either
sexually or asexually, using methods well known in the art, to
produce successive generations of transformed plants.
[0164] In a further aspect of the present invention there is
provided a plant cell, plant, plant seed or other plant part,
including, eg. transformed with, a vector of the present
invention.
[0165] The plant cell, plant, plant seed or other plant part may be
from any suitable species, including monocotyledons, dicotyledons
and gymnosperms. In a preferred embodiment the plant cell, plant,
plant seed or other plant part is from a monocotyledon, preferably
a grass species, more preferably a ryegrass (Lolium species) or
fescue (Festuca species), even more preferably perennial ryegrass
(Lolium perenne), including cultivars for forage and turf
applications.
[0166] The present invention also provides a plant, plant seed, or
other plant part derived from a plant cell of the present
invention.
[0167] The present invention also provides a plant, plant seed or
other plant part derived from a plant of the present invention.
[0168] In a still further aspect of the present invention there is
provided a recombinant plant genome including a regulatory element
according to the present invention.
[0169] In a preferred embodiment of this aspect of the invention
the recombinant plant genome further includes an exogenous gene
operatively linked to said regulatory element.
[0170] In a further aspect of the present invention there is
provided a method for expressing an exogenous gene in plant cells,
said method including introducing into said plant cells an
effective amount of a regulatory element and/or a vector according
to the present invention.
[0171] By "an effective amount" is meant an amount sufficient to
result in an identifiable phenotypic change in said plant cells or
a plant, plant seed or other plant part derived therefrom. Such
amounts can be readily determined by an appropriately skilled
person, taking into account the type of plant cell, the route of
administration and other relevant factors. Such a person will
readily be able to determine a suitable amount and method of
administration. See, for example, Maniatis et al, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, the entire disclosure of which is incorporated
herein by reference.
[0172] The present invention will now be more fully described with
reference to the accompanying Examples and drawings. It should be
understood, however, that the description following is illustrative
only and should not be taken in any way as a restriction on the
generality of the invention described above.
[0173] In the Figures
[0174] FIG. 1 shows plasmid maps of the three cDNAs encoding
perennial ryegrass 4CL homologues.
[0175] FIG. 2 shows the nucleotide (Sequence ID No: 1) and amino
acid (Sequence ID No: 2) sequences of Lp4CL1.
[0176] FIG. 3 shows the nucleotide (Sequence ID No: 3) and amino
acid (Sequence ID No: 4) sequences of Lp4CL2.
[0177] FIG. 4 shows the nucleotide (Sequence ID No: 5) and amino
acid (Sequence ID No: 6) sequences of Lp4CL3.
[0178] FIG. 5 shows amino acid sequence alignment of deduced
proteins encoded by Lp4CL1 (Sequence ID No: 2), Lp4CL2 (Sequence ID
No: 4) and Lp4CL3 (Sequence ID No: 6).
[0179] FIG. 6 shows northern hybridisation analysis of developing
perennial ryegrass using Lp4CL1, Lp4CL2 and Lp4CL3 as hybridisation
probes. SR: roots from seedlings (3-5 d post-germination), SS:
shoots from seedlings (3-5 d post-germination), ML: leaves from
12-week-old plants, MS: stems from 12-week-old plants. Blots were
washed in 0.2.times.SSPE, 0.1% SDS at 65.degree. C. Lp4CL1, Lp4CL2
and Lp4CL3 do not cross hybridise at this stringency. Sizes are
given in kb.
[0180] FIG. 7 shows northern hybridisation analysis showing the
time course of expression of 4CL mRNA in wounded perennial ryegrass
leaves. Sizes are given in kb.
[0181] FIG. 8 shows genomic Southern hybridisation analysis using
Lp4CL1, Lp4CL2 and Lp4CL3 as hybridisation probes. 10 .mu.g of
digested perennial ryegrass genomic DNA or 20 .mu.g of digested
tall fescue genomic DNA were separated on a 1.0% agarose gel,
transferred to Hybond N.sup.+ membranes and then hybridised with
.sup.32P labelled Lp4CL1, Lp4CL2 or Lp4CL3 probes. The ryegrass
Lp4CL1, Lp4CL2 and Lp4CL3 genes reveal homologous sequences in tall
fescue and indicate that the ryegrass 4CL genes can be used to
isolate and to manipulate the expression of the tall fescue
(Festuca arundinacea) 4CL genes.
[0182] FIG. 9 shows restriction map of LpCCR1. An L. perenne
seedling cDNA library constructed in Uni-ZAP.TM. (Stratagene) was
screened in a solution containing 10.times.PIPES, 50% deionised
formamide and 10% SDS at 42.degree. C. Filters were washed at room
temperature, three times in 0.1% SDS, 2.times.SSPE and then twice
in 0.1% SDS, 0.2.times.SSPE. The location of the probe used for
northern and Southern hybridisation analyses is indicated by the
black line labelled LpCCR531.
[0183] FIG. 10 shows the nucleotide (Sequence ID No: 7) and amino
acid (Sequence ID No: 8) sequences of LpCCR1.
[0184] FIG. 11 shows Southern hybridisation analysis of DNA from
double haploid (DH) perennial ryegrass using LpCCR1 as
hybridisation probe. 10 .mu.g of DH genomic DNA was digested with
Dral, BamHI, EcoRI, EcoRV, HindIII or XbaI, separated on a 1%
agarose gel and then capillary blotted onto nylon membrane
(Amersham Hybond-N). The membrane was probed with the digoxigenin
(DIG) labelled LpCCR531 fragment at 25 ng/ml in the hybridisation
solution. Hybridisation was in 4.times.SSC, 50% formamide, 0.1%
N-Lauroyl-sarcosine, 0.02% SDS, 2% Blocking solution at 42.degree.
C. The membrane was washed twice for five minutes in 2.times.SSC,
0.1% SDS at room temperature, then twice for fifteen minutes in
0.5.times.SSC, 0.1% SDS at 68.degree. C. Molecular weight was
determined by comparison to a DIG-labelled marker (Roche Molecular
Biochemicals).
[0185] FIG. 12 shows northern hybridisation analysis of RNA samples
from different organs and developmental stages of perennial
ryegrass using LpCCR1 probe. Roots from seedlings (3-5 d
post-germination), shoots from seedlings (3-5 d post-germination),
roots from seedlings (7-10 d post-germination), leaves from
seedlings (7-10 d post-germination), roots from 6 and 10 week old
plants, leaves from 6 and 10 week old plants, stems from 6 and 10
week old plants, whole seedling from 11 day old Phalaris and 7 day
old Festuca.
[0186] Total RNA was isolated using Trizol (GibcoBRL) and 15 .mu.g
was separated on a 1.2% Agarose gel containing 6% formamide and
then capillary blotted onto nylon membrane (Amersham Hybond-N). The
membrane was stained with 0.2% methylene blue/0.3M sodium acetate
to visualise the marker and ensure that RNA was evenly loaded. 50
ng LpCCR531 was random-labelled with .sup.32P-dCTP (Amersham
Megaprime) and hybridisation conditions were 4.times.SSC, 50%
formamide, 0.5% SDS, 5.times.denhardt solution, 5% dextrane
sulphate, 0.1% Herring sperm DNA at 42.degree. C. over-night. The
ryegrass LpCCR1 gene reveal homologous transcripts in tall fescue
and Phalaris, thus indicating that the ryegrass CCR gene can be
used to manipulate the expression of the tall fescue (Festuca
arundinacea) and Phalaris CCR endogenous genes.
[0187] FIG. 13 shows the nucleotide (Sequence ID No: 9) and amino
acid (Sequence ID No: 10) sequences of LpCAD1.
[0188] FIG. 14 shows the nucleotide (Sequence ID No: 11) and amino
acid (Sequence ID No: 12) sequences of LpCAD2.
[0189] FIG. 15 shows a plasmid map of a cDNA clone encoding
perennial ryegrass CAD homologue LpCAD1.
[0190] FIG. 16 shows northern hybridisation analysis of RNA samples
from different organs and developmental stages of perennial
ryegrass using A) LpCAD1 and B) LpCAD2 as hybridisation probes.
Roots from seedlings 3-5 d post-germination, 7-10 d
post-germination, 6 weeks and 10 weeks, Shoots from seedlings 3-5 d
post-germination and 7-10 d post-germination, Leaves from 6 week
old and 10 week old plants, stem tissue from 6 and 10 week old
plants. RNA isolated, from Phalaris and Festuca 11 and 7 day old
seedlings. The ryegrass CAD genes reveal homologous transcripts in
tall fescue and Phalaris, thus indicating that the ryegrass CAD
gene can be used to manipulate the expression of the tall fescue
and Phalaris CAD endogenous genes.
[0191] FIG. 17 shows genomic Southern hybridisation analysis. 10
.mu.g of perennial ryegrass genomic DNA digested with a range of
restriction enzymes was separated on a 0.8% agarose gel,
transferred to Hybond N and then hybridised with a DIG labelled A)
LpCAD1, and B) LpCAD2 hybridisation probe.
[0192] FIG. 18 shows the nucleotide sequence of the LpOmt1 promoter
(Sequence ID No: 13).
[0193] FIG. 19 shows a plasmid map of plant transformation vector
carrying the reporter .beta.-glucuronidase (GUS) gene (gusA) under
control of the perennial ryegrass LpOmt1 promoter.
[0194] FIG. 20 (upper image) shows PCR analysis of transgenic
tobacco plants containing the gusA gene under the control of the
perennial ryegrass LpOMT1 promoter (upper figure). PCR reactions
using gusA-specific primers were performed. FIG. 20 (lower images)
show histochemical GUS assays, demonstrating xylem-specific gusA
expression (A and B) and gusA expression in glandular leaf
trichomes (C and D) in transgenic tobacco plants containing the
gusA gene under the control of the perennial ryegrass LpOMT1
promoter.
[0195] FIG. 21 shows the isolation of the LpCCR1 genomic clone 1.
A) Southern hybridization analysis of CCR genomic clone
.lamda.Lp6.1.1a digested with XbaI, NcoI, SalI, XhoI, XhoI/SalI DNA
was separated on a 0.8% agarose gel, transferred to Hybond N and
hybridized with a DIG labelled CCR1 probe. B) Map showing the
genomic gene organisation of LpCCR1 clone 1 based on sequence
results. C) Comparison of plant CCR exon size and number in
different plant species (Lolium perenne, Lp., Eucalyptus gunni,
Eg., Eucalyptus saligna, Es., Populus balsamifera, Pb.)
[0196] FIG. 22 shows the isolation of the LpCCR1 genomic clone 2.
A) Southern hybridization analysis of CCR genomic clone
.lamda.Lp6.1.1a digested with XbaI, NcoI, SalI, XhoI, XhoI/SalI DNA
was separated on a 0.8% agarose gel, transferred to Hybond N and
hybridized with 200 bp of the CCR1 promoter (FIG. 21B). B) Map
showing the promoter region of LpCCR1 clone 2 based on sequence
results.
[0197] FIG. 23 shows the isolation of an Lp4CL genomic clone. A)
Southern hybridisation analysis of 4CL genomic clone .lamda.Lp4CL2
digested with BamHI, KpnI or SalI. DNA was separated on a 0.8%
agarose gel, transferred to Hybond N and hybridized with a DIG
labelled 4CL1 hybridisation probe. B) 10 .mu.l of a standard PCR
reaction using forward and reverse oligonucleotides designed to
positions outlined on C). The PCR products were separated on a 0.8%
agarose gel and stained with ethidium bromide. C) Map showing the
genomic gene organisation of .lamda.Lp4CL2 based on sequence and
PCR results.
[0198] FIG. 24 shows the isolation of an Lp4CL genomic clone. A)
Southern hybridisation analysis of 4CL genomic clone .lamda.Lp4CL2
digested with BamHI, KpnI, SalI. DNA was separated on a 0.8%
agarose gel, transferred to Hybond N and hybridized with a DIG
labelled 4CL1 probe. B) Map showing the genomic gene organisation
of Lp4CL2 clone 1 and the promoter region of clone 2.
[0199] FIG. 25 shows plasmid map of plant transformation vector
carrying the gusA gene under control of the perennial ryegrass
Lp4CL2 promoter (Lp4CL2::gusA).
[0200] FIG. 26 shows nucleotide (Sequence ID No: 14) and amino acid
(Sequence ID No: 15) sequences of genomic clone CAD2 cv Barlano
(Intron 1 and first 111 by of the coding region are missing).
[0201] FIG. 27 shows nucleotide (Sequence ID No: 16) and amino acid
(Sequence ID No:15) sequences of coding sequence deduced from
genomic clone CAD2 cv Barlano (region in bold is missing from the
genomic clone).
[0202] FIG. 28 shows the isolation of LpCAD2 genomic clone. A)
Southern hybridization analysis of CAD genomic clone .lamda.LpCAD2
digested with BamHI, EcoRI, KpnI, SalI or XbaI. DNA was separated
on a 0.8% agarose gel, transferred to Hybond N and hybridized with
a DIG labelled CAD2 hybridisation probe. B) Map showing the genomic
gene organisation of .lamda.LpCAD2 based on sequence results.
[0203] FIG. 29 shows A) Sense and antisense Lp4CL1, Lp4CL2 and
Lp4CL3 transformation vectors under control of the CaMV 35S
promoter; B) Sense and antisense Lp4CL1, Lp4CL2 and Lp4CL3
transformation vectors under control of the maize ubiquitin
promoter.
[0204] FIG. 30 shows A) Sense and antisense LpCCR1 transformation
vectors under control of the CaMV 35S promoter; B) Sense and
antisense LpCCR1 transformation vectors under control of the maize
ubiquitin promoter.
[0205] FIG. 31 shows A) Sense and antisense LpCAD1 transformation
vectors under control of the CaMV 35S promoter; B) Sense and
antisense LpCAD1 transformation vectors under control of the maize
ubiquitin promoter.
[0206] FIG. 32 shows molecular analysis of Lp4CL1-transgenic
tobacco. A) Plasmid map of transformation vector carrying a
chimeric sense Lp4CL1 gene. B) PCR analysis of independent
transgenic tobacco clones using Lp4CL1 specific primers. C)
Southern hybridization analysis of independent transgenic tobacco
plants using an Lp4CL1 specific probe. D) Northern hybridization
analysis of independent transgenic tobacco plants using an Lp4CL1
specific probe.
[0207] FIG. 33 shows molecular analysis of LpCCR1-transgenic
tobacco. A) Plasmid map of transformation vectors carrying a
chimeric sense and antisense LpCCR1 gene. B) PCR analysis of
independent sense transgenic tobacco clones using LpCCR1 specific
primers.
[0208] FIG. 34 shows protocol for suspension culture-independent
production of transgenic perennial ryegrass plants. A) Isolated
zygotic embryos, plated on MSM5 medium, day 0; B) Embryogenic
callus formation and proliferation, 6-8 weeks after embryo
isolation; C) Embryogenic calli arranged on high osmotic MSM3Plus
medium prior to biolistic transformation; D) Histochemical GUS
assay showing GUS expressing foci 3-4 days post-bombardment of
chimeric gusA gene; E) Selection of embryogenic calli on MSM3
medium containing 100 mg/l paromomycin (Pm), 2 weeks after
microprojectile bombardment; F) Regeneration of Pm resistant shoots
on MSK medium containing 100 mg/l Pm, 4 weeks after microprojectile
bombardment; G) In vitro plant regeneration from PM resistant
embryogenic calli, 6 weeks after microprojectile bombardment; H)
Transgenic perennial ryegrass plants 28 weeks after embryo
isolation.
[0209] FIG. 35 shows molecular analysis of transgenic perennial
ryegrass plants carrying sense and antisense LpOmt1 transgenes.
Plasmid maps of vectors used for the co-transformation of perennial
ryegrass embryogenic calli; pHP23 carrying a chimeric neomycin
phosphotransferase (npt2) selectable marker gene; pUbiomt1 carrying
a maize ubiquitin promoter driven sense LpOmt1 gene; pUbitmo1
carrying a maize ubiquitin promoter driven antisense LpOmt1 gene
(top). PCR analysis using npt2-specific primers of 5 independent
transgenic perennial ryegrass plants from biolistic transformation
with sense and antisense LpOmt1 vectors (upper centre). Southern
hybridization analysis with an omt1 hybridization probe of 7
independent perennial ryegrass plants co-transformed with sense
(lanes 1-3) and antisense (lanes 4-7) LpOmt1 vectors (lower centre
left). Southern hybridisation analysis with an npt2 hybridisation
probe of independent perennial ryegrass plants (lower centre
right). Northern hybridisation analysis of perennial ryegrass
plants co-transformed with antisense LpOmt1 vector (bottom).
C=negative control untransformed perennial ryegrass; P=positive
plasmid control.
[0210] FIG. 36 shows biochemical analysis of LpOmt1-transgenic
perennial ryegrass. OMT activity of leaf samples from selected
independent LpOmt1-transgenic perennial ryegrass plants (EII8,
EII11, EII14 and EII15) was determined and compared to
untransformed perennial ryegrass negative control plant L. perenne
cv. Ellett (wild type). Mean values and standard deviations of
replicate assays are shown.
[0211] FIG. 37 shows PCR screening of transgenic ryegrass plants.
PCR analysis using npt2-specific primers of 8 independent
transgenic perennial ryegrass plants from biolistic transformation
with antisense LpUbi4CL2 vector.
[0212] FIG. 38 shows the nucleotide sequence of genomic clone 4CL2
from perennial ryegrass (Sequence ID No: 17).
[0213] FIG. 39 shows the nucleotide sequence of genomic clone CCR1
from perennial ryegrass (Sequence ID No: 18).
[0214] FIG. 40 shows the map location of Lp4CL1, Lp4CL3, LpCAD1,
LpCAD2, LpCCR1, LpOMT1 and LpOMT2 (in bold) within the genetic
linkage map of perennial ryegrass.
[0215] FIG. 41. Illustration of the Gateway-derived expression
vectors used for generating the constructs for expressing perennial
ryegrass lignin biosynthetic genes.
[0216] FIG. 42. Vector details of Gateway.TM. Entry clone for the
LpCAD3 cDNA.
[0217] FIG. 43. Vector details of Gateway.TM. Entry clone for the
promoter LpCAD2.
[0218] FIG. 44. Vector details of Gateway.TM. Entry clone for the
terminator LpCAD2.
[0219] FIG. 45. Plasmid map of Construct 1,
LpCAD2p::LpCAD3::LpCAD2t in vector pAUX3132.
[0220] FIG. 46. Plasmid map of Construct 2, LpCCR1::LpCCR1::LpCCR1
in vector pAUX3169.
[0221] FIG. 47. Sequence of LpCCR1 gene (SEQ ID No: 19) and
modified forward primer (SEQ ID No: 20) that imparts a single base
deletion in the LpCCR1 gene.
[0222] FIG. 48. Plasmid map of Construct 3,
LpCCR1::LpCCR1(fs)::LpCCR1 in vector pAUX3169.
[0223] FIG. 49. Vector details for pAcH1 construct that was used as
the plant selectable marker containing the expression construct
Act1D::hph::35S.
[0224] FIG. 50. Production of transgenic perennial ryegrass from
microprojectile bombardment of embryogenic calli derived from
immature inflorescences. A) Excised immature inflorescence of
perennial ryegrass; 2-3 mm; B-E) Induction and proliferation of
embryogenic calli; 1-8 weeks after inflorescence excision. F).
Distribution of embryogenic calli on high osmotic medium LP3-OS
medium prior to biolisitic transformation; G) Biolistic
transformation device, PDS-1000/He; H-I) Growth and development of
hygromycin-resistant shoots, 30-75 days post bombardment; J) Growth
and development of hygromycin-resistant shoots in vitro; K)
Hygromycin-resistant plants established in soil and grown under
containment glasshouse conditions.
[0225] FIG. 51. Production of transgenic perennial ryegrass from
microprojectile bombardment of embryogenic calli derived from
seedling meristems. A) In vitro shoot culture for basal meristem
isolation; regenerated from seedling meristem-derived calli; B)
Distribution of basal meristematic material on callus initiation
medium; C-E) Induction and proliferation of embryogenic calli from
shoot meristems of Lolium perenne; F) Distribution of embryogenic
calli on high osmotic medium prior to biolistic transformation; G)
Biolistic transformation device, PDS-1000/He; H-I) Growth and
development of hygromycin-resistant shoots, 30-84 days post
bombardment; J) Growth and development of hygromycin-resistant
shoots in vitro; K) Hygromycin-resistant plants established in soil
and grown under containment glasshouse conditions.
[0226] FIG. 52. Flowchart describing the transformation method used
to generate transgenic perennial ryegrass containing the expression
construct of interest and the selectable marker gene (hph).
[0227] FIG. 53. Amplification of the hygromycin phosphotransferase
(hph) gene by Q-PCR in samples of genomic DNA extracted from
putative transgenic perennial ryegrass regenerated after
co-bombardment with plasmids pAcH1 and
pAUX3132-LpCAD2::LpCAD3::LpCAD2.
[0228] FIG. 54. Southern analysis of genomic DNA digested with Eco
R1 (R1) and separated by agarose gel electrophoresis and the
transgene detected with either hph or Ubi promoter probes. All six
putative transgenic plants were confirmed to contain both hph and
the gene-of-interest, hpLpCCR1.
[0229] FIG. 55. Maule staining of cross-sectioned internodes from
wild type and transgenic pUbi::hpCCR1::35 S ryegrass at R1 and R2
stage shows a strong decrease of reddish colour in transgenic
plants which may suggest a decrease in S lignin content compared to
wild type plants.
[0230] FIG. 56. Total lignin content of perennial ryegrass
internodes at the R1 developmental stage shows a progressive
reduction in lignin content from internode 1 (base) to internode 5
(top).
[0231] FIG. 57. Example of a gas chromatogram (GC-MS) showing
separation and identification of G-lignin and S-lignin monomers
after thioacidolysis derivatisation of lignin extracted from wild
type perennial ryegrass.
EXAMPLE 1
[0232] Isolation and characterisation of three 4-Coumarate
CoA-Ligase (4CL) cDNAs from Lolium perenne
Materials and Methods
Plant Material
[0233] Plants and embryogenic cell suspensions of perennial
ryegrass (Lolium perenne L.) cv Ellet and tall fescue (Festuca
arundinacea Schreb.) cv Triumph were established and maintained as
previously described (Heath et al., 1998). Wounding experiments
were performed with 10-day-old seedlings of perennial ryegrass (cv
Ellet) as previously described (Heath et al., 1998).
Screening of a cDNA Library
[0234] A cDNA library prepared with RNA isolated from perennial
ryegrass seedlings (Heath et al., 1998) was screened with a
[.sup.32P]dCTP-labelled rice partial 4CL probe. The rice 4CL probe
and consisted of a 844 by 4CL specific sequence inserted into
PUC119. This insert has 93% sequence identity with a rice 4CL cDNA
sequence (Genbank, L43362, bases 453-1300). cDNA inserts were
excised and recircularized using the ExAssist helper phage with
SOLR strain (Stratagene) as described by the manufacturer.
DNA Sequencing
[0235] cDNA clones were digested with 8 restriction enzymes (BamHI,
EcoRI, KpnI, NotI, PstI, SalI, XbaI, XhoI) and selected clones were
sequenced on both strands by the dideoxy chain termination method
using M13 forward and reverse primers. For sequencing the internal
regions of Lp4CL1, Lp4CL2 and Lp4CL3 synthetic oligonucleotide
primers were designed from the DNA sequences previously determined.
Sequencing was performed using the ABI dye terminator kit and
automatic sequencer. Nucleotide sequences were aligned using the
SeqEd program (ABI) and further analysis was performed using the
HIBIO DNASIS vs2 program (Hitachi Software Engineering).
Genomic DNA Blot Analysis
[0236] Genomic DNA was isolated from single genotype-derived cell
suspensions of perennial ryegrass and tall fescue according to
Lichtenstein and Draper (1985). Ten of perennial ryegrass DNA and
20 .mu.g of tall fescue DNA was digested with each of the
restriction enzymes HindIII and XbaI, separated on 1% agarose gels,
and transferred to Hybond N+membranes according to the
manufacturer's instructions (Amersham). Probes consisted of
BamHI/KpnI fragments of Lp4CL1 (1771 bp), Lp4CL2 (2034 bp) or
Lp4CL3 (2080 bp) labelled using the Megaprime labelling kit
(Amersham) and [.sup.32P]dCTP. Hybridization was performed at
65.degree. C. in 5.times.SSPE, 5.times.Denhardt's solution, 0.5%
(w/v) SDS, and 200 .mu.g/mL denatured herring sperm DNA. Membranes
were washed three times in 2.times.SSPE, 0.1% SDS for 10 min at
25.degree. C. and then twice in 0.1 X SSPE, 0.1% SDS for 20 min at
65.degree. C.
RNA Blot Analysis
[0237] Total RNA (10 .mu.g) was separated on 1.2% formaldehyde gels
and transferred to Hybond N (Amersham) membranes according to the
manufacturers instructions. Membranes were stained with 0.2%
methylene blue to confirm correct loading and transfer of RNA.
Hybridisation was performed at 42.degree. C. in 5.times.SSPE,
5.times.Denhart's solution, 0.5% SDS, 50% deionized formamide, 200
.mu.g/mL denatured herring sperm DNA. Preparation of probes and
washing of membranes was as for DNA blot analysis except for the
tall fescue Northern blot when the final two washes were performed
with 0.1.times.SSPE, 0.1% SDS for 10 min at 42.degree. C.
Results
[0238] Isolation and Sequence Analysis of Perennial Ryegrass 4CL
cDNAs
[0239] A cDNA library prepared from RNA extracted from perennial
ryegrass seedlings was screened with a rice 4CL hybridization probe
and ten cDNAs were isolated from 2.times.10.sup.5 pfu. The cDNAs
were characterised by restriction analysis with 8 restriction
enzymes. All clones were full length (approximately 2.0-2.2 kb)
with poly(A) tails and could be separated into three groups: Lp4CL1
(four clones) Lp4CL2 (five clones) and Lp4CL3 (one clone). Plasmid
maps for Lp4CL1, Lp4CL2 and Lp4CL3 are shown (FIG. 1). Lp4CL1,
Lp4CL2 and Lp4CL3 were fully sequenced (FIGS. 2, 3 and 4,
respectively).
[0240] Lp4CL1 is 2284 by long with an open reading frame (ORF) of
1710 bp, a 5' noncoding region of 322 by and a 3' noncoding region
of 252 by including a poly(A) tail. Lp4CL2 is 1992 by long with an
ORF of 1668 bp, a 5' noncoding region of 61 by and a 3' noncoding
region of 263 by including a poly(A) tail. Lp4CL3 is 2038 by long
with an ORF of 1671 bp, a 5' noncoding region of 112 by and a 3'
noncoding region of 255 by including a poly(A) tail.
[0241] Within the coding region, Lp4CL1 has 70% nucleic acid
sequence identity with both Lp4CL2 and Lp4CL3, while Lp4CL2 has 79%
sequence identity with Lp4CL3. There is little sequence homology in
the 3' noncoding regions between clones (52-55%).
Amino Acid Sequence Comparisons
[0242] The putative proteins encoded by the three cDNAs consist of
570 amino acids [60290 u (Da)] for Lp4CL1, 556 amino acids (59238
u) for Lp4CL2 and 557 amino acids (59735 u) for Lp4CL3. The deduced
amino acid sequences of Lp4CL1, Lp4CL2 and Lp4CL3 are shown (FIG.
5). Lp4CL2 and Lp4CL3 share 79% amino acid sequence identity,
Lp4CL1 and Lp4CL2 have 61% amino acid sequence identity, while
Lp4CL1 and Lp4CL3 have only 58% amino acid sequence identity.
Regions of high sequence homology are more prevalent in the central
and c-terminal regions of the enzyme. For example the sequence
identity between amino acids 208 to 568 of each enzyme is 85% for
Lp4CL2 and Lp4CL3, 72% for Lp4CL1 and Lp4CL2 and 67% for Lp4CL1 and
Lp4CL3.
[0243] Lp4CL1, Lp4CL2 and Lp4CL3 share several common regions with
other plant 4CLs. In particular, they contain the putative
AMP-binding domain and the conserved GEICIRG motif, except for
Lp4CL3 where the second isoleucine has been replaced with valine
(FIG. 5). It has been proposed that domain II is associated with
the catalytic activity of 4CL. Also, four Cys residues conserved in
plant 4CLs are conserved in Lp4CL1, Lp4CL2 and Lp4CL3 (FIG. 5).
These results suggest that the L. perenne cDNAs encode three
divergent 4CL enzymes that are likely to have originated from three
different 4CL genes.
Expression of Perennial Ryegrass 4CL Genes
[0244] Lp4CL1, Lp4CL2 and Lp4CL3 were used as hybridization probes
in Northern blots with RNA prepared from different organs of
perennial ryegrass at two developmental stages. All three probes
hybridized to a single mRNA species of approximately 2.2-2.3 kb.
Lp4CL1, Lp4CL2 and Lp4CL3 were expressed at both seedling and
mature stages of development and in all organs tested. For Lp4CL2
and Lp4CL3 the strongest signal was found in RNA samples from
seedling roots and mature stems (FIG. 6).
[0245] Lp4CL1, Lp4CL2 and Lp4CL3 were also used as hybridization
probes in Northern blots with RNA prepared from tall fescue. All
three probes hybridized to a similar mRNA species (2.3 kb) as that
in perennial ryegrass (FIG. 6). The strongest signal was found in
RNA samples from mature stems with weaker signals in RNA from roots
and seedling shoots. No expression of Lp4CL1, Lp4CL2 or Lp4CL3 was
observed in leaves. The three probes varied in their ability to
hybridize to the corresponding homologues in tall fescue, with
Lp4CL3 resulting in the highest signal and Lp4CL1 hybridizing only
weakly.
[0246] To determine whether 4CL could be induced under stress
conditions, leaves of perennial ryegrass seedlings were wounded. No
increase in the transcript level upon wounding was observed with
Lp4CL1, Lp4CL2 or Lp4CL3 (FIG. 7).
Genomic organization of perennial ryegrass 4CL genes
[0247] Perennial ryegrass DNA was digested with two restriction
enzymes, HindIII or XbaI. Restriction sites for these enzymes are
not present in the cDNA sequence of Lp4CL1, Lp4CL2 or Lp4CL3. When
Lp4CL1, Lp4CL2 or Lp4CL3 was used as a probe, several DNA
hybridizing fragments of varying intensity were revealed (FIG. 8).
Each probe hybridized to a unique set of fragments, suggesting that
Lp4CL1, Lp4CL2 and Lp4CL3 represent three different genes.
Furthermore, Lp4CL1 and Lp4CL2 hybridized to 2 to 3 major fragments
per digest which may represent either alleles of the same gene or
indicate the presence of more than one gene in each class. The
Lp4CL1, Lp4CL2 and Lp4CL3 probes also revealed several different
size hybridizing DNA fragments in genomic Southern blots from tall
fescue under high stringency conditions (FIG. 8), suggesting that
three similar 4CL genes are present in F. arundinacea.
EXAMPLE 2
Isolation and Characterisation of a Cinnamoyl CoA Reductase (CCR)
cDNA From Lolium Perenne
[0248] A total of 500,000 phage were screened from a cDNA library
constructed from ten-day-old etiolated L. perenne seedlings using a
maize CCR probe. Ninety-three positive plaques were observed in the
primary screen and five were subsequently analysed by restriction
enzyme digestion. Four out of the five were identical. One of the
four identical cDNAs, LpCCR1, was selected for further analysis
(FIG. 9).
Nucleic Acid Sequence Analysis of Perennial Ryegrass CCR cDNA
[0249] The full nucleotide sequence of LpCCR1 was obtained and the
amino acid sequence predicted (FIG. 10). LpCCR1 is a 1395 by cDNA
with 149 by of 5' non-coding region and 160 by of 3' non-coding
region. An open reading frame of 1086 by encodes a protein of 362
amino acids. The composition of the coding region was found to be
68% G+C rich. Codon usage was also examined and found to be biased
towards XXC/G codons (94%), with XCG and XUA codons accounting for
only 9% and 0.55% respectively. G+C richness and bias towards G and
C in the third position of a codon triplet are previously reported
characteristics of monocot genes.
Genomic Organization of Perennial Ryegrass CCR Gene
[0250] The number of CCR genes present in the ryegrass genome was
determined by Southern blot analysis of genomic DNA from double
haploid plants, using as probe a fragment of the LpCCR1 cDNA
(LpCCR531, FIG. 9). Double haploid DNA reduces the complexity
associated with allelic variation. Genomic DNA was cut with enzymes
that do not cut the cDNA internally; DraI, BamHI, EcoRI, EcoRV,
HindIII and XbaI, and the membrane was hybridised and washed under
medium-stringency conditions. A single strongly hybridising band
was evident in each lane (FIG. 11) indicating that there is a
single copy of the LpCCR1 gene in the perennial ryegrass
genome.
Expression of Perennial Ryegrass CCR Gene
[0251] To investigate the expression profile of the CCR gene in
ryegrass, northern hybridisation analysis was carried out with
total RNA extracted from roots and shoots at seedling growth stages
(0.5-1 cm and 4-6 cm shoots) and roots, stem and leaves at mature
growth stages (6 and 10 weeks). Seedlings were grown on filter
paper in the dark at 25.degree. C. and then transferred to soil and
glasshouse conditions (25.degree. C.) until the 6 and 10-week
stages. Whole seedling total RNA from Festuca and Phalaris was
included in the northern analysis. Hybridisation with LpCCR531
(FIG. 9) was performed at medium-stringency and the membrane was
then washed at high-stringency. A transcript of approximately 1.5
kb was detected in all tissues, the level of expression varying
with maturity and from one tissue type to another (FIG. 12). The
LpCCR1 transcript appears to be more abundant in roots and stem
than shoots and leaves. In the stem, transcript abundance increases
from 6-weeks to 10-weeks; indicating that transcription in stem
tissue is up-regulated as the plant matures. Expression was found
predominantly in tissues such as stems and roots that are forming
secondary cell walls indicating that LpCCR1 is constitutively
involved in lignification.
EXAMPLE 3
Isolation and Characterisation of Cinnamyl Alcohol Dehydrogenase
(CAD) cDNAs from Lolium Perenne
[0252] A 558 by cinnamyl alcohol dehydrogenase (CAD) fragment was
amplified from cDNA synthesised from total RNA prepared from
perennial ryegrass seedlings. The conserved amino acid domains
between Pinus radiata, Medicago sativa, Aralia cordata, Eucalyptus
botryoides and Arabidopsis thaliana CADs were used to design
oligonucleotides for the amplification of the perennial ryegrass
CAD. The forward oligonucleotide was designed to the conserved
amino acid domain CAGVTVYS and the reverse oligonucleotide to the
conserved domain DVRYRFV. The 551 by PCR fragment was cloned and
sequenced to confirm that it corresponded to a perennial ryegrass
CAD PCR fragment. A cDNA library prepared from RNA extracted from
perennial ryegrass seedlings was screened with the 551 bp PCR
fragment specific for perennial ryegrass CAD. Eight cDNAs were
isolated and separated into six groups by restriction digest
analysis. One representative clone each from two groups (LpCAD1,
LpCAD2) were selected for further characterisation.
Nucleic Acid Sequence Analysis of Perennial Ryegrass CAD cDNAs
[0253] The complete sequence of the perennial ryegrass CAD
homologue LpCAD1 was determined (FIG. 13). The 1325 by clone had a
poly (A) tail, typical start and stop codons and the open reading
frame (ORF) of this clone coded for a putative protein of 408 amino
acids.
[0254] The complete nucleotide sequence of the perennial ryegrass
CAD homologue LpCAD2 was also determined (FIG. 14).
Expression Of Perennial Ryegrass CAD Genes
[0255] A northern hybridisation analysis with RNA samples isolated
from perennial ryegrass at different developmental stages
hybridised with the full length LpCAD1 1325 by cDNA (FIG. 15) was
performed to determine patterns of organ and developmental
expression. The probe hybridised to a single mRNA species of
approximately 1.6 kb. The LpCAD1 transcript was expressed in all
tissue tested: roots, shoots, stem and leaves (FIG. 16A). The
LpCAD1 transcript was most abundant in root tissue and the mature
stem, this expression pattern is typical of a gene involved in the
lignification of plant cell walls. Intergeneric homologies were
revealed in Festuca and Phalaris.
[0256] A similar northern hybridisation analysis was performed with
LpCAD2 (FIG. 16B), however the transcript was found to be most
abundant in mature stem tissue and the shoots.
Genomic Organization of Perennial Ryegrass CAD Genes
[0257] A Southern hybridisation analysis using DNA samples isolated
from a perennial ryegrass double haploid plant digested with DraI,
BamHI, EcoRI, EcoRV, HindIII and XbaI and hybridised with a 500 by
LpCAD1 probe was performed. The hybridisation pattern at high
stringency revealed the presence of two prominent bands for most
digests indicating that LpCAD1 belongs to a small gene family and
exists a muliticopy gene in perennial ryegrass (FIG. 17A).
[0258] A similar Southern hybridization analysis was performed with
LpCAD2 (FIG. 17B) the hybridisation pattern at high stringency
revealed the presence of one or two prominent bands for most
digests indicating that LpCAD2 exists as a single copy gene or a
member of a small gene family in perennial ryegrass (FIG. 17B).
EXAMPLE 4
Isolation and Characterisation of Genomic Clones and Promoters for
O-Methyltransferase (OMT), Cinnamoyl-CoA Reductase (CCR), 4
Coumarate CoA-Ligase (4CL) and Cinnamyl Alcohol Dehydrogenase (CAD)
from Lolium Perenne
[0259] Genomic clones and promoters of O-methyltransferase (OMT),
cinnamoyl-CoA reductase (CCR), 4 coumarate CoA-ligase (4CL) and
cinnamyl alcohol dehydrogenase (CAD) were isolated from a perennial
ryegrass genomic library using the corresponding cDNAs as
hybridisation probes.
Isolation and Characterisation of Genomic Clones and Promoters for
Perennial Ryegrass O-Methyltransferase (OMT)
[0260] A perennial ryegrass genomic library was screened with the
cDNA clone, LpOmt1, (Heath et al. 1998) encoding
O-methyltransferase (OMT). The sequence of the 5' untranslated
region and the coding region was found to be identical to that of
the LpOmt1 cDNA previously isolated. The entire 4.8 kb genomic
clone was fully sequenced (FIG. 18).
[0261] To further characterise the promoters, transcriptional
fusions of the promoter sequence to the .beta.-glucuronidase (GUS)
coding sequence (gusA) have been generated (FIG. 19). Direct gene
transfer experiments to tobacco protoplasts were performed with the
corresponding chimeric genes to transgenically express them in a
heterologous system for in planta expression pattern analysis by
histochemical GUS assays. A set of transgenic tobacco plants
carrying a chimeric gusA gene under the control of the 5'
regulatory region of the LpOmt1 promoter was generated to assess
the potential use of the LpOmt1 promoter for xylem-specificity and
targeted downregulation of genes encoding key lignin biosynthetic
enzymes.
[0262] The transgenic tobacco plants generated using the LpOmt1
promoter driven chimeric gusA transformation vector were screened
by PCR and histochemical GUS assays.
[0263] A PCR screening was undertaken using gusA specific primers
for the initial identification of transgenic tobacco plants (FIG.
20). PCR positive tobacco plants were screened by histochemical GUS
assays for in planta expression pattern analysis (FIG. 20).
Isolation and Characterisation of Genomic Clones and Promoters for
Perennial Ryegrass Cinnamoyl-CoA Reductase (CCR)
[0264] A CCR genomic clone from perennial ryegrass was isolated
containing 6.5 kb of promoter and the entire gene organisation
(intron/exon boundaries). The CCR promoter can be used for targeted
expression of foreign genes in transgenic plants.
[0265] A perennial ryegrass genomic library was screened with the
cDNA clone LpCCR1 which codes for the lignin biosynthetic enzyme,
cinnamyl-CoA reductase (CCR). Four different genomic clones were
identified based on restriction digest analysis. Clone 6.1.1a was
selected for further analysis. A 6.42 kb XhoI fragment from clone
6.1.1a, which hybridized strongly to the LpCCR1 cDNA probe, was
subcloned into pBluescriptSK (FIG. 21A). Sequence analysis revealed
that the 6.42 kb XhoI fragment contained the entire LpCCR1 gene and
200 by of promoter region. The intron/exon boundaries are
illustrated in FIG. 21B, the location and the size of the exons
appear to be conserved in other CCRs from different species (FIG.
21C).
[0266] To isolate the promoter region of LpCCR1, the Southern blot
containing digested phage genomic DNA isolated from clone
.lamda.Lp6.1.1a was reprobed with the 200 bp promoter region. The
probe hybridized strongly to a 6.5 kb SalI fragment. This genomic
fragment LpCCR1 clone 2, was subcloned into pBluescriptSK and
sequenced (FIG. 22A). Sequence results revealed that the 6.5 kb
SalI fragment contained 6.5 kb of promoter (FIG. 22B). The full
sequence of LpCCR1 genomic clone containing the promoter and entire
gene sequence (exons and introns) was obtained and is shown on FIG.
39.
Isolation and Characterisation of Genomic Clones and Promoters for
Perennial Ryegrass 4 Coumarate CoA-Ligase (4CL)
[0267] A 4CL2 genomic clone from perennial ryegrass was isolated
containing 2.5 kb of promoter and partial gene organisation
(intron/exon boundaries). The 4CL2 promoter can be used for
targeted expression of foreign genes in transgenic plants. The 2.5
kb promoter has been fused to the reporter gene gusA for expression
analysis.
[0268] A perennial ryegrass genomic library was screened with an
Lp4CL cDNA probe. After tertiary screening positive 4CL genomic
clones were obtained and characterised by restriction digest and
Southern hybridisation analysis (FIG. 23A).
[0269] Sequence analysis revealed that the isolated 4CL genomic
clone (4CL2) from perennial ryegrass had 100% nucleotide identity
to the Lp4CL2 cDNA clone. To further characterise this 5 kb
.lamda.Lp4CL2 genomic clone and to confirm that it corresponds to
the cDNA of Lp4CL2, a number of PCR reactions using primers
designed to the cDNA were used. PCR results confirmed that the 5 kb
genomic fragment was a partial genomic clone corresponding to the
Lp4CL2 cDNA (FIG. 23B). Using primer combinations F1 and R1 the
entire 4.8 kb genomic fragment was amplified. To determine the
location of introns additional PCR reactions using the primer
combinations F1/R2 and F2/R1 were performed, a 1 kb and 3.5 kb
bands were amplified respectively. The location and size of the
introns could be determined from these results, and further
confirmed by sequence analysis. This large 5 kb genomic fragment
contains 4 small exons representing the coding sequence of Lp4CL2
between 508 by and 1490 by (FIG. 23C).
[0270] The genomic clone 1, Lp4CL2 contained no promoter region. To
isolate the promoter region of Lp4CL2, the Southern blot containing
digested phage genomic DNA isolated from clone .lamda.Lp4CL2 was
reprobed with a 300 by EcoRI/BglI isolated from the 5' end of the
cDNA clone Lp4CL2. The 300 by probe hybridised strongly to a 2.5 kb
BamHI fragment. This genomic fragment Lp4CL2 clone 2, was subcloned
into pBluescriptSK and sequenced (FIG. 24A). Sequence results
revealed that the 2.5 kb BamHI fragment contained the 508 by of the
5' ORF of Lp4CL2 missing from genomic clone 1 and 2.0 kb of
promoter region (FIG. 24B). The full sequence of the Lp4CL2 genomic
clone containing the promoter and partial gene sequence (exons and
introns) was obtained and is shown on FIG. 39.
[0271] The promoter from Lp4CL2 was thus isolated and used for the
production of a chimeric gusA reporter gene (FIG. 25).
Isolation and Characterisation of Genomic Clones and Promoters for
Perennial Ryegrass Cinnamyl Alcohol Dehydrogenase (CAD)
[0272] A CAD genomic clone from perennial ryegrass was isolated
containing the gene organisation (intron/exon boundaries) minus
intron 1 containing the first 111 by of the CAD coding region. The
genomic clone has allowed the identification of a G at position 851
by in the coding region of the CAD2 genomic clone isolated from
perennial ryegrass cv. Barlano which is absent in the CAD2 cDNA
clone isolated from perennial ryegrass cv. Ellett. The SNP (single
nucleotide polymorphism) found to exist between the 2 cultivars has
the potential utility as a molecular marker for herbage quality,
dry matter digestibility, mechanical stress tolerance, disease
resistance, insect pest resistance, plant stature and leaf and stem
colour.
[0273] Results below show the isolation of the genomic clone and
sequence analysis of deduced coding sequence from the genomic clone
CAD2 from perennial ryegrass cv. Barlano compared to the truncated
cDNA CAD2 from the cv Ellett. The missing G in the perennial
ryegrass cv. Ellett has been highlighted (FIGS. 26 and 27).
[0274] A perennial ryegrass genomic library was screened with a
probe corresponding to the 5' end of the LpCAD2 cDNA clone, which
codes for the lignin biosynthetic enzyme cinnamyl alcohol
dehydrogenase. Ten positive plaques were identified and isolated in
the primary library screening. After a secondary and tertiary
screening, two positive plaques were obtained and corresponding
positive genomic clones were further characterised by restriction
digest and Southern hybridization analyses. Both genomic clones
were found to be identical based on restriction digest analyses.
One clone, named .lamda.LpCAD2 was chosen for further Southern
hybridization analyses. A 4.5 kb BamHI fragment which hybridized
strongly to the LpCAD2 cDNA probe was subcloned into pBluescriptSK
and sequenced (FIG. 28A). Sequence analysis revealed that the 4.5
kb BamHI fragment was a partial genomic clone of LpCAD2. This large
4.5 kb genomic fragment contains 4 small exons representing the
coding sequence of LpCAD2 between 213 by and the stop codon at 1213
bp, and the location of the intron/exon boundaries are illustrated
in FIG. 28B.
EXAMPLE 5
Development of Transformation Vectors Containing Chimeric Genes
with 4CL, CCR and CAD cDNA Sequences from Perennial Ryegrass
[0275] To alter the expression of the key enzymes involved in
lignin biosynthesis 4CL, CCR and CAD, through antisense and/or
sense suppression technology and for over-expression of these key
enzymes in transgenic plants, a set of sense and antisense
transformation vectors was produced. Transformation vectors
containing chimeric genes using perennial ryegrass 4CL, CCR and CAD
cDNAs in sense and antisense orientations under the control of
either the CaMV 35S or the maize ubiquitin promoter were generated
(FIGS. 29, 30 and 31).
EXAMPLE 6
Production and Characterisation of Transgenic Tobacco Plants
Expressing Chimeric 4CL, CCR and CAD Genes from Perennial
Ryegrass
[0276] A set of transgenic tobacco plants carrying chimeric 4CL,
CCR and CAD genes from perennial ryegrass were produced and
analysed.
[0277] Transformation vectors with Lp4CL1, Lp4CL2 and Lp4CL3 full
length cDNA sequences in sense and antisense orientations under the
control of either the CaMV 35S or the maize ubiquitin promoters
were generated. Transformation vectors with LpCCR1 cDNA in both
sense and antisense orientation under the control of either the
CaMV 35S and maize ubiquitin promoters were generated.
Transformation vectors with 1325 by full length LpCAD1 cDNA in
sense and 1051 by partial LpCAD1 cDNA in antisense orientation
under the control of either the CaMV 35S and maize ubiquitin
promoters were generated.
[0278] Direct gene transfer experiments to tobacco protoplasts were
performed using these transformation vectors.
[0279] The production and molecular analysis of transgenic tobacco
plants carrying the perennial ryegrass Lp4CL1 and LpCCR1 cDNAs
under the control of the constitutive CaMV 35S promoter is
described here in detail.
[0280] A set of transgenic tobacco plants generated using the
Lp4CL1 sense transformation vector was screened by PCR and
subjected to Southern and northern hybridization analyses.
[0281] A PCR screening was undertaken using npt2 and Lp4CL1
specific primers for the initial identification of transgenic
tobacco plants. Independent transgenic tobacco plants were
identified to be co-transformed with both the selectable marker
npt2 and the Lp4CL1 chimeric genes (FIG. 32).
[0282] Southern hybridisation analysis was performed with DNA
samples from PCR positive transgenic tobacco plants to demonstrate
the integration of the chimeric Lp4CL1 transgene in the tobacco
plant genome. Independent transgenic tobacco plants carried between
1 and 5 copies of the Lp4CL1 transgene. No cross-hybridization was
observed between the endogenous tobacco 4CL gene and the perennial
ryegrass hybridization probe used (FIG. 32).
[0283] Northern hybridization analysis using total RNA samples
prepared from the transgenic tobacco plants carrying the chimeric
sense Lp4CL1 transgene and probed with the Lp4CL1-specific
hybridization probe revealed the presence of a 1.2 kb Lp4CL1
transcript strongly expressed in one Lp4CL1-transgenic tobacco
plant analysed (FIG. 32).
[0284] The sense and antisense transformation vectors of LpCCR1
under the control of the CaMV 35S promoter were introduced into
tobacco protoplasts via direct gene transfer. A set of transgenic
tobacco plants was generated and screened by PCR with specific
primers to identify transgenic tobacco plants carrying chimeric
LpCCR1 transgene. The molecular analysis of LpCCR1-transgenic
tobacco plants is shown (FIG. 33).
EXAMPLE 7
Production and Characterisation of Transgenic Perennial Ryegrass
Plants Expressing Chimeric OMT, 4CL, CCR and CAD Genes from
Perennial Ryegrass
[0285] An improved transformation method was developed for the
production of transgenic perennial ryegrass plants by biolistic
transformation of embryogenic cells. Transgenic perennial ryegrass
plants were generated using chimeric OMT, 4CL, CCR and CAD genes
from perennial ryegrass and the improved transformation method.
Improved Method for the Production of Transgenic Perennial Ryegrass
Plants
[0286] This improved procedure utilises embryogenic calli produced
from mature seed-derived embryos as direct targets for biolistic
transformation without requiring the establishment of embryogenic
cell suspensions. The protocol relies on a continuous supply of
isolated zygotic embryos for callus induction. Transgenic ryegrass
plants can be regenerated 24-28 weeks after embryo isolation (FIG.
34). Isolated embryos are plated onto MSM5 medium to produce
embryogenic calli suitable as targets for biolistic transformation
within 8 weeks. The embryogenic calli, treated on high-osmoticum
medium MSM3 Plus prior to microprojectile bombardment, are selected
on MSM3 medium containing 100 mg/l paromomycin (Pm) for 2 weeks
before being transferred onto MSK with 100 mg/l Pm for further 4
weeks until differentiation of Pm resistant shoot appear.
Regenerated shoots are transferred on to fresh selective media MSK
with 100 mg/l Pm for a further 4 weeks (FIG. 34).
Production of Transgenic Perennial Ryegrass Plants Expressing
Chimeric OMT, 4CL, CCR and CAD Genes from Perennial Ryegrass
[0287] Transgenic perennial ryegrass (Lolium perenne) plants were
generated using chimeric ryegrass OMT, 4CL, CCR and CAD genes by
biolistic transformation of embryogenic calli. Examples of the
production and detailed molecular analysis of these transgenic
ryegrass plants are described.
[0288] Transgenic perennial ryegrass plants for OMT down-regulation
were produced using biolistic transformation of embryogenic calli
and plant transformation vectors pUbiomt1 and pUbitmo1 carrying
LpOmt1 cDNA sequence in sense and antisense orientation under
control of the constitutive maize ubiquitin promoter. These
transgenic perennial ryegrass plants for down-regulated OMT
activity were regenerated from paromomycin resistant calli obtained
from biolistic transformation using microprojectilies coated with
two plasmids; pHP23 (carrying the chimeric npt2 gene as the
selectable marker) and either the sense or antisense LpOmt1
transformation vector driven by the maize Ubi promoter.
[0289] Transgenic perennial ryegrass plants were subjected to a
polymerase chain reaction (PCR) screening using npt2-specific
primers. Independent npt2 PCR-positive transgenic perennial
ryegrass plants obtained from biolistic transformation of
embryogenic calli-generated from approximately 60,000 isolated
mature seed-derived embryos--using LpOmt1 sense (pUbiomt1) and
LpOmt1 antisense (pUbitmo1) transformation vectors were identified
[16 pUbiomt1 transgenic plants and 27 pUbitmo1 transgenic plants]
(FIG. 35).
[0290] Southern hybridization analysis was performed with
undigested and HindIII -digested DNA samples prepared from the PCR
positive transgenic perennial ryegrass plants, to demonstrate their
transgenic nature and the integration of the chimeric npt2 and
LpOmt1 transgenes. Independent transgenic perennial ryegrass plants
co-transformed with both, the selectable marker npt2 gene and
LpOmt1 chimeric genes, were identified (FIG. 35). In most
instances, the transgenic perennial ryegrass plants recovered
contained multiple copies of the selectable marker gene including
rearranged transgene copies. No npt2-hybridizing bands were
detected in the untransformed negative control.
[0291] Samples of HindIII-digested genomic DNA were included in the
analysis when the LpOmt1 gene-specific hybridization probe (omt1)
was used. The omt1probe hybridized to a number of bands in DNA
samples corresponding to both, the transgenic plants and the
untransformed negative control. The omt1-hybridizing bands shared
in all samples correspond to endogenous LpOmt1 gene sequences
represented as a small multigene family in the perennial ryegrass
genome (Heath et al. 1998). The different omt1-hybridizing bands
evident in the samples from the transgenic plants and absent in the
untransformed negative control sample correspond to antisense
(tmo1) and sense (omt1) LpOmt1 transgene integration events (FIG.
35).
[0292] Northern hybridization analysis using strand-specific LpOmt1
probes allowed the identification of transgenic perennial ryegrass
plants expressing the antisense LpOmt1 transgene (FIG. 35).
[0293] The OMT activity of selected antisense and sense LpOmt1
transgenic perennial ryegrass plants was determined. Biochemical
assays for OMT activity were initially established in untransformed
plants (such as tobacco and perennial ryegrass). The assays utilise
radiolabelled S-adenosylmethionine as the methyl donor for the
OMT-catalysed conversion of caffeic acid into ferulic acid. The
production of radioactive ferulic acid is measured and allows the
OMT activity to be determined.
[0294] The OMT activity of selected LpOmt1-transgenic perennial
ryegrass plants (L. perenne cv. Ellett) was determined.
Significantly altered OMT activity in individual transformation
events was observed (FIG. 36). The manipulation of OMT activity in
transgenic perennial ryegrass plants due to the expression of the
chimeric ryegrass LpOmt1 gene was thus demonstrated.
[0295] Transgenic perennial ryegrass plants were recovered, using
biolistic transformation of embryogenic calli, for the manipulation
of the expression of genes encoding the key lignin biosynthetic
enzyme, 4CL. The plant transformation vectors pUbi4CL2 and pUbi2LC4
carrying chimeric Lp4CL2 cDNA sequences in sense and antisense
orientation, respectively, driven by the constitutive maize
ubiquitin (Ubi) promoter were used. Perennial ryegrass plants for
4CL manipulation were regenerated from Pm-resistant calli obtained
from biolistic transformation of embryogenic calli using
microprojectiles coated with the plasmids pHP23, carrying a
chimeric npt2 gene as selectable marker gene and the antisense
pUbi2LC4.
[0296] Transgenic perennial ryegrass plants were subjected to a
polymerase chain reaction (PCR) screening using npt2-specific
primers. Independent npt2 PCR-positive transgenic perennial
ryegrass plants were obtained from biolistic transformation of
embryogenic calli (FIG. 37).
[0297] Transgenic perennial ryegrass plants were also recovered,
using biolistic transformation of embryogenic calli, for the
manipulation of the expression of genes encoding the key lignin
biosynthetic enzymes, CCR and CAD.
EXAMPLE 8
Genetic Mapping of Perennial Ryegrass OMT, 4CL, CCR and CAD
Genes
[0298] Lp4CL1, Lp4CL3, LpCAD1, LpCAD2, LpCCR1, LpOMT1 and LpOMT2
clones were PCR amplified and radio-labelled for use as probes to
detect restriction fragment length polymorphisms (RFLPs). RFLPs
were mapped using 110 progeny individuals of the p150/112 perennial
ryegrass reference population restricted with the enzymes described
in Table 3 below.
TABLE-US-00003 TABLE 3 Mapping of RFLPs Enzyme Polymorphic mapped
Linkage Clones in p150/112 with Locus group Lp4CL1 Y Dral Lp4CL1 2
Lp4CL3 Y EcoRV Lp4CL3 6 LpCAD1 Y EcoRV LpCAD1 2 LpCAD1.2.1 Y EcoRI
LpCAD2a 7 LpCAD2b -- LpCAD2c 2 LpCCR1 Y EcoRI LpCCR1 7 LpOMT1 Y
Dral LpOMT1 7 LpOMT2 Y EcoRV LpOMT2 6
[0299] Lp4CL1, Lp4CL3, LpCAD1, LpCAD2, LpCCR1, LpOMT1 and LpOMT2
loci mapped to the linkage groups as indicated in Table 3 and in
FIG. 40. These gene locations can now be used as candidate genes
for quantitative trait loci for lignin biosynthesis associated
traits such as herbage quality, dry matter digestibility,
mechanical stress tolerance, disease resistance, insect pest
resistance, plant stature and leaf and stem colour.
EXAMPLE 9
Sense Suppression
DNA Sequence Elements and Construct Production
[0300] Three constructs were engineered for development of
transgenic perennial ryegrass with modified lignin biosynthesis,
using sense suppression technology. The individual components of
the sequence elements are listed in Table 4. The promoters and
terminators used in construct production originated from perennial
ryegrass genomic sequences. The genes were derived from perennial
ryegrass cDNA sequences. The origin of the pAUX plasmid vectors has
been described previously (Goderis et al., 2002).
TABLE-US-00004 TABLE 4 Components used in the generation of
constructs for perennial ryegrass transformation. Vector Construct
No. backbone Promoters Genes Terminators 1 pAUX3132 LpCAD2 LpCAD3
LpCAD2 2 pAUX3169 LpCCR1 LpCCR1 LpCCR1 3 pAUX3169 LpCCR1 LpCCR1(fs)
LpCCR1
[0301] The constructs were produced using Gateway.TM. technology
(Invitrogen). The Gateway.TM. cloning system consists of one vector
backbone and several auxiliary vectors based on pUC18 (Goderis et
al., 2002). The multisite recombination cassette was assembled in
the auxiliary vectors utilizing the multi-cloning site, flanked by
homing endonuclease sites (FIG. 41). Homing endonucleases are rare
cutting restriction enzymes minimising the risk of accidental
restriction within the expression cassettes if excision of the
expression cassette is required.
[0302] The respective promoter, cDNA and terminator sequences were
amplified by PCR using primers incorporating the appropriate AttB
recombination sequences and cloned into separate Gateway.TM. Entry
vectors. For example, three Entry clones were required for the
generation of the LpCAD expression vector (Construct 1); the LpCAD3
cDNA (FIG. 42), the LpCAD2 promoter (FIG. 43) and the LpCAD2
terminator (FIG. 44). These were then combined with pAUX3132 for
the multi-recombination reaction and generation of the expression
cassette pAUX3132-LpCAD2::LpCAD3::LpCAD2 (FIG. 45).
[0303] For Construct 2, Entry clones with the individual
components; LpCCR1 promoter, LpCCR1 cDNA, and LpCCR1 terminator
were generated using the same PCR cloning strategy. The Entry
clones were combined in a recombination cloning reaction with base
vector pAUX3169 to produce the final construct
pAUX3169-LpCCR1::LpCCR1::LpCCR1 (FIG. 46).
[0304] For Construct 3, an alternative silencing strategy was
employed involving a frame-shift based approach. This method
involves the deletion of a single base pair, just downstream of the
start site, which is introduced using a forward primer which has
the single base deletion (FIG. 47). This construct works via sense
suppression, as the transcript produced will not encode the correct
protein and no functional protein will be produced.
[0305] The Entry clones with individual components LpCCR1 promoter,
LpCCR1(fs) cDNA, and LpCCR1 terminator were generated and combined
in a recombination cloning reaction with base vector pAUX3169 to
produce the final construct pAUX3169-LpCCR1::LpCCR1(fs)::LpCCR1
(FIG. 48).
[0306] The plant selectable marker which facilitates selection of
putative transgenic ryegrass on the antibiotic Hygromycin B is
contained on a separate plasmid, pAcH1. This plasmid utilizes the
rice Actin1D promoter to drive in planta expression of the
hygromycin phosphotransferase (hph) gene. The pAcH1 plasmid has
been used previously in the transformation of forage grasses
(Spangenberg et al., 1995).
Transformation Protocols
[0307] The protocol developed and established is based on the
biolistic transformation of embryogenic calli induced from immature
inflorescences isolated from an in planta maintained vernalised
collection of perennial ryegrass, or seedling meristems derived
from in vitro seedling cultures. Illustrations of the different
stages in both processes, from the isolation of explants for the
induction and proliferation of embryogenic calli for genetic
transformation to the recovery of transgenic plants are shown in
FIGS. 50 and 51. Both genetic transformation methods allow for a
sustainable, readily-available source of donor plant materials
which are highly competent for plant regeneration and genetic
transformation and are compatible with biolistic transformation
techniques. A general outline of the process involved in
transformation is described in FIG. 52.
Molecular Analysis of Putative Transgenic Plants
[0308] Molecular analysis of putative transgenic perennial ryegrass
plants has been conducted using primers for Q-PCR. The following
primers were designed: [0309] 1. Primers specific for the hph gene
[0310] 2. Primers across the CAD2 promoter-CAD3 gene junction
[0311] 3. Primers specific for the pAUX3169 vector (as primers
specific for the CCR1 junctions could also amplify endogenous
genomic sequences).
[0312] An example of Q-PCR run for detection of hph in extracted
genomic DNA is shown in FIG. 53.
[0313] The results summarising the number of transgenic perennial
ryegrass plants for each Construct is shown in Table 5.
TABLE-US-00005 TABLE 5 Summary of transformation progress for
perennial ryegrass lines harbouring constructs for the modification
of lignin biosynthesis. No. Putative No. hph No. GOI Construct
Vector Transgenics positive positive 1
pAUX3132-LpCAD2::LpCAD3-LpCAD2 180 65 25 2
pAUX3169-LpCCR1::LpCCR1::LpCCR1 90 67 38 3
pAUX3169-LpCCR1::LpCCR1(fs)::LpCCR1 322 185 141 Total 592 317
204
Down-Regulation of CAD and CCR Expression by RNA Interference and
Sense Suppression
[0314] In order to modify the expression level of LpCCR1 in
perennial ryegrass, an RNA-mediated posttranscriptional gene
silencing strategy was employed (RNA interference). The maize
Ubiquitin (Ubi) promoter was used to drive expression of a LpCCR1
hairpin (hp) construct containing the variable region of 3' UTR in
transgenic perennial ryegrass. Immature inflorescence-derived calli
of perennial ryegrass were used as a target for biolistic
transformation. hpLpCCR1 transgenic ryegrass plants were confirmed
by Southern analysis (FIG. 54).
[0315] In the same manner, CAD and CCR expression is modified in
perennial ryegrass using constructs 1, 2 and 3 (sense
suppression).
Analysis of Lignin in Transgenic Plants
[0316] Lignin content and composition is visualised by specialized
staining methods, including Maule histochemical staining which can
differentiate between G-lignin and S-lignin monomers (Moore et al.,
1991). Maule staining of flowering stems from different internodes
was conducted for wild type and Ubi::hpLpCCR1 transgenic perennial
ryegrass. The results demonstrate that there is significantly less
lignin accumulating in stems at both the early reproductive (R1)
and mid-reproductive (R2) stages (FIG. 55). Furthermore, there is
an acropetal (base to apex) decrease in the relative amount of
total lignin in the different internodes.
[0317] In the same manner, lignin content and composition is
analysed in transgenic perennial ryegrass lines harbouring
constructs 1, 2 and 3.
[0318] Additional lignin analytical methods includes isolation of
cell wall material by successive hot water, ethanol and
chloroform/methanol extractions (Fukushima and Hatfield, 2001)
followed by determination of total lignin content/dry weight, using
acetyl bromide method (Liyama and Wallis, 1990) (FIG. 56).
[0319] Further lignin monomer analysis to determine the G/S ratio
is performed by thioacidoylysis cleavage method (Rolando et al.,
1992) and quantification using a gas chromatography (GC-MS) (FIG.
57).
REFERENCES
[0320] Fukushima, R. S. and R. D. Hatfield (2001). "Extraction and
isolation of lignin for utilization as a standard to determine
lignin concentration using acetyl bromide spectrophotometric
method." J. Agri. Food Chem. 49: 3133-3139. [0321] Goderis, I., M.
De Bolle, I. Francois, P. Wouters, W. Broekaert and B. Cammue
(2002). "A set of modular plant transformation vectors allowing
flexible insertion of up to six expression units." Plant Mol Biol
50: 17-27. [0322] Heath et al (1988) cDNA cloning and differential
expression of three caffeic acid O-methyltransferase homologues
from perennial ryegrass (Lolium perenne). Journal of Plant
Physiology 153:649-657 [0323] Lichtenstein, C, And J. Draper (1985)
Genetic engineering of plants. In: D. M. Glover (ed.), DNA Cloning,
Vol. 2, pp. 67-119, IRL Press, Washington. [0324] Liyama, K. and A.
F. A. Wallis (1990). "Determination of lignin in herbaceous plants
by an improved acetyl bromide procedure." J Sci Food Agric 51:
145-161. [0325] Moore, K. J., L. E. Moser, K. P. Vogel, S. S.
Waller, Johnson B. E. and P. J. F. (1991). "Describing and
quantifying growth stages of perennial forage grasses." Agron. J.
83: 1073-1077. [0326] Rolando, C., B. Monties and C. Lapierre
(1992). Thioacidolysis. Methods in Lignin Chemistry S. Y. Lin and
C. W. Dence, Springer-Verlag: pp. 334-349. [0327] Spangenberg, G.,
Z. Y. Wang, X. L. Wu, J. Nagel, V. A. Iglesias and I. Potrykus
(1995). "Transgenic tall fescue and red fescue plants from
microprojectile bombardment of embryogenic suspension cells." J
Plant Physiol 145: 693-701.
[0328] Finally, it is to be understood that various alterations,
modifications and/or additions may be made without departing from
the spirit of the present invention as outlined herein.
[0329] It will also be understood that the term "comprises" (or its
grammatical variants) as used in this specification is equivalent
to the term "includes" and should not be taken as excluding the
presence of other elements or features.
[0330] Documents cited in this specification are for reference
purposes only and their inclusion is not an acknowledgement that
they form part of the common general knowledge in the relevant art.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20100287660A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20100287660A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References