U.S. patent application number 17/091776 was filed with the patent office on 2021-02-25 for als inhibitor herbicide tolerant beta vulgaris mutants.
The applicant listed for this patent is BAYER INTELLECTUAL PROPERTY GMBH, KWS SAAT AG. Invention is credited to Juergen BENTING, Guenter DONN, Ruediger HAIN, Bernd HOLTSCHULTE, Rudolf JANSEN, Nathalie KNITTEL-OTTLEBEN, Andreas LOOCK, Clemens SPRINGMANN.
Application Number | 20210054360 17/091776 |
Document ID | / |
Family ID | 1000005197678 |
Filed Date | 2021-02-25 |
United States Patent
Application |
20210054360 |
Kind Code |
A1 |
HAIN; Ruediger ; et
al. |
February 25, 2021 |
ALS INHIBITOR HERBICIDE TOLERANT BETA VULGARIS MUTANTS
Abstract
The present invention relates to an ALS inhibitor herbicide
tolerant Beta vulgaris plant and parts thereof comprising a
mutation of an endogenous acetolactate synthase (ALS) gene, wherein
the ALS gene encodes an ALS polypeptide containing an amino acid
different from tryptophan at a position 569 of the ALS
polypeptide.
Inventors: |
HAIN; Ruediger; (Frankfurt,
DE) ; BENTING; Juergen; (Leichlingen, DE) ;
DONN; Guenter; (Karlsruhe, DE) ; KNITTEL-OTTLEBEN;
Nathalie; (Kriftel, DE) ; HOLTSCHULTE; Bernd;
(Einbeck, DE) ; LOOCK; Andreas; (Einbeck, DE)
; SPRINGMANN; Clemens; (Bad Gandersheim, DE) ;
JANSEN; Rudolf; (Einbeck, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BAYER INTELLECTUAL PROPERTY GMBH
KWS SAAT AG |
Monheim
Einbeck |
|
DE
DE |
|
|
Family ID: |
1000005197678 |
Appl. No.: |
17/091776 |
Filed: |
November 6, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13821969 |
Mar 8, 2013 |
10865406 |
|
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PCT/EP2011/067925 |
Oct 13, 2011 |
|
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17091776 |
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61394463 |
Oct 19, 2010 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/01 20130101;
A01H 1/06 20130101; A01H 1/04 20130101 |
International
Class: |
C12N 15/01 20060101
C12N015/01; A01H 1/06 20060101 A01H001/06; A01H 1/04 20060101
A01H001/04 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 15, 2010 |
EP |
10187751.2 |
Claims
1. An ALS inhibitor herbicide tolerant Beta vulgaris plant and
parts thereof comprising a mutation of an endogenous acetolactate
synthase (ALS) gene, wherein the ALS gene encodes an ALS
polypeptide containing an amino acid different from tryptophan at a
position 569 of the ALS polypeptide.
2. The Beta vulgaris plant and parts thereof according to claim 1,
in which the ALS polypeptide has at position 569 the amino acid
alanine, glycine, isoleucine, leucine, methionine, phenylalanine,
proline, valine, or arginine instead of the naturally encoded amino
acid tryptophan.
3. The Beta vulgaris plant and parts thereof according to claim 1,
wherein the ALS gene encodes an ALS polypeptide containing an amino
acid different from tryptophan at a position 569 of the ALS
polypeptide sequence as set forth in SEQ ID NO: 2.
4. The Beta vulgaris plant and parts thereof according to claim 1,
in which the amino acid is leucine and in which the endogenous ALS
gene is identical to the nucleotide sequence defined by SEQ ID NO:
3.
5. The Beta vulgaris plant and parts thereof according to claim 1,
which are tolerant to one or more ALS-inhibitor herbicides
belonging to the group consisting of sulfonylurea herbicides,
sulfonylaminocarbonyltriazolinone herbicides, imidazolinone
herbicides, triazolopyrimidine herbicides, and
pyrimidinyl(thio)benzoate herbicides.
6. The Beta vulgaris plant and parts thereof according to claim 1,
which are homozygous for the mutation of the endogenous
acetolactate synthase (ALS) gene.
7. The parts of the Beta vulgaris plant according to claim 1,
wherein the parts are organs, tissues, and cells of the plant, and
preferably seeds.
8. Method for the manufacture of the Beta vulgaris plant and the
parts thereof of claim 1, comprising the following steps: (a)
exposing calli from B. vulgaris, to about 10.sup.-7 M-10.sup.-9 M
of an ALS inhibitor herbicide, preferably foramsulfuron; (b)
selecting cell colonies which can grow in the presence of up to
3.times.10.sup.-6 M of an ALS inhibitor herbicide, preferably
foramsulfuron; (c) regenerating shoots in presence of an ALS
inhibitor herbicide, preferably foramsulfuron; (d) selecting
regenerated plantlets with an ALS inhibitor herbicide, preferably
foramsulfuron, iodosulfuron-methyl-sodium and/or a mixture of both,
wherein the dose of foramsulfuron is preferably equivalent to 7-70
g a.i./ha and the dose of iodosulfuron-methyl-sodium is preferably
equivalent to 1-10 g a.i./ha.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/821,969, filed 8 Mar. 2013, which is a
National Stage entry of International Application No.
PCT/EP2011/067925, filed 13 Oct. 2011, which claims priority to
U.S. Provisional Application No. 61/394,463, filed 19 Oct. 2010 and
European Application No. 10187751.2, filed 15 Oct. 2010. The
disclosure of the priority applications are incorporated in their
entirety herein by reference.
REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT
FILE (.TXT)
[0002] Pursuant to the EFS-Web legal framework and 37 CFR
.sctn..sctn. 1.821-825 (see MPEP .sctn. 2442.03(a)), a Sequence
Listing in the form of an ASCII-compliant text file (entitled
"Sequence_Listing_2923343-035001_ST25.txt" created on 3 Nov. 2020,
and 26,010 bytes in size) is submitted concurrently with the
instant application, and the entire contents of the Sequence
Listing are incorporated herein by reference.
DESCRIPTION OF RELATED ART
[0003] The present invention relates to ALS inhibitor herbicide
tolerant Beta vulgaris plants and parts thereof as well as a method
for their manufacture.
[0004] Cultivated forms of Beta vulgaris (as defined in Ford-Lloyd
(2005) Sources of genetic variation, Genus Beta. In: Biancardi E,
Campbell L G, Skaracis G N, De Biaggi M (eds) Genetics and Breeding
of Sugar Beet. Science Publishers, Enfield (NH), USA, pp. 25-33)
are important agricultural crops in temperate and subtropical
regions. For example, about 20% of the world sugar production is
based on sugar beet. Because beet seedlings and juvenile plants
during their first 6-8 weeks of their life are susceptible for
strong competition caused by fast growing weeds, which outcompete
the young crop plants, reliable weed control measures are
imperative in these crop areas.
[0005] Since more than 40 years, herbicides are the preferred tools
to control weeds in cultured beets. The products used for this
purpose, like phenmedipham, desmediphan and metamitron allow to
suppress the growth of weeds in beet fields without damaging the
crop. Nevertheless under adverse environmental conditions the
efficacy of these products leaves room for improvements, especially
if noxious weeds like Chenopodium album, Amaranthus retroflexus
and/or Tripleurospermum inodorata germinate over an extended period
of time.
[0006] Innovative herbicidal active ingredients are highly
desirable in order to improve the weed control options in beet.
Such compounds should act against a broad weed spectrum, preferably
from weed germination until full development of the weed plants,
without affecting the beet crop irrespective of its developmental
stage. Via the classical herbicide screening approach no selective
herbicidal active ingredient was discovered for beet during the
past decades which fulfils all these stringent properties in an
agronomically superior way.
[0007] Some chemicals inhibit the enzyme "acetohydroxyacid
synthase" (AHAS), also known as "acetolactate synthase" (ALS [EC
4.1.3.18]). ALS is the site of action of five structurally diverse
herbicide families belonging to the class of ALS inhibitor
herbicides, like (a) sulfonylurea herbicides (Beyer E. M et al.
(1988), Sulfonylureas in Herbicides: Chemistry, Degradation, and
Mode of Action; Marcel Dekker, New York, 1988, 117-189), (b)
sulfonylaminocarbonyltriazolinone herbicides (Pontzen, R.,
Pflanz.-Nachrichten Bayer, 2002, 55, 37-52), (c) imidazolinone
herbicides (Shaner, D. L., et al., Plant Physiol., 1984, 76,
545-546; Shaner, D. L., and O'Connor, S. L. (Eds.) The
Imidazolinone Herbicides, CRC Press, Boca Raton, Fla., 1991), (d)
triazolopyrimidine herbicides (Kleschick, W. A. et al., Agric. Food
Chem., 1992, 40, 1083-1085), and (e) pyrimidinyl(thio)benzoate
herbicides (Shimizu, T. J., Pestic. Sci., 1997, 22, 245-256;
Shimizu, T. et al., Acetolactate Syntehase Inhibitors in Herbicide
Classes in Development, Boger, P., Wakabayashi, K., Hirai, K.,
(Eds.), Springer Verlag, Berlin, 2002, 1-41).
[0008] ALS is involved in the conversion of two pyruvate molecules
to an acetolactate molecule and carbon dioxide. The reaction uses
thyamine pyrophosphate in order to link the two pyruvate molecules.
The resulting product of this reaction, acetolactate, eventually
becomes valine, leucine and isoleucine (Singh (1999) "Biosynthesis
of valine, leucine and isoleucine", in Plant Amino Acids, Singh, B.
K., ed., Marcel Dekker Inc. New York, N.Y., pp. 227-247).
[0009] Inhibitors of the ALS interrupt the biosynthesis of valine,
leucine and isoleucine in plants. The consequence is an immediate
depletion of the respective amino acid pools causing a stop of
protein biosynthesis leading to a cessation of plant growth and
eventually the plant dies, or--at least--is damaged.
[0010] ALS inhibitor herbicides are widely used in modern
agriculture due to their effectiveness at moderate application
rates and relative non-toxicity in animals. By inhibiting ALS
activity, these families of herbicides prevent further growth and
development of susceptible plants including many weed species. In
order to provide plants with an increased tolerance to even high
concentrations of ALS inhibitor herbicides that may be required for
sufficient weed control, additional ALS-inhibiting
herbicide-resistant breeding lines and varieties of crop plants, as
well as methods and compositions for the production and use of ALS
inhibiting herbicide-resistant breeding lines and varieties, are
needed.
[0011] A broad variety of ALS inhibitor herbicides enable a farmer
to control a wide range of weed species independently of their
growth stages, but these highly efficient herbicides cannot be used
in beet because conventional beet plants/commercial beet varieties
are highly susceptible against these ALS inhibitor herbicides.
Nevertheless, these ALS inhibitor herbicides show an excellent
herbicidal activity against broadleaf and grass weed species. The
first herbicides having the mode of action of inhibiting the ALS
were developed for their use in agriculture already 30 years ago.
Nowadays, active ingredients of this class of herbicides exhibit a
strong weed control and are widely used in maize and cereals as
well as in dicotyledonous crops, except beet.
[0012] The only ALS inhibitor herbicide that is known today to be
applied in post-emergent application schemes in beet is Debut. This
herbicide (containing triflusulfuron-methyl as the active
ingredient plus specific formulation compounds) is degraded by
beets before it can inhibit the beet endogenous ALS enzyme but it
has severe gaps in weed control in beet growing areas.
[0013] Since ALS inhibitor herbicides were introduced into
agriculture it was observed that susceptible plant species,
including naturally occurring weeds, occasionally develop
spontaneous tolerance to this class of herbicides. Single base pair
substitutions at specific sites of the ALS gene usually lead to
more or less resistant ALS enzyme variants which show different
levels of inhibition by the ALS inhibitor herbicides.
[0014] Plants conferring mutant ALS alleles therefore show
different levels of tolerance to ALS inhibitor herbicides,
depending on the chemical structure of the ALS inhibitor herbicide
and the site of the point mutation in the ALS gene.
[0015] For example, Hattori et al. (1995), Mol. Gen. Genet. 246:
419-425, describes a single mutation in the Trp 557 codon in a
Brassica napus cell line (according to the numbering of the
Arabidopsis thaliana sequence that is used in the literature in
order to compare all ALS/AHAS mutants this refers to position
"574")--which equals position 569 of the beet ALS sequence. These
authors observed resistance to several members of sub-classes of
ALS inhibitor herbicides, like sulfonylureas, imidazolinones and
triazolopyrimidines.
[0016] Beet mutants were described conferring a point mutation in
the Ala 122 codon which led to a certain tolerance to the ALS
inhibitor herbicide subclass of imidazolinones (WO 98/02526) but
which is not sufficient for weed control in agricultural
application schemes. No cross-tolerance to other ALS inhibitor
herbicide classes were described by employing this mutant.
Furthermore, beet plants conferring a second point mutation in the
Pro 197 codon showed a moderate tolerance to ALS inhibitor
herbicides belonging to members of the subclass of sulfonylurea
herbicides. Also double mutants of these two were described (WO
98/02527). However, none of these mutants were used for the market
introduction of beet varieties because the level of herbicide
tolerance to ALS inhibitor herbicides was not sufficiently high in
these mutants to be exploited agronomically.
[0017] Stougaard et al. (1990), J. Cell Biochem., Suppl. 14E, 310
describe the isolation of ALS mutants in a tetraploid sugar beet
cell culture. Two different ALS genes (ALS I and ALS II) were
isolated which differed at amino acid position 37 only. Mutant 1
contained in its ALS I gene 2 mutations, while mutant 2 contained 3
mutations in its ALS II gene. After the mutations were separated to
resolve which mutation would confer resistance against an ALS
inhibitor, it was revealed that ALS synthesized from a recombinant
E. coli was herbicide resistant if it contained a point mutation in
the Trp 574 codon (according to the numbering of the Arabidopsis
thaliana sequence that is used in the literature in order to
compare all ALS mutants)--which equals position 569 of the beet ALS
sequence, leading to a replacement of the amino acid "Trp" by the
amino acid "Leu". Stougaard et al did not show in sugar beet that
the mutation at position 569 of any of the sugar beet ALS genes is
sufficient in order to obtain an acceptable level of tolerance to
ALS inhibitor herbicides. Moreover, Stougaard et al did not
regenerate or handle sugar beet plants comprising a mutation,
including Trp->Leu mutation at position 569 of sugar beet
ALS.
[0018] Knowing this, Stougaard et al. constructed plant
transformation vectors containing different ALS genes for use in
plant transformation. However, up to now, no further
data--especially not concerning the effects of the application of
ALS inhibitor herbicides to plants and/or agricultural areas
comprising this mutation in Beta vulgaris plants have been
disclosed by these or other authors either in genetically
engineered or mutant plants over more than 20 years,
thereafter.
[0019] WO 99/57965 generally describes sulfonylurea resistant sugar
beet plants and methods for obtaining them by EMS
(Ethylmethanesulfonate) mutagenesis. However, apart from the
research that is required to obtain such mutants, this publication
does neither provide such plants, nor describes any specific
location in the ALS gene that may be relevant for obtaining ALS
inhibitor herbicide tolerant mutants, nor discloses any correlated
agronomical use of such. Furthermore, there is a strong likelihood
that--by employing such strong mutagenic compound like EMS--various
further mutations may occur elsewhere in the genome and which might
lead to disadvantages up to non-fertility and/or growth retardation
of such obtained plants. Moreover, due to its chemical interaction
with the DNA, the EMS application may have gaps of inducing
specific mutations, like converting the triplet TGG into TTG,
because EMS always converts a guanosine into an adenosine.
[0020] In some weed species as Amaranthus, the Trp 574 Leu mutation
could be detected in plant populations which were repeatedly
exposed to ALS inhibitor herbicides. These Trp 574 Leu mutants show
a high level of tolerance to several chemical classes of ALS
inhibitor herbicides, like those selected from the group consisting
of sulfonylureas and sulfonylaminocarbonyltriazolinones.
[0021] WO 2008/124495 discloses ALS double and triple mutants.
According to WO 2009/046334, specific mutations in the ALS gene
were provided. However, agronomically exploitable herbicide
tolerant Beta vulgaris mutants containing such mutations according
to WO 2009/046334 have not been obtained so far.
[0022] Moreover, in view of the fact that, for example, sugar beet
accounts for about 20% of the world sugar production, it would also
be highly desirable to have available sugar beet plants which have
a growth advantage versus highly potent weeds. It would thus be
highly desirable to have available, with respect to the ALS gene,
non-transgenic Beta vulgaris plants including sugar beet plants
which are tolerant to ALS inhibitor herbicides. Hence, there is a
need for such non-transgenic Beta vulgaris plants, in particular
sugar beet plants which are tolerant to ALS inhibitor herbicides at
an agronomically exploitable level of ALS inhibitor herbicides.
[0023] Thus, the technical problem is to comply with this need.
SUMMARY
[0024] The present invention addresses this need and thus provides
as a solution to the technical problem an ALS inhibitor herbicide
tolerant Beta vulgaris plant and parts thereof comprising a
mutation of an endogenous acetolactate synthase (ALS) gene, wherein
the ALS gene encodes an ALS polypeptide containing an amino acid
different from tryptophan at a position 569 of the ALS
polypeptide.
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
[0025] Seeds according to present invention have been deposited
with the NCIMB, Aberdeen, UK, under Number NCIMB 41705 on Mar. 12,
2010.
[0026] By applying various breeding methods, high yielding
commercial varieties highly competitive in all specific markets
with the add-on of a robust ALS inhibitor herbicide tolerance can
be developed subsequently by using the originally obtained mutant
plants.
[0027] It must be noted that as used herein, the singular forms
"a", "an", and "the", include plural references unless the context
clearly indicates otherwise. Thus, for example, reference to "a
reagent" includes one or more of such different reagents and
reference to "the method" includes reference to equivalent steps
and methods known to those of ordinary skill in the art that could
be modified or substituted for the methods described herein.
[0028] All publications and patents cited in this disclosure are
incorporated by reference in their entirety. To the extent the
material incorporated by reference contradicts or is inconsistent
with this specification, the specification will supersede any such
material.
[0029] Unless otherwise indicated, the term "at least" preceding a
series of elements is to be understood to refer to every element in
the series. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
present invention.
[0030] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integer or step. The word "comprise" and its
variations on the one side and "contain" and its analogous
variations on the other side can be used interchangeably without a
preference to any of them.
[0031] In the present invention, beet plants were obtained which
comprise an altered endogenous ALS gene (also referred to as "AHAS"
gene), carrying a point mutation in the Trp 569 codon (in relation
to the Beta vulgaris ALS amino acid reference sequence shown in SEQ
ID NO: 2; this equals position 574 of the referenced Arabidopsis
thaliana sequence as shown in SEQ ID NO: 6) and which point
mutation was obtained by several circles of selection on
specifically elected ALS inhibitor herbicides.
[0032] Due to the fact that the B. vulgaris plants of the present
invention were obtained by isolating spontaneous mutant plant
cells, which were directly regenerated to fully fertile beet plants
having a point mutation as described herein in more detail. These
plants are non-transgenic as far as the ALS gene is concerned.
[0033] Moreover, the plants of the present invention themselves as
well as their offspring are fertile and thus useful for breeding
purposes without any further manipulation that may cause stress
induced further alterations of the genetic background. Such plants
obtained according to the selection procedure applied herein can
directly be employed in order to generate beet varieties and/or
hybrids conferring agronomically useful levels of tolerance to ALS
inhibitor herbicides, thus allowing innovative weed control
measures in beet growing areas.
[0034] When used herein, the term "transgenic" means that a
gene--which can be of the same or a different species--has been
introduced via an appropriate biological carrier, like
Agrobacterium tumefaciens or by any other physical means, like
protoplast transformation or particle bombardment, into a plant and
which gene is able to be expressed in the new host environment,
namely the genetically modified organism (GMO).
[0035] In accordance to the before definition, the term
"non-transgenic" means exactly the contrary, i.e. that no
introduction of the respective gene has occurred via an appropriate
biological carrier or by any other physical means. However, a
mutated gene can be transferred through pollination, either
naturally or via a breeding process to produce another
non-transgenic plant concerning this specific gene.
[0036] An "endogenous" gene means a gene of a plant which has not
been introduced into the plant by genetic engineering
techniques.
[0037] The term "sequence" when used herein relates to nucleotide
sequence(s), polynucleotide(s), nucleic acid sequence(s), nucleic
acid(s), nucleic acid molecule, peptides, polypeptides and
proteins, depending on the context in which the term "sequence" is
used.
[0038] The terms "nucleotide sequence(s)", "polynucleotide(s)",
"nucleic acid sequence(s)", "nucleic acid(s)", "nucleic acid
molecule" are used interchangeably herein and refer to nucleotides,
either ribonucleotides or deoxyribonucleotides or a combination of
both, in a polymeric unbranched form of any length. Nucleic acid
sequences include DNA, cDNA, genomic DNA, RNA, synthetic forms and
mixed polymers, both sense and antisense strands, or may contain
non-natural or derivatized nucleotide bases, as will be readily
appreciated by those skilled in the art.
[0039] When used herein, the term "polypeptide" or "protein" (both
terms are used interchangeably herein) means a peptide, a protein,
or a polypeptide which encompasses amino acid chains of a given
length, wherein the amino acid residues are linked by covalent
peptide bonds. However, peptidomimetics of such
proteins/polypeptides wherein amino acid(s) and/or peptide bond(s)
have been replaced by functional analogs are also encompassed by
the invention as well as other than the 20 gene-encoded amino
acids, such as selenocysteine. Peptides, oligopeptides and proteins
may be termed polypeptides. The term polypeptide also refers to,
and does not exclude, modifications of the polypeptide, e.g.,
glycosylation, acetylation, phosphorylation and the like. Such
modifications are well described in basic texts and in more
detailed monographs, as well as in the research literature. The
polypeptide (or protein) that is preferably meant herein is the B.
vulgaris ALS polypeptide (or ALS protein) [SEQ ID NO: 2].
[0040] Amino acid substitutions encompass amino acid alterations in
which an amino acid is replaced with a different
naturally-occurring amino acid residue. Such substitutions may be
classified as "conservative`, in which an amino acid residue
contained in the wild-type ALS protein is replaced with another
naturally-occurring amino acid of similar character, for example
GlyAla. ValIleLeu, AspGlu, LysArg, AsnGln or PheTrpTyr.
Substitutions encompassed by the present invention may also be
"non-conservative", in which an amino acid residue which is present
in the wild-type ALS protein is substituted with an amino acid with
different properties, such as a naturally-occurring amino acid from
a different group (e.g. substituting a charged or hydrophobic amino
acid with alanine. "Similar amino acids", as used herein, refers to
amino acids that have similar amino acid side chains, i.e. amino
acids that have polar, non-polar or practically neutral side
chains. "Non-similar amino acids", as used herein, refers to amino
acids that have different amino acid side chains, for example an
amino acid with a polar side chain is non-similar to an amino acid
with a non-polar side chain. Polar side chains usually tend to be
present on the surface of a protein where they can interact with
the aqueous environment found in cells ("hydrophilic" amino acids).
On the other hand, "non-polar" amino acids tend to reside within
the center of the protein where they can interact with similar
non-polar neighbours ("hydrophobic" amino acids"). Examples of
amino acids that have polar side chains are arginine, asparagine,
aspartate, cysteine, glutamine, glutamate, histidine, lysine,
serine, and threonine (all hydrophilic, except for cysteine which
is hydrophobic). Examples of amino acids that have non-polar side
chains are alanine, glycine, isoleucine, leucine, methionine,
phenylalanine, proline, and tryptophan (all hydrophobic, except for
glycine which is neutral).
[0041] Generally, the skilled person knows, because of his common
general knowledge and the context when the terms ALS, ALSL, AHAS or
AHASL are used, as to whether the nucleotide sequence or nucleic
acid, or the amino acid sequence or polypeptide, respectively, is
meant.
[0042] The term "gene" when used herein refers to a polymeric form
of nucleotides of any length, either ribonucleotides or
desoxyribonucleotides. The term includes double- and
single-stranded DNA and RNA. It also includes known types of
modifications, for example, methylation, "caps", substitutions of
one or more of the naturally occurring nucleotides with an analog.
Preferably, a gene comprises a coding sequence encoding the herein
defined polypeptide. A "coding sequence" is a nucleotide sequence
which is transcribed into mRNA and/or translated into a polypeptide
when placed or being under the control of appropriate regulatory
sequences. The boundaries of the coding sequence are determined by
a translation start codon at the 5'-terminus and a translation stop
codon at the 3'-terminus. A coding sequence can include, but is not
limited to mRNA, cDNA, recombinant nucleic acid sequences or
genomic DNA, while introns may be present as well under certain
circumstances. When used herein the term "Beta vulgaris" is
abbreviated as "B. vulgaris".
[0043] Furthermore, the term "beet" is used herein. Said three
terms are interchangeably used and should be understood to fully
comprise the cultivated forms of Beta vulgaris as defined in
Ford-Lloyd (2005) Sources of genetic variation, Genus Beta. In:
Biancardi E, Campbell L G, Skaracis G N, De Biaggi M (eds) Genetics
and Breeding of Sugar Beet. Science Publishers, Enfield (NH), USA,
pp 25-33. Similarly, for example, the term "Arabidopsis thaliana"
is abbreviated as "A. thaliana". Both terms are interchangeably
used herein.
[0044] The term "position" when used in accordance with the present
invention means the position of either an amino acid within an
amino acid sequence depicted herein or the position of a nucleotide
within a nucleotide sequence depicted herein. The term
"corresponding" as used herein also includes that a position is not
only determined by the number of the preceding nucleotides/amino
acids.
[0045] The position of a given nucleotide in accordance with the
present invention which may be substituted may vary due to
deletions or additional nucleotides elsewhere in the ALS
5'-untranslated region (UTR) including the promoter and/or any
other regulatory sequences or gene (including exons and introns).
Similarly, the position of a given amino acid in accordance with
the present invention which may be substituted may vary due to
deletion or addition of amino acids elsewhere in the ALS
polypeptide.
[0046] Thus, under a "corresponding position" in accordance with
the present invention it is to be understood that nucleotides/amino
acids may differ in the indicated number but may still have similar
neighbouring nucleotides/amino acids. Said nucleotides/amino acids
which may be exchanged, deleted or added are also comprised by the
term "corresponding position".
[0047] In order to determine whether a nucleotide residue or amino
acid residue in a given ALS nucleotide/amino acid sequence
corresponds to a certain position in the nucleotide sequence of SEQ
ID NO: 1 or the amino acid sequence of SEQ ID NO: 2, the skilled
person can use means and methods well-known in the art, e.g.,
alignments, either manually or by using computer programs such as
BLAST (Altschul et al. (1990), Journal of Molecular Biology, 215,
403-410), which stands for Basic Local Alignment Search Tool or
ClustalW (Thompson et al. (1994), Nucleic Acid Res., 22, 4673-4680)
or any other suitable program which is suitable to generate
sequence alignments.
[0048] SEQ ID NO: 1 is the nucleotide sequence encoding Beta
vulgaris wild type ALS. SEQ ID NO: 2 is the Beta vulgaris amino
acid sequence derived from SEQ ID NO: 1. Accordingly, the codon at
position 1705-1707 of the nucleotide sequence of SEQ ID NO: 1
encodes the amino acid at position 569 (i.e. the amino acid "Trp"
according to the three letter code or "W" according to the one
letter code) of SEQ ID NO: 2.
[0049] In the alternative to determine whether a nucleotide residue
or amino acid residue in a given ALS nucleotide/amino acid sequence
corresponds to a certain position in the nucleotide sequence of SEQ
ID NO: 1, the nucleotide sequence encoding A. thaliana wild type
ALS shown in SEQ ID NO: 5 can be used. SEQ ID NO: 6 is the A.
thaliana amino acid sequence derived from SEQ ID NO: 5.
[0050] Accordingly, the codon at position 1720-1722 of the
nucleotide sequence of SEQ ID NO: 5 encodes the amino acid at
position 574 (i,e, the amino acid "Trp" according to the three
letter code or "W" according to the one letter code) of SEQ ID NO.
6.
[0051] If the A. thaliana wild type ALS nucleotide sequence shown
in SEQ ID NO: 5 is used as reference sequence (as it is done in
most of the relevant literature and, therefore, is used to enable
an easier comparison to such known sequences), the codon encoding
an amino acid different from tryptophan is at a position
corresponding to position 1720-1722 of the nucleotide sequence of
the A. thaliana ALS gene shown in SEQ ID NO: 5.
[0052] However, SEQ ID NO: 1 is preferred as the reference
nucleotide sequence and SEQ ID NO: 2 is preferred as the reference
amino acid sequence in all of the subsequent disclosures.
[0053] The following table provides an overview on the reference
sequences used herein when the position of the point mutation in a
nucleotide sequence or the substitution in an amino acid sequence
is determined:
TABLE-US-00001 SEQ ID NO: Type of Sequence Species 1 nucleotide
sequence B. vulgaris 2 amino acid sequence B. vulgaris 3 nucleotide
sequence B. vulgaris (mutated) 4 amino acid sequence B. vulgaris
(mutated) 5 nucleotide sequence A. thaliana 6 amino acid sequence
A. thaliana
[0054] Thus, in any event, the equivalent position could still be
determined through alignment with a reference sequence, such as SEQ
ID NO: 1 or 5 (nucleotide sequence) or SEQ ID NO: 2 or 6 (amino
acid sequence).
[0055] In view of the difference between the B. vulgaris wild-type
ALS gene and the ALS gene comprised by a B. vulgaris plant of the
present invention, the ALS gene (or polynucleotide or nucleotide
sequence) comprised by a B. vulgaris plant of the present invention
may also be regarded as a "mutant ALS gene", "mutant ALS allele",
"mutant ALS polynucleotide" or the like. Thus, throughout the
specification, the terms "mutant allele", "mutant ALS allele",
"mutant ALS gene" or "mutant ALS polynucleotide" are used
interchangeably.
[0056] Unless indicated otherwise herein, these terms refer to a
nucleotide sequence that comprises a codon encoding an amino acid
different from tryptophan at a position corresponding to position
1705-1707 of the nucleotide sequence of the B. vulgaris ALS gene
shown in SEQ ID NO: 1. When set in relation to the A. thaliana
reference sequence shown in SEQ ID NO: 5, the position of the codon
is 1720-1722.
[0057] Likewise, these terms refer to a nucleotide sequence that
encodes an ALS protein having at a position corresponding to
position 569 of the amino acid sequence of the Beta vulgaris ALS
protein shown in SEQ ID NO: 2 an amino acid different from
tryptophan. When set in relation to the A. thaliana reference
sequence shown in SEQ ID NO: 6, the position is 574.
[0058] An "amino acid different from tryptophan" (indicated by
"Trp" in the three letter code or "W" in the equivalently used one
letter code) includes any naturally-occurring amino acid different
from tryptophan. These naturally-occurring amino acids include
alanine (A), arginine (R), asparagine (N), aspartate (D), cysteine
(C), glutamine (Q), glutamate (E), glycine (G), histidine (H),
isoleucine (I), leucine (L), lysine (K), methionine (M),
phenylalanine (F), proline (P), serine (S), threonine (T), tyrosine
(Y) or valine (V).
[0059] However, preferably, the amino acid different from
tryptophan (belonging to the group of neutral-polar amino acids) is
an amino acid with physico-chemical properties different from
tryptophan, i.e. belonging to any of the amino acids showing
neutral-nonpolar, acidic, or basic properties. More preferably, the
amino acid different from tryptophan is selected from the group
consisting of alanine, glycine, isoleucine, leucine, methionine,
phenylalanine, proline, valine, and arginine. Even more preferably,
said amino acid is a neutral-nonpolar amino acid such as alanine,
glycine, isoleucine, leucine, methionine, phenylalanine, proline or
valine. Particularly preferred said amino acid is alanine, glycine,
isoleucine, leucine, valine. Even more preferred is glycine and
leucine. Most preferably, it is leucine.
[0060] In contrast, unless indicated otherwise, the terms
"wild-type allele," "wild-type ALS allele", "wild-type ALS gene" or
"wild-type ALS polynucleotide" refer to a nucleotide sequence that
encodes an ALS protein that lacks the W569 substitution in relation
to SEQ ID NO: 2 (or W574 substitution in relation to SEQ ID NO: 6).
These terms also refer to a nucleotide sequence comprising at a
position corresponding to position 1705-1707 of the nucleotide
sequence of the B. vulgaris ALS gene shown in SEQ ID NO: 1, a codon
encoding an amino acid different from tryptophan.
[0061] Such a "wild-type allele", "wild-type ALS allele",
"wild-type ALS gene" or "wild-type ALS polynucleotide" may, or may
not, comprise mutations, other than the mutation that causes the
W569 substitution.
[0062] In essence, as regards the ALS gene, the only difference
between a wild-type B. vulgaris plant and the B. vulgaris plant of
the present invention is preferably (and specifically) that at a
position as specified herein (in particular at a position
corresponding to position 1705-1707 of the nucleotide sequence of
the B. vulgaris ALS gene shown in SEQ ID NO: 1), the B. vulgaris
plant of the present invention comprises a codon encoding an amino
acid different from tryptophan, preferably the codon encodes an
amino acid as specified herein elsewhere. However, as mentioned
above, further differences such as additional mutations may be
present between wild-type and the mutant ALS allele as specified
herein. Yet, these further differences are not relevant as long as
the difference explained before is present.
[0063] Consequently, the W569 substitution (or W574 substitution
when the A. thaliana ALS amino acid sequence of SEQ ID NO: 6 is
used as reference) is a result of an alteration of the codon at a
position corresponding to position 1705-1707 of the nucleotide
sequence shown in SEQ ID NO: 1 (or at a position corresponding to
position 1720-1722 of the nucleotide sequence shown in SEQ ID NO:
5, respectively).
[0064] Preferably, the substitution at position 569 is a W-+L
substitution, wherein "L" is encoded by any of the codons "CTT",
"CTC", "CTA", "CTG", "TTA" or "TTG".
[0065] Most preferably, the substitution at position 569 is a
W.fwdarw.L substitution, because of a transversion of the "G"
nucleotide at a position corresponding to position 1706 of the
nucleotide sequence shown in SEQ ID NO: 1 (or at a position
corresponding to position 1721 of the nucleotide sequence shown in
SEQ ID NO: 5, respectively), to a "T" nucleotide. Accordingly, the
codon at a position corresponding to position 1705-1707 of the
nucleotide sequence shown in SEQ ID NO: 1 (or at a position
corresponding to position 1720-1722 of the nucleotide sequence
shown in SEQ ID NO: 5, respectively) is changed from "TGG" to
"TTG". While the codon "TGG" encodes tryptophan, the codon "TTG"
encodes leucine.
[0066] Hence, in the most preferred embodiment, the present
invention provides a Beta vulgaris plant comprising in the
nucleotide sequence of the endogenous ALS gene, the codon TTG
(encoding leucine) at a position corresponding to position
1705-1707 of the nucleotide sequence of the B. vulgaris ALS mutant
gene shown in SEQ ID NO: 1, said nucleotide sequence comprising (or
less preferably consisting of) SEQ ID NO: 3.
[0067] The B. vulgaris plants encoding an ALS polypeptide having at
a position corresponding to position 569 of the amino acid sequence
of the Beta vulgaris ALS protein shown in SEQ ID NO: 2 an amino
acid different from tryptophan, preferably comprise in the
nucleotide sequence of the endogenous ALS gene a codon encoding an
amino acid different from tryptophan at a position corresponding to
position 1705-1707 of the nucleotide sequence of the B. vulgaris
ALS gene shown in SEQ ID NO: 1.
[0068] The term B. vulgaris "ALS" or "AHAS" gene also includes B.
vulgaris nucleotide sequences which are at least 90, 95, 97, 98, or
99% identical to the B. vulgaris ALS nucleotide sequence of SEQ ID
NO: 1 or 3, wherein these 60, 70, 80, 90, 95, 97, 98, or 99%
identical nucleotide sequences comprise at a position corresponding
to position 1705-1707 of the nucleotide sequence of SEQ ID NO: 1 a
codon encoding an amino acid different from tryptophan.
[0069] Likewise, these at least 90, 95, 97, 98, or 99% identical
nucleotide sequences encode an ALS polypeptide comprising at a
position corresponding to position 569 of SEQ ID NO: 2 an amino
acid different from tryptophan. Said identical nucleotide sequences
encode an ALS protein which retains the activity as described
herein, more preferably the thus-encoded ALS polypeptide is
tolerant to one or more ALS inhibitor herbicides as described
herein. Said term also includes allelic variants and homologs
encoding an ALS polypeptide which is preferably tolerant to one or
more ALS inhibitor herbicides as described herein.
[0070] In order to determine whether a nucleic acid sequence has a
certain degree of identity to the nucleotide sequences of the
present invention, the skilled person can use means and methods
well-known in the art, e.g., alignments, either manually or by
using computer programs such as those mentioned further down below
in connection with the definition of the term "hybridization" and
degrees of homology.
[0071] For example, BLAST, which stands for Basic Local Alignment
Search Tool (Altschul, Nucl. Acids Res. 25 (1997), 3389-3402;
Altschul, J. Mol. Evol. 36 (1993), 290-300; Altschul, J. Mol. Biol.
215 (1990), 403-410), can be used to search for local sequence
alignments. BLAST produces alignments of both nucleotide and amino
acid sequences to determine sequence similarity. Because of the
local nature of the alignments, BLAST is especially useful in
determining exact matches or in identifying similar sequences. The
fundamental unit of BLAST algorithm output is the High-scoring
Segment Pair (HSP). An HSP consists of two sequence fragments of
arbitrary but equal lengths whose alignment is locally maximal and
for which the alignment score meets or exceeds a threshold or
cutoff score set by the user. The BLAST approach is to look for
HSPs between a query sequence and a database sequence, to evaluate
the statistical significance of any matches found, and to report
only those matches which satisfy the user-selected threshold of
significance. The parameter E establishes the statistically
significant threshold for reporting database sequence matches. E is
interpreted as the upper bound of the expected frequency of chance
occurrence of an HSP (or set of HSPs) within the context of the
entire database search. Any database sequence whose match satisfies
E is reported in the program output.
[0072] Analogous computer techniques using BLAST (Altschul (1997),
loc. cit.; Altschul (1993), loc. cit.; Altschul (1990), loc. cit.)
are used to search for identical or related molecules in nucleotide
databases such as GenBank or EMBL. This analysis is much faster
than multiple membrane-based hybridizations. In addition, the
sensitivity of the computer search can be modified to determine
whether any particular match is categorized as exact or similar.
The basis of the search is the product score which is defined
as:
% sequence identity .times. % maximum BLAST score 100
##EQU00001##
and it takes into account both the degree of similarity between two
sequences and the length of the sequence match. For example, with a
product score of 40, the match will be exact within a 1-2% error;
and at 70, the match will be exact. Similar molecules are usually
identified by selecting those which show product scores between 15
and 40, although lower scores may identify related molecules.
[0073] The term B. vulgaris "ALS" or "AHAS" polypeptide also
includes amino acid sequences which are at least 90, 95, 97, 98, or
99% identical to the ALS amino acid sequence of SEQ ID NO: 2 or 4,
wherein these at least 90, 95, 97, 98, or 99% identical amino acid
sequences comprising at a position corresponding to position 569 of
SEQ ID NO: 2 an amino acid different from tryptophan. Said
identical amino acid sequences retain the activity of ALS as
described herein, more preferably the ALS polypeptide is tolerant
to ALS inhibitor herbicides as described herein. ALS activity, if
required, can be measured in accordance with the assay described in
Singh (1991), Proc. Natl. Acad. Sci. 88:4572-4576.
[0074] However, the ALS nucleotide sequences referred to herein
encoding an ALS polypeptide preferably confer tolerance to one or
more ALS inhibitor herbicides (or, vice versa, less sensitivity to
an ALS inhibitor herbicide) as described herein. This is because of
the point mutation leading to an amino acid substitution as
described herein.
[0075] Accordingly, tolerance to an ALS inhibitor herbicide (or,
vice versa, less sensitivity to an ALS inhibitor herbicide) can be
measured by comparison of ALS activity obtained from cell extracts
from plants containing the mutated ALS sequence and from plants
lacking the mutated ALS sequence in the presence of an
ALS-inhibitor herbicide, like it is described in Singh et al (1988)
[J. Chromatogr., 444, 251-261].
[0076] However, a more preferred activity assay for the ALS
polypeptide encoded by a nucleotide sequence comprising a codon
encoding an amino acid different from tryptophan at a position
corresponding to position 1705-1707 of the nucleotide sequence of
the B. vulgaris ALS gene shown in SEQ ID NO: 1 can be done as
follows:
[0077] The coding sequence of a Beta vulgaris wild-type and a
mutant B. vulgaris plant is cloned into, for example, Novagen
pET-32a(+) vectors and the vectors are transformed into, for
example, Escherichia coli AD494 according to the instructions of
the manufacturer. Bacteria are preferably grown at 37.degree. C. in
medium under selection pressure such as in LB-medium containing 100
mg/l carbenicillin and 25 mg/l kanamycin, are induced with, for
example, 1 mM isopropyl-s-D-thiogalactopyranoside at an OD.sub.600
of preferably about 0.6, cultivated for about 16 hours at
preferably 18.degree. C. and harvested by centrifugation. Bacterial
pellets are resuspended in 100 mM sodium phosphate buffer pH 7.0
containing 0.1 mM thiamine-pyrophosphate, 1 mM MgCl.sub.2, and 1
.mu.M FAD at a concentration of 1 gram wet weight per 25 ml of
buffer and disrupted by sonification. The crude protein extract
obtained after centrifugation is used for ALS activity
measurements.
[0078] ALS assays are then carried out in, for example, 96-well
microtiter plates using a modification of the procedure described
by Ray (1984), Plant Physiol., 75, 827-831. The reaction mixture
contains preferably 20 mM potassium phosphate buffer pH 7.0, 20 mM
sodium pyruvate, 0.45 mM thiamine-pyrophosphate, 0.45 mM
MgCl.sub.2, 9 .mu.M FAD, ALS enzyme and various concentrations of
ALS inhibitors in a final volume of about 90 .mu.l.
[0079] Assays are initiated by adding enzyme and terminated after
preferably 75 min incubation at 30.degree. C. by the addition of 40
.mu.l 0.5 M H.sub.2SO.sub.4. After about 60 min at room temperature
about 80 .mu.l of a solution of 1.4% .alpha.-naphthol and 0.14%
creatine in 0.7 M NaOH is added and after an additional about 45
min incubation at room temperature the absorbance is determined at
540 nm. pI50-values for inhibition of ALS were determined as
described by Ray (1984)), Plant Physiol., 75, 827-831, using the
XLFit Excel add-in version 4.3.1 curve fitting program of ID
Business Solutions Limited, Guildford, UK.
[0080] When plants are used, ALS activity is preferably determined
in cell extracts or leaf extracts of wild type and B. vulgaris cell
extracts or leaf extracts of the obtained mutant in the presence of
various concentrations of ALS-inhibitor herbicides, preferably
sulfonylurea herbicides or sulfonylamino-carbonyltriazolinone
herbicides, more preferably in the presence of various
concentrations of the ALS inhibitor herbicide "foramsulfuron". ALS
is thus preferably extracted from sugar beet leaves or sugar beet
tissue cultures as described by Ray (1984) in Plant Physiol
75:827-831.
[0081] It is preferred that the B. vulgaris plants of the present
invention are less sensitive to an ALS inhibitor, more preferably
it is at least 100 times less sensitive, more preferably, 500
times, even more preferably 1000 times and most preferably less
than 2000 times. Less sensitive when used herein may, vice versa,
be seen as "more tolerable" or "more resistant". Similarly, "more
tolerable" or "more resistant" may, vice versa, be seen as "less
sensitive".
[0082] For example, the B. vulgaris plants of the present invention
and in particular the B. vulgaris plant described in the appended
Examples are/is at least 2000 times less sensitive to the ALS
inhibitor herbicide foramsulfuron (a member of the ALS inhibitor
subclass "sulfonylurea herbicides") compared to the wild type
enzyme.
[0083] Preferably, the B. vulgaris plants of the present invention
are less sensitive to various members of ALS inhibitor herbicides,
like sulfonylurea herbicides, sulfonylamino-carbonyltriazolinone
herbicides, and imidazolinone herbicides. Sulfonylurea herbicides
and sulfonylaminocarbonyltriazolinone herbicides against which said
plants are less sensitive are preferably selected. In a particular
preferred embodiment, the B. vulgaris plants of the present
invention are less sensitive to the ALS inhibitor herbicide
formasulfuron (sulfonylurea herbicide) either alone or in
combination with one or more further ALS inhibitor herbicides
either from the subclass of the sulfonyurea-herbicides or any other
sub-class of the ALS inhibitor herbicides.
[0084] Hence, the B. vulgaris plants of the present invention which
are preferably less sensitive to an ALS inhibitor herbicide can
likewise also be characterized to be "more tolerant" to an ALS
inhibitor" (i.e. an ALS inhibitor tolerant plant).
[0085] Thus, an "ALS inhibitor tolerant" plant refers to a plant,
in particular a B. vulgaris plant that is more tolerant to at least
one ALS inhibitor herbicide at a level that would normally inhibit
the growth of a normal or wild-type plant, preferably the ALS
inhibitor herbicide controls a normal or wild-type plant. Said
normal or wild-type plant does not comprise in the nucleotide
sequence of any allele of the endogenous ALS gene, a codon encoding
an amino acid different from tryptophan at a position corresponding
to position 1705-1707 of the nucleotide sequence of the B. vulgaris
ALS gene shown in SEQ ID NO: 1.
[0086] Said nucleotide sequence may generally also be characterized
to be an "ALS inhibitor herbicide tolerant" nucleotide sequence. By
"ALS inhibitor herbicide tolerant nucleotide sequence" is intended
a nucleic acid molecule comprising a nucleotide sequence comprising
at least the mutation that results in a codon encoding an amino
acid different from tryptophan relative to an ALS protein which
does not have at a position corresponding to position 569 of the
amino acid sequence of the B. vulgaris ALS protein shown in SEQ ID
NO: 2 an amino acid different from tryptophan, wherein said at
least one mutation results in the expression of a less sensitive to
an ALS inhibitor herbicide ALS protein.
[0087] By "herbicide-tolerant ALS protein", it is intended that
such an ALS protein displays higher ALS activity, relative to the
ALS activity of a wild-type ALS protein, in the presence of at
least one ALS inhibitor herbicide that is known to interfere with
ALS activity and at a concentration or level of said herbicide that
is known to inhibit the ALS activity of the wild-type ALS
protein.
[0088] Similarly, the terms "ALS-inhibitor herbicide(s)" or simply
"ALS-inhibitor(s)" are used interchangeably. As used herein, an
"ALS-inhibitor herbicide" or an "ALS inhibitor" is not meant to be
limited to single herbicide that interferes with the activity of
the ALS enzyme. Thus, unless otherwise stated or evident from the
context, an "ALS-inhibitor herbicide" or an "ALS inhibitor" can be
a one herbicide or a mixture of two, three, four, or more
herbicides known in the art, preferably as specified herein, each
of which interferes with the activity of the ALS enzyme.
[0089] Surprisingly, it was found that even the single point
mutation according to the present invention confers agronomically
useful and stable levels of ALS inhibitor herbicide tolerance in B.
vulgaris plants as well as in their offsprings, particularly, if
homozygocity is established. Compared to herbicide tolerant Beta
vulgaris plants of the same genetic background in which such
mutation is only heterozygously present, the herbicide tolerant
Beta vulgaris plants which are homozygous for the mutation revealed
a better agronomical level of ALS inhibitor herbicide
tolerance.
[0090] Therefore, present invention relates to an ALS inhibitor
herbicide tolerant Beta vulgaris plant having a mutation of the
endogenous acetolactate synthase (ALS) gene, wherein the ALS gene
encodes an ALS polypeptide containing an amino acid different from
tryptophan at a position 569 of the ALS polypeptide. The respective
mutation can be heterozygously present, and can preferably be the
sole mutation of the ALS gene. More preferably, the respective
mutation can be homozygously present, and most preferably, the
respective mutation is homozygously present as the sole mutation of
the endogenous ALS gene.
[0091] It could also not be expected that only one single mutation
of an ALS gene in Beta vulgaris would be sufficient, since, for
example, WO 2010/037061 teaches that double or triple mutants in
the ALS gene are necessary to confer the agromically useful
ALS-inhibitor herbicide tolerance.
[0092] Therefore, B. vulgaris plants and parts thereof which are
heterozygous for the mutation are less preferred, but are still
covered by the present invention and may be sufficient for certain
application schemes and/or certain environment conditions. Also
covered by the present invention are plants containing at least in
one allele of the endogenous ALS gene, a codon encoding an amino
acid different from tryptophan, preferably leucine at a position
corresponding to position 1705-1707 of the nucleotide sequence of
the B. vulgaris ALS gene shown in SEQ ID NO: 1, and containing one
(in case of diploidy) or more further alleles (in case of
polyploidy) having one or more further mutations in the endogenous
ALS gene.
[0093] Accordingly, when used herein the term "heterozygous" or
"heterozygously" means that a plant of the present invention has
different alleles at a particular locus, in particular at the ALS
gene locus.
[0094] "Homozygous" or "homozygously" indicates that a plant of the
present invention has two copies of the same allele on different
DNA strands, in particular at the ALS gene locus.
[0095] As used herein unless clearly indicated otherwise, the term
"plant" intended to mean a plant at any developmental stage.
[0096] It is preferred that the Beta vulgaris plant of the present
invention is orthoploid or anorthoploid. An orthoploid plant may
preferably be haploid, diploid, tetraploid, hexaploid, octaploid,
decaploid or dodecaploid, while an anorthoploid plant may
preferably be triploid or pentaploid.
[0097] Parts of plants may be attached to or separate from a whole
intact plant. Such parts of a plant include, but are not limited
to, organs, tissues, and cells of a plant, and preferably
seeds.
[0098] Accordingly, the B. vulgaris plant of the present invention
is non-transgenic as regards an endogenous ALS gene. Of course,
foreign genes can be transferred to the plant either by genetic
engineering or by conventional methods such as crossing. Said genes
can be genes conferring herbicide tolerances, preferably conferring
herbicide tolerances different from ALS inhibitor herbicide
tolerances, genes improving yield, genes improving resistances to
biological organisms, and/or genes concerning content
modifications.
[0099] In a further aspect, the present invention relates to a
method for the manufacture of the Beta vulgaris plant and the parts
thereof, comprising the following steps: [0100] (a) exposing calli,
preferably from sugar beet, to about 10.sup.7 M-10.sup.-9 M of an
ALS inhibitor herbicide, preferably foramsulfuron; [0101] (b)
selecting cell colonies which can grow in the presence of up to
3.times.10.sup.-6 M of an ALS inhibitor herbicide, preferably
foramsulfuron [CAS RN 173159-57-4]; [0102] (c) regenerating shoots
in presence of an ALS inhibitor herbicide, preferably
foramsulfuron; [0103] (d) selecting regenerated plantlets with an
ALS inhibitor herbicide, preferably foramsulfuron,
iodosulfuron-methyl-sodium [CAS RN 144550-36-7] and/or a mixture of
both, wherein the dose of foramsulfuron is preferably equivalent to
7-70 g a.i./ha and the dose of iodosulfuron-methyl-sodium is
preferably equivalent to 1-10 g a.i./ha.
[0104] In a further aspect, the regenerated plantlets obtained
according to the processes (a) to (d) above, can be employed for
further manufacture of Beta vulgaris plants by applying the
following steps (e) to (m): [0105] (e) vegetative micropropagation
of individual plantlets of step (d) to rescue different positive
variants by establishing a cell line (clone) of each ALS inhibitor
herbicide tolerant plantlet; [0106] (f) longterm storage of each
established clone in the vegetative state; [0107] (g) transfer of
cloned plants of each clone from the long term storage into the
greenhouse; [0108] (h) vernalisation and adaptation in
vernalisation chambers to induce flowering; [0109] (i) transfer of
vernalised plants to growth rooms (controlled temperature and
light); [0110] (j) select best pollen shedding plants of best
flowering clones for crossing with emasculated plants of an elite
but ALS inhibitor herbicide sensitive line to overcome the negative
impact of somaclonal variation on the generative fertility (male
and female) of plantlets of step (d); [0111] (k) backcross to elite
line until fertility is restored and finally self heterozygous
plants to reach the homozygous state; [0112] (l) produce
testcrosses with an ALS inhibitor herbicide-sensitive partner and
selfed seed of each backcrossed line for field evaluations; [0113]
(m) applying agronomically relevant dose rates of different ALS
inhibitor herbicides to select the best performing line, preferably
in its homozygous state.
[0114] The lines obtained according to above steps (a) to (m) form
the basis for the development of commercial varieties following
procedures known in the breeding community supported by molecular
breeding techniques (like marker assisted breeding or marker
assisted selection) for speeding up the processes and to secure the
correct selection of plants to either obtain the mutation in its
homozygous form or in case of containing one or more mutations at
various locations of the ALS encoding endogenous gene to perform
the correct selection of heterozygous plants that do contain at
least at one of the alleles the W569 mutation according to present
invention. (For review, see Bertrand C. Y. et al. (2008), Phil.
Trans. R. Soc, B., 363, 557-572) Calli are obtained by means and
methods commonly known in the art, for example, as described in the
appended Examples.
[0115] Seeds obtained under step (m), above, have been deposited
with the NCIMB, Aberdeen, UK, under Number NCIMB 41705.
[0116] In a further aspect, the present invention relates to a
method for producing an herbicide tolerant Beta vulgaris plant and
parts thereof comprising (i) a mutation of an endogenous
acetolactate synthase (ALS) gene, wherein the ALS gene encodes an
ALS polypeptide containing an amino acid different from tryptophan
at a position 569 of the ALS polypeptide, and (ii) an additional
mutation in the endogenous ALS gene, comprising the following
steps: [0117] (a) producing an ALS inhibitor herbicide tolerant
Beta vulgaris plant comprising a mutation of an endogenous
acetolactate synthase (ALS) gene, wherein the ALS gene encodes an
ALS polypeptide containing an amino acid different from tryptophan
at a position 569 of the ALS polypeptide (parent A); [0118] (b)
crossing parent A with a Beta vulgaris plant (parent B) containing
one or more further mutations in the endogenous ALS gene at
positions differing from amino acid position 569; [0119] (c)
obtaining a Beta vulgaris progeny that is heterozygous for the ALS
gene mutation of amino acid position 569 and to one or more of any
further ALS gene mutations encoded by parent B; [0120] (d) wherein
the breeding process is controlled by [0121] (i) the application of
marker assisted breeding and/or microsequencing techniques, and/or
[0122] (ii) the application of agronomically relevant doses of one
or more ALS inhibitor herbicides to which the generated progeny
according to step (c) are tolerant.
[0123] Accordingly, it is envisaged that the present invention also
relates to B. vulgaris plants obtainable by the aforementioned
methods of manufacture.
[0124] In a non-limiting example, sugar beet plants of the present
invention were obtained by performing the following non-limiting
protocol. Without being bound by theory, the same protocol may be
used for obtaining B. vulgaris plants other than sugar beet.
[0125] Sugar beet cell cultures were initiated from seedlings of a
diploid sugar beet genotype 7T9044 (as, for example, described by
Alexander Dovzhenko, PhD Thesis, Title: "Towards plastid
transformation in rapeseed (Brassica napus L.) and sugarbeet (Beta
vulgaris L.)", Ludwig-Maximilians-Universitat Munchen, Germany,
2001). Sugar beet seeds were immersed for 60 seconds in 70%
ethanol, then rinsed twice in sterile water with 0.01% detergent
and then incubated for 1-4 hours in 1% NaOCl bleach. Thereafter the
seeds were washed 3 times with sterile H.sub.2O and the seeds were
stored in sterile water overnight at 4.degree. C. The embryos were
then isolated using forceps and scalpel.
[0126] The freshly prepared embryos were immersed in 0.5% NaOCl for
30 min and then washed 3 times in sterile water. After the last
washing step they were placed on hormone free MS agar medium
(Murashige and Skoog (1962), Physiol. Plantarum, 15, 473-497).
Those embryos which developed into sterile seedlings were used for
the initiation of regenerable sugar beet cell cultures.
[0127] Cotyledons as well as hypocotyls were cut into 2-5 mm long
segments and then cultivated on agar (0.8%) solidified MS medium
containing either 1 mg/l Benzylaminopurine (BAP) or 0.25 mg/l
Thidiazuron (TDZ). 4 weeks later the developing shoot cultures were
transferred onto fresh agar medium of the same composition and then
sub-cultured in monthly intervals. The cultures were kept at
25.degree. C. under dim light at a 12 h/12 h light/dark cycle.
[0128] After 7-10 subcultures the shoot cultures which were grown
on the thidiazuron containing medium formed a distinct callus type,
which was fast growing, soft and friable. The colour of this callus
type was yellowish to light green. Some of these friable calli
consistently produced chlorophyll containing shoot primordia from
embryo-like structures. These fast growing regenerable calli were
used for the selection of ALS-inhibitor herbicide tolerant sugar
beet mutants.
[0129] When this callus type was exposed to 10.sup.-9 M of the
sulfonylurea foramsulfuron (CAS RN 173159-57-4), the cells
survived, but produced less than 50% of the biomass of their
siblings on medium devoid of the inhibitor. On medium containing
3.times.10.sup.-8 M foramsulfuron no growth was detectable. For
large scale mutant selection experiments 10.sup.-7 M foramsulfuron
was chosen. Surviving and growing cell colonies were numbered and
transferred after 4-6 weeks onto fresh medium containing
3.times.10.sup.-7 M of the inhibitor. One of these cell colonies
was able to grow not only at this concentration of the inhibitor
but even in presence of 3.times.10.sup.-6 M of foramsulfuron. From
this clone (SB574TL), shoots were regenerated in presence of the
ALS-inhibitor herbicide and then the shoots were transferred to MS
medium containing 0.05 mg/l Naphthalene acetic acid (NAA).
[0130] Within 4-12 weeks the shoots formed roots and then they were
transferred into sterile plant containers filled with wet,
sterilized perlite, watered with half strength MS inorganic
ingredients. Alternatively the plantlets were transferred directly
from the agar solidified medium in a perlite containing soil
mixture in the greenhouse. During the first 10-15 days after
transfer into soil containing substrate the plants were kept in an
environment with high air humidity. During and after they were
weaned to normal greenhouse air humidity regimes the plants were
kept in the greenhouse under artificial light (12 h) at
20+-3.degree. C./15+-2.degree. C. day/night temperatures.
[0131] 3-5 weeks later, the regenerated plants from the above
obtained foramsulfuron tolerant cell culture (SB574TL) as well as
from the wild type cell cultures were treated with foramsulfuron,
iodosulfuron-methyl-sodium (CAS RN 144550-3-7) and a mixture of
both active ingredients. The herbicide doses tested were equivalent
to 7-70 g a.i./ha for foramsulfuron and 1-10 g a.i./ha for
iodosulfuron-methyl-sodium. Regenerated plants from this tolerant
cell line tolerated even the highest herbicide doses
(foramsulfuron, iodosulfuron-methyl-sodium and their mixtures in
the ratio 7:1 whereas even the lowest doses killed the wild type
plants.
[0132] Offsprings were tested as follows (in a non-limiting
way):
[0133] Based on SB574TL, F2 and F3 seeds of experimental hybrids
conferring the resistance allele in the heterozygous state as well
as F4-F6 seeds conferring the mutant allele in the homozygous state
were sown in the field and treated with foramsulfuron,
iodosulfuron-methyl-sodium as well as with mixtures of both ALS
inhibitor herbicides when the plants developed 3-5 rosette leaves.
The homozygous seedlings tolerated mixtures of 35 g
foramsulfuron/ha+7 g iodosulfuron-methyl-sodium/ha without growth
retardation or any signs of visible damage. In several cases,
heterozygous lines showed signs of retarded growth and some leaf
chlorosis at these rates, but they recovered within 3-5 weeks,
whereas the conventional sugar beet seedlings were killed by the
ALS inhibitor herbicides.
[0134] The ALS mutants were characterized as follows:
[0135] Extraction and nucleic acid sequence analysis of the
obtained mutant was performed by LGC Genomics GmbH, Berlin, Germany
according to amended standard protocols.
[0136] The nucleic acid sequence obtained from the sugar beet
mutant SB574TL is shown in SEQ ID NO: 3. SEQ ID NO: 4 represents
the corresponding amino acid sequence, whereas SEQ ID NO: 1 was
obtained after sequencing the wild type sugar beet plant that was
taken as the starting material. SEQ ID NO: 2 represents the
corresponding amino acid sequence of the wild type sugar beet.
[0137] Comparison of all these sequences shows up that there is
only the mutation at position 574 but no other change took place at
any other part of this endogenous ALS gene.
TABLE-US-00002 (1) SEQ ID No 1
ATGGCGGCTACCTTCACAAACCCAACATTTTCCCCTTCCTCAACTCCATTAACCAAAACC (1)
SEQ ID No 3
ATGGCGGCTACCTTCACAAACCCAACATTTTCCCCTTCCTCAACTCCATTAACCAAAACC (61)
SEQ ID No 1
CTAAAATCCCAATCTTCCATCTCTTCAACCCTCCCCTTTTCCACCCCTCCCAAAACCCCA (61)
SEQ ID No 3
CTAAAATCCCAATCTTCCATCTCTTCAACCCTCCCCTTTTCCACCCCTCCCAAAACCCCA (121)
SEQ ID No 1
ACTCCACTCTTTCACCGTCCCCTCCAAATCTCATCCTCCCAATCCCACAAATCATCCGCC (121)
SEQ ID No 3
ACTCCACTCTTTCACCGTCCCCTCCAAATCTCATCCTCCCAATCCCACAAATCATCCGCC (181)
SEQ ID No 1
ATTAAAACACAAACTCAAGCACCTTCTTCTCCAGCTATTGAAGATTCATCTTTCGTTTCT (181)
SEQ ID No 3
ATTAAAACACAAACTCAAGCACCTTCTTCTCCAGCTATTGAAGATTCATCTTTCGTTTCT (241)
SEQ ID No 1
CGATTTGGCCCTGATGAACCCAGAAAAGGGTCCGATGTCCTCGTTGAAGCTCTTGAGCGT (241)
SEQ ID No 3
CGATTTGGCCCTGATGAACCCAGAAAAGGGTCCGATGTCCTCGTTGAAGCTGTTGAGCGT (301)
SEQ ID No 1
GAAGGTGTTACCAATGTGTTTGCTTACCCTGGTGGTGCATCTATGGAAATCCACCAAGCT (301)
SEQ ID No 3
GAAGGTGTTACCAATGTGTTTGCTTACCCTGGTGGTGCATCTATGGAAATCCACCAAGCT (361)
SEQ ID No 1
CTCACACGCTCTAAAACCATCCGCAATGTCCTCCCTCGCCATGAACAAGGCGGGGTTTTC (361)
SEQ ID No 3
CTCACACGCTCTAAAACCATCCGCAATGTCCTCCCTCGCCATGAACAAGGCGGGGTTTTC (421)
SEQ ID No 1
GCCGCCGAGGGATATGCTAGAGCTACTGGAAAGGTTGGTGTCTGCATTGCGACTTCTGGT (421)
SEQ ID No 3
GCCGCCGAGGGATATGCTAGAGCTACTGGAAAGGTTGGTGTCTGCATTGCGACTTCTGGT (481)
SEQ ID No 1
CCTGGTGCTACCAACCTCGTATCAGGTCTTGCTGACGCTCTCCTTGATTCTGTCCCTCTT (481)
SEQ ID No 3
CCTGGTGCTACCAACCTCGTATCAGGTCTTGCTGACGCTCTCCTTGATTCTGTCCCTCTT (541)
SEQ ID No 1
GTTGCCATCACTGGCCAAGTTCCACGCCGTATGATTGGCACTGATGCTTTTCAGGAGACT (541)
SEQ ID No 3
GTTGCCATCACTGGCCAAGTTCCACGCCGTATGATTGGCACTGATGCTTTTCAGGAGACT (601)
SEQ ID No 1
CCAATTGTTGAGGTGACTTAGGTCTATTACTAAGCATAATTATTTAGTTTTGGATGTAGAG (601)
SEQ ID No 3
CCAATTGTTGAGGTGACAAGGTCTATTAGTAAGCATAATTATTTAGTTTTGGATGTAGAG (661)
SEQ ID No 1
GATATTCCTAGAATTGTTAAGGAAGCCTTTTTTTTAGCTAATTCTGGTAGGCCTGGACCT (661)
SEQ ID No 3
GATATTCCTAGAATTGTTAAGGAAGCCTTTTTTTTAGCTAATTCTGGTAGGCCTGGACCT (721)
SEQ ID No 1
GTTTTGATTGATCTTCCTAAAGATATTCAGCAGCAATTGGTTGTTCCTGATTGGGATAGG (721)
SEQ ID No 3
GTTTTGATTGATCTTCCTAAAGATATTCAGCAGCAATTGGTTGTTCCTGATTGGGATAGG (781)
SEQ ID No 1
CCTTTTAAGTTGGGTGGGTATATGTCTAGGCTGCCAAAGTCCAAGTTTTCGAGAATGAG (781)
SEQ ID No 3
CCTTTTAAGTTGGGTGGGTATATGTCTAGGCTGCCAAAGTCCAAGTTTTCGACGAATGAG (841)
SEQ ID No 1
GTTGGACTTCTTGAGCAGATTGTGAGTTGATGAGTGAGTCGAAGLAAGCCTGTCTTGTAT (841)
SEQ ID No 3
GTTGGACTTCTTGAGCAGATTGTGAGTTGATGAGTGAGTCGAAGLAAGCCTGTCTTGTAT (901)
SEQ ID No 1
GTGGGAGGTGGGTGTTTGAATTCTAGTGAGGAGTTGAGGAGATTTGTTGAGTTGACAGGG (901)
SEQ ID No 3
GTGGGAGGTGGGTGTTTGAATTCTAGTGAGGAGTTGAGGAGATTTGTTGAGTTGACAGGG (961)
SEQ ID No 1
ATTCCGGTGGCTAGTACTTTGATGGGGTTGGGGTCTTACCCTTGTAATGATGAACTGTCT (961)
SEQ ID No 3
ATTCCGGTGGCTAGTACTTTGATGGGGTTGGGGTCTTACCCTTGTAATGATGAACTGTCT (1021)
SEQ ID No 1
CTTCATATGTTGGGGATGCACGGGACTGTTTATGCCAATTATGCGGTGGATAAGGCGGAT (1021)
SEQ ID No 3
CTTCATATGTTGGGGATGCACGGGACTGTTTATGCCAATTATGCGGTGGATAAGGCGGAT (1081)
SEQ ID No 1
TTGTTGCTTGCTTTCGGGGTTAGGTTTGATGATCGTGTGACCGGGAAGCTCGAGGCGTTT (1081)
SEQ ID No 3
TTGTTGCTTGCTTTCGGGGTTAGGTTTGATGATCGTGTGAGCGGGAAGCTCGAGGCGTTT (1141)
SEQ ID No 1
GCTAGCCGTGCTAAGATTGTGCATATTGATATTGACTCTGCTGAGATTGGGAAGAACAAG (1141)
SEQ ID No 3
GCTAGCCGTGCTAAGATTGTGCATATTGATATTGACTCTGCTGAGATTGGGAAGAACAAG (1201)
SEQ ID No 1
CAGCCCCATGTGTCCATTTGTGCTGATGTTAAATTGGCATTGCGGGGTATGAATAAGATT (1201)
SEQ ID No 3
CAGCCCCATGTGTCCATTTGTGCTGATGTTAAATTGGCATTGCGGGGTATGAATAAGATT (1261)
SEQ ID No 1
CTGGAGTCTAGAATAGGGAAGCTGAATTTGGATTTCTCCAAGTGGAGAGAAGAATTAGGT (1261)
SEQ ID No 3
CTGGAGTCTAGAATAGGGAAGCTGAATTTGGATTTCTCCAAGTGGAGAGAAaAATTAGGT (1321)
SEQ ID No 1
GAGCAGAAGAAGGAATTCCCACTGAGTTTTAAGACATTTGGGGATGCAATTCCTCCACAA (1321)
SEQ ID No 3
GAGCAGAAGAAGGAATTCCCACTGAGTTTTAAGACATTTGGGGATGCAATTCCTCCACAA (1381)
SEQ ID No 1
TATGCCATTCAGGTGCTTGATGAGTTGACCAATGGTAATGCTATTATAAGTACTGGTGTT (1381)
SEQ ID No 3
TATGCCATTCAGGTGCTTGATGAGTTGACCAATGGTAATGCTATTATAAGTACTGGTGTT (1441)
SEQ ID No 1
GGGCAGCACCAAATCTGGGCTGCGCAGCATTAAAAGTACAGAAACCCTCGCCAATGGCTG (1441)
SEQ ID No 3
GGGCAGCACCAAATGTGGGCTGCGCAGCATTAAAAGTACAGAAACCCTCGCCAATGGCTG (1501)
SEQ ID No 1
ACCTCTGGTGGGTTGGGGGCTATGGGGTTTGGGCTACCAGCCGCCATTGGAGCTGCAGTT (1501)
SEQ ID No 3
ACCTCTGGTGGGTTGGGGGCTATGGGGTTTGGGCTACCAGCCGCCATTGGAGCTGCAGTT (1561)
SEQ ID No 1
GCTCGACCAGATGCAGTGGTTGTCGATATTGATGGGGATGGCACTTTTATTATGAATGTT (1561)
SEQ ID No 3
GCTCGACCAGATGCAGTGGTTGTCGATATTGATGGGGATGGCAGTTTTATTATGAATGTT (1621)
SEQ ID No 1
CAAGAGTTGGCTACAATTAGGGTGGAAAATCTCCCAGTTAAGATAATGCTGCTAAACAAT (1621)
SEQ ID No 3
CAAGAGTTGGCTACAATTAGGGTGGAAAATCTCCCAGTTAAGATAATGCTGCTAAACAAT (1681)
SEQ ID No 1
CAACATTTAGGTATGGTTGTCCAATGGGAAGATAGGTTCTATAAAGCTAACCGGGCACAT (1681)
SEQ ID No 3
CAACATTTAGGTATGGTTGTCCAATTGGAAGATAGGTTCTATAAAGCTAACCGGGCACAT (1741)
SEQ ID No 1
ACATACCTTGGAAACCCTTCCAAATCTGCTGATATCTTCCCTGATATGCTCAAATTCGCT (1741)
SEQ ID No 3
ACATACCTTGGAAACCCTTCCAAATCTGCTGATATCTTCCCTGATATGCTCAAATTCGCT (1801)
SEQ ID No 1
GAGGCATGTGATATTCCTTCTGCCCGTGTTAGCAACGTGGCTGATTTGAGGGCCGCCATT (1801)
SEQ ID No 3
GAGGCATGTGATATTCCTTCTGCCCGTGTTAGCAACGTGGCTGATTTGAGGGCCGCCATT (1861)
SEQ ID No 1
CAAACAATGTTGGATACTCCAGGGCCGTACCTGCTCGATGTGATTGTACCGCATCAAGAG (1861)
SEQ ID No 3
CAAACAATGTTGGATACTCCAGGGCCGTACCTGCTCGATGTGATTGTACCGCATCAAGAG (1921)
SEQ ID No 1
CATGTGTTGCCTATGATTCCAAGTGGTGCCGGTTTCAAGGATACCATTACAGAGGGTGAT (1921)
SEQ ID No 3
CATGTGTTGCCTATGATTCCAAGTGGTGCCGGTTTCAAGGATACCATTACAGAGGGTGAT (1981)
SEQ ID No 1 GGAAGAACCTCTTATTGA (1981) SEQ ID No 3
GGAAGAACCTCTTATTGA (1) SEQ ID No. 2
MAATFTNPTFSPSSTPLTHTLKSQSSISSTLPFSTPPKTPTPLFHRPLQISSSQSHKSSA (1)
SEQ ID No. 4
MAATFTNPTFSPSSTPLTHTLKSQSSISSTLPFSTPPKTPTPLFHRPLQISSSQSHKSSA (61)
SEQ ID No. 2
IKTQTQAPSSPAIEDSSFVSRFGPDEPRKGSDVLVEALEREGVTNVFAYPGGASMEIHQA (61)
SEQ ID No. 4
IKTQTQAPSSPAIEDSSFVSRFGPDEFRKGSDVLVEALEREGVTNVFAYPGGASMEIHQA (121)
SEQ ID No. 2
LTRSKTIRNVLPRHEQGGVFAAEGYARATGKVGVCIATSGPGATNLVSGLADALLDSVPL (121)
SEQ ID No. 4
LTRSKTIRNVLPRHEQGGVFAAEGYARATGKVGVCIATSGPGATNLVSGLADALLDSVPL (181)
SEQ ID No. 2
VAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDVEDIPRIVKEAFFLANSGRPGP (181)
SEQ ID No. 4
VAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDVEDIPRIVKEAFFLANSGRPGP (241)
SEQ ID No. 2
VLIDLPKDIQQQLVVPDWDRPFKLGGYMSRLPKSKFSTNEVGLLEQIVRLMSESKKPVLY (241)
SEQ ID No. 4
VLIDLPKDIQQQLVVPDWDRPFKLGGYMSRLPKSKFSTNEVGLLEQIVRLMSESKKPVLY (301)
SEQ ID No. 2
VGGGCLNSSEELRRFVELTGIPVASTLMGLGSYPCNDELSLHMLGMHGTVYANYAVDKAD (301)
SEQ ID No. 4
VGGGCLNSSEELRRFVELTGIPVASTLMGLGSYPCNDELSLHMLGMHGTVYANYAVDKAD (361)
SEQ ID No. 2
LLLAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPKVSICADVKLALRGMNKI (361)
SEQ ID No. 4
LLLAFGVRFDDRVTGKLEAFASPAKIVHIDIDSAEIGKNKQPHVSICADVKLALRGMNKI (421)
SEQ ID No. 2
LESRIGKLNLDFSKWREELGEQKKEFPLSFKTFGDAIPPQYAIQVLDELTNGNAIISTGV (421)
SEQ ID No. 4
LESRIGKLNLDFSKWRESLGEQKKEFPLSFKTFGDAIPPQYAIQVLDELTNGNAIISTGV (481)
SEQ ID No. 2
GQHQMWAAQHYKYRNPRQWLTSGGLGAMGFGLPAAIGAAVARPDAVVVDIDGDGSFIMNV (481)
SEQ ID No. 4
GQHQMWAAQKYKYRNPRQWLT3GGLGAMGFGLPAAIGAAVARPDAVVVDIDGDGSFIMNV (541)
SEQ ID No. 2
QELATIRVENLFVKIMLLNNQHLGMVVQWEDRFYKANRAHTYLGNPSKSADIFFDMLKFA (541)
SEQ ID No. 4
QELATIRVENLFVKIMLLNNQHLGMVVQLEDRFYKANRAHTYLGNPSKSADIFFDMLKFA (601)
SEQ ID No. 2
EACDIPSARVSNVADLRAAIQTMLDTPGPYLLDVIVPHQEHVLPMIPSGAGFKDTITEGD (601)
SEQ ID No. 4
EACDIPSARVSNVADLRAAIQTMLDTPGPYLLDVIVPHQEHVLPMIPSGAGFKDTITEGD (661)
SEQ ID No. 2 GRTSY- (661) SEQ ID No. 4 GRTSY-
[0138] Yet, it is generally preferred that the B. vulgaris plants
of the present invention and parts thereof are agronomically
exploitable. "Agronomically exploitable" means that the B. vulgaris
plants and parts thereof are useful for agronomical purposes. For
example, the B. vulgaris plants should serve for the purpose of
being useful for sugar production, bio fuel production (such as
biogas, biobutanol), ethanol production, betaine and/or uridine
production. The term "agronomically exploitable" when used herein
also includes that the B. vulgaris plants of the present invention
are preferably less sensitive against an ALS-inhibitor herbicide,
more preferably it is at least 100 times less sensitive, more
preferably, 500 times, even more preferably 1000 times and most
preferably less than 2000 times. The ALS inhibitor herbicide is one
or more described herein, preferably it is foramsulfuron either
alone or in combination with one or more further ALS-inhibitor
herbicide(s) either from the sub-class of the sulfonyurea
herbicides or any other sub-class of the ALS-inhibitor herbicides,
most preferably it is foramsulfuron in combination with a further
sulfonylurea herbicide and/or an ALS-inhibitor of the
sulfonylaminocarbonyltriazolinone herbicide sub-class.
[0139] Preferably, agronomically exploitable B. vulgaris plants,
most preferably sugar beet plants, of the present invention are
fully fertile, more preferably have wild-type fertility. Fertility
is of utmost importance for a B. vulgaris plant of the present
invention in order to be agronomically exploitable.
[0140] An example for an agronomically exploitable B. vulgaris
plant is sugar beet. A sugar beet plant of the present invention
when cultivated in an area of one hectare yields (about 80,000 to
90,000 sugar beets) should preferably serve for the production of
at least 4 tons of sugar.
[0141] Alternatively, a sugar beet plant of the present invention
should preferably contain a sugar content between 15-20%,
preferably at least 17% so as to be agronomically exploitable.
Thus, sugar beet plants that contain a sugar content between
15-20%, preferably at least 17% are a preferred embodiment of the
present invention.
[0142] Plants of the present invention can be identified using any
genotypic analysis method. Genotypic evaluation of the plants
includes using techniques such as Isozyme Electrophoresis,
Restriction Fragment Length Polymorphisms (RFLPs), Randomly
Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase
Chain Reaction (AP-PCR), Allele-specific PCR (AS-PCR), DNA
Amplification Fingerprinting (DAF), Sequence Characterized
Amplified Regions (SCARs), Amplified Fragment Length Polymorphisms
(AFLPs), Simple Sequence Repeats (SSRs) which are also referred to
as "Microsatellites". Additional compositions and methods for
analyzing the genotype of the plants provided herein include those
methods disclosed in U.S. Publication No. 2004/0171027, U.S.
Publication No. 2005/02080506, and U.S. Publication No.
2005/0283858.
[0143] Another aspect of the present invention is the use of the
Beta vulgaris plant described herein and/or the harvestable parts
or propagation material described herein for the
manufacture/breeding of Beta vulgaris plants. Methods for the
manufacture/breeding of B. vulgaris plants are described herein
elsewhere. Such manufacture/breeding methods may be used to
generate B. vulgaris plants of the present invention further
comprising novel plant traits such as stress-resistance, like but
not limited to drought, heat, cold, or salt stress and the
like.
[0144] In a still further aspect, the present invention envisages
the use of the herbicide tolerant Beta vulgaris plant described
herein and/or harvestable parts or propagation material derived
thereof in a screening method for the selection of ALS inhibitor
herbicides.
[0145] A better understanding of the present invention and of its
many advantages will be had from the following examples, offered
for illustrative purposes only, and are not intended to limit the
scope of the present invention in any way.
Example 1: Mutant Isolation
[0146] Sugar beet cell cultures were initiated from seedlings of a
diploid sugar beet genotype 7T9044 (as, for example, described by
Alexander Dovzhenko, PhD Thesis, Title: "Towards plastid
transformation in rapeseed (Brassica napus L.) and sugarbeet (Beta
vulgaris L.)", Ludwig-Maximilians-Universitat Munchen, Germany,
2001).
[0147] Sugar beet seeds were immersed for 60 seconds in 70%
ethanol, then rinsed twice in sterile water with 0.01% detergent
and then incubated for 1-4 hours in 1% NaOCl bleach. Thereafter the
seeds were washed 3 times with sterile H.sub.2O and the seeds were
stored in sterile H.sub.2O overnight at 4.degree. C. The embryos
were then isolated using forceps and scalpel.
[0148] The freshly prepared embryos were immersed in 0.5% NaOCl for
30 min and then washed 3 times in sterile H.sub.2O. After the last
washing step they were placed on hormone free MS agar medium
(Murashige and Skoog (1962), Physiol. Plantarum, 15, 473-497).
Those embryos which developed into sterile seedlings were used for
the initiation of regenerable sugar beet cell cultures.
[0149] Cotyledons as well as hypocotyls were cut into 2-5 mm long
segments and then cultivated on agar (0.8%) solidified MS agar
medium containing either 1 mg/l Benzylaminopurin (BAP) or 0.25 mg/l
Thidiazuron (TDZ). 4 weeks later the developing shoot cultures were
transferred onto fresh MS agar medium of the same composition and
then sub-cultured in monthly intervals. The cultures were kept at
25.degree. C. under dim light at a 12 h/12 h light/dark cycle.
[0150] After 7-10 days, subcultures the shoot cultures which were
grown on the thidiazuron containing medium formed a distinct callus
type, which was fast growing, soft and friable. The colour of this
callus type was yellowish to light green. Some of these friable
calli consistently produced chlorophyll containing shoot primordia
from embryo-like structures. These fast growing regenerable calli
were used for the selection of ALS inhibitor herbicide tolerant
sugar beet mutants.
[0151] When this callus type was exposed to 10.sup.-9 M of the ALS
inhibitor herbicide foramsulfuron (belonging to the sulfonylurea
subclass, see above), the cells survived, but produced less than
50% of the biomass of their siblings on medium devoid of the
inhibitor. On medium containing 3.times.10.sup.-8 M foramsulfuron
no growth was detectable. For large scale mutant selection
experiments, 10.sup.-7 M foramsulfuron was chosen. Surviving and
growing cell colonies were numbered and transferred after 4-6 weeks
onto fresh medium containing 3.times.10.sup.-7 M of the inhibitor.
One of these cell colonies was able to grow not only at this
concentration of the inhibitor but even in presence of
3.times.10.sup.-8 M of foramsulfuron.
[0152] From this clone (SB574TL), shoots were regenerated in
presence of the ALS inhibitor herbicide, and then the shoots were
transferred to MS medium containing 0.05 mg/l naphthalene acetic
acid (NAA).
[0153] Within 4-12 weeks the shoots formed roots and then they were
transferred into sterile plant containers filled with wet,
sterilized perlite, watered with half strength MS inorganic
ingredients. Alternatively the plantlets were transferred directly
from the agar solidified medium in a perlite containing soil
mixture in the greenhouse. During the first 10-15 days after
transfer into soil containing substrate the plants were kept in an
environment with high air humidity. During and after they were
weaned to normal greenhouse air humidity regimes the plants were
kept in the greenhouse under artificial light (12 h) at
20+-3.degree. C./15+-2.degree. C. day/night temperatures. 3-5 weeks
later, the regenerated plants from the above obtained foramsulfuron
tolerant cell culture (SB574TL) as well as from the wild type cell
cultures were treated with foramsulfuron,
iodosulfuron-methyl-sodium (CAS RN 144550-3-7) and a mixture of
both active ingredients. The herbicide doses tested were equivalent
to 7-70 g a.i./ha for foramsulfuron and 1-10 g a.i./ha for
iodosulfuron-methyl-sodium. Regenerated plants from this tolerant
cell line tolerated even the highest herbicide doses
(foramsulfuron, iodosulfuron-methyl-sodium and their mixtures in
the ratio 7:1 whereas even the lowest doses killed the wild type
plants.
Example 2: Test of Offsprings
[0154] Based on SB574TL, F2 and F3 seeds of experimental hybrids
conferring the resistance allele in the heterozygous state as well
as F4-F6 seeds conferring the mutant allele in the homozygous state
were sown in the field and treated with foramsulfuron,
iodosulfuron-methyl-sodium as well as with mixtures of both ALS
inhibitor herbicides when the plants developed 3-5 rosette leaves.
The homozygous seedlings tolerated mixtures of 35 g
foramsulfuron/ha+7 g iodosulfuron-methyl-sodium/ha without growth
retardation or any signs of visible damage. Heterozygous lines
showed signs of retarded growth and some leaf chlorosis at these
rates, but they recovered within 3-5 weeks, whereas the
conventional sugar beet seedlings were killed by the ALS inhibitor
herbicides.
Example 3: Molecular Characterization of the Obtained Sugar Beet
Mutant (SB574TL)
[0155] Extraction and nucleic acid sequence analysis of the
obtained mutant was performed by LGC Genomics GmbH, Berlin, Germany
according to amended standard protocols.
[0156] The nucleic acid sequence obtained from the sugar beet
mutant SB574TL is shown under SEQ ID NO: 3 with SEQ ID NO: 4
representing the corresponding amino acid sequence, whereas SEQ ID
NO: 1 was obtained after sequencing the wild type sugar beet plant
that was taken as the starting material. SEQ ID NO: 2 represents
the corresponding amino acid sequence of the wild type sugar
beet.
[0157] Comparison of all these sequences clearly show up that there
is only one mutation at position 569 but no other change took place
at any other part of this endogenous ALS gene of this sugar beet
plant material.
Example 4: Enzyme Activity Measurements
[0158] The coding sequences of Beta vulgaris wild-type and
W574L-mutant (SB574TL) ALS gene were cloned into Novagen pET-32a(+)
vectors and the vectors transformed into Escherichia coli AD494
according to the instructions of the manufacturer. Bacteria were
grown at 37.degree. C. in LB-medium (Luria-Broth-medium) containing
100 mg/l carbenicillin and 25 mg/l kanamycin, induced with 1 mM
isopropyl-b-D-thiogalactopyranoside at an OD.sub.600 of 0.6,
cultivated for 16 hours at 18.degree. C. and harvested by
centrifugation. Bacterial pellets were resuspended in 100 mM sodium
phosphate buffer pH 7.0 containing 0.1 mM thiamine-pyrophosphate, 1
mM MgCl.sub.2, and 1 .mu.M FAD at a concentration of 1 gram wet
weight per 25 ml of buffer and disrupted by sonification. The crude
protein extract obtained after centrifugation was used for ALS
activity measurements.
[0159] ALS assays were carried out in 96-well microtiter plates
using a modification of the procedure described by Ray (1984). The
reaction mixture contained 20 mM potassium phosphate buffer pH 7.0,
20 mM sodium pyruvate, 0.45 mM thiamine-pyrophosphate, 0.45 mM
MgCl.sub.2, 9 .mu.M FAD, ALS enzyme and various concentrations of
ALS inhibitors in a final volume of 90 .mu.l. Assays were initiated
by adding enzyme and terminated after 75 min incubation at
30.degree. C. by the addition of 40 .mu.l 0.5 M H.sub.2SO.sub.4.
After 60 min at room temperature 80 .mu.l of a solution of 1.4%
a-naphthol and 0.14% creatine in 0.7 M NaOH was added and after an
additional 45 min incubation at room temperature the absorbance was
determined at 540 nm. pI50-values for inhibition of ALS were
determined as described by Ray (1984), using the XLFit Excel add-in
version 4.3.1 curve fitting program of ID Business Solutions
Limited.
[0160] In total, the mutant enzyme was at least 2000 times less
sensitive against the ALS inhibitor foramsulfuron than the wild
type enzyme.
Example 5: Enzyme Activity Measurements (from Plants)
[0161] ALS was extracted from sugar beet leaves or sugar beet
tissue cultures as described by Ray (1984), Plant Physiol
75:827-831.
[0162] ALS activity was determined in leaf extracts of wild type
and sugar beets and leaf extracts of the obtained SB574TL in
presence of various concentrations of foramsulfuron as described in
Example 4.
[0163] In total, the mutant enzyme was at least 2000 times less
sensitive against the ALS inhibitor foramsulfuron than the wild
type enzyme.
Example 6 Field Trials by Employing Homozygous ALS Inhibitor
Herbicide Tolerant Sugar Beet Plants
[0164] Based on SB574TL, F4-F6 seeds conferring the mutant allele
of the endogenous ALS gene in the homozygous state were applied for
further testing Plant seeds of the homozygous SB574TL mutant plants
and those of the traditional variety KLARINA (commonly available
ALS inhibitor sensitive reference sugar beet varieties, not having
the respective mutation at position 569 in its ALS protein.) were
sown in the field and grew up to various growth stages according to
the BBCH standard (as defined in the monographie
"Entwicklungsstadien mono- und dikotyler Pflanzen", 2nd edition,
2001, ed. Uwe Meier, Biologische Bundesanstalt for Land und
Forstwirtschaft).
[0165] Afterwards the plants were treated with the respective ALS
inhibitor herbicides as specified in Tables 1 below and which
identical to those being employed during the selection
procedure.
[0166] The water quantity applied in the various applications
equaled 200 l/ha. At 8, 14, and 28 days (as indicated in Table 1)
after application (DAA) of the respective ALS inhibitor
herbicide(s), the damages (phytotoxicity/phyto) on the different
sugar beet plants were scored according to the scale from 0% to
100%. In this context, "0%" means "no phytotoxicity/phyto" and
"100%" means plants were completely killed.
TABLE-US-00003 TABLE 1 SB574TL SB574TL SB574TL Variety based sugar
based sugar based sugar characteristic KLARINA beet KLARINA beet
KLARINA beet Stage of BBCH 14 BBCH 14 BBCH 14 BBCH 14 BBCH 14 BBCH
14 application Rating % phyto % phyto % phyto % phyto % phyto %
phyto Application- 8 days 8 days 14 days 14 days 28 days 28 days
Assessment interval Active substance gai/ha Foramsulfuron 25 85 0
83 0 86 0 g/ha Foramsulfuron 50 90 0 92 0 94 0 g/ha Iodosulfuron- 7
90 0 97 0 100 0 methyl-sodium g/ha
Sequence CWU 1
1
611998DNABeta vulgaris 1atggcggcta ccttcacaaa cccaacattt tccccttcct
caactccatt aaccaaaacc 60ctaaaatccc aatcttccat ctcttcaacc ctcccctttt
ccacccctcc caaaacccca 120actccactct ttcaccgtcc cctccaaatc
tcatcctccc aatcccacaa atcatccgcc 180attaaaacac aaactcaagc
accttcttct ccagctattg aagattcatc tttcgtttct 240cgatttggcc
ctgatgaacc cagaaaaggg tccgatgtcc tcgttgaagc tcttgagcgt
300gaaggtgtta ccaatgtgtt tgcttaccct ggtggtgcat ctatggaaat
ccaccaagct 360ctcacacgct ctaaaaccat ccgcaatgtc ctccctcgcc
atgaacaagg cggggttttc 420gccgccgagg gatatgctag agctactgga
aaggttggtg tctgcattgc gacttctggt 480cctggtgcta ccaacctcgt
atcaggtctt gctgacgctc tccttgattc tgtccctctt 540gttgccatca
ctggccaagt tccacgccgt atgattggca ctgatgcttt tcaggagact
600ccaattgttg aggtgacaag gtctattact aagcataatt atttagtttt
ggatgtagag 660gatattccta gaattgttaa ggaagccttt tttttagcta
attctggtag gcctggacct 720gttttgattg atcttcctaa agatattcag
cagcaattgg ttgttcctga ttgggatagg 780ccttttaagt tgggtgggta
tatgtctagg ctgccaaagt ccaagttttc gacgaatgag 840gttggacttc
ttgagcagat tgtgaggttg atgagtgagt cgaagaagcc tgtcttgtat
900gtgggaggtg ggtgtttgaa ttctagtgag gagttgagga gatttgttga
gttgacaggg 960attccggtgg ctagtacttt gatggggttg gggtcttacc
cttgtaatga tgaactgtct 1020cttcatatgt tggggatgca cgggactgtt
tatgccaatt atgcggtgga taaggcggat 1080ttgttgcttg ctttcggggt
taggtttgat gatcgtgtga ccgggaagct cgaggcgttt 1140gctagccgtg
ctaagattgt gcatattgat attgactctg ctgagattgg gaagaacaag
1200cagccccatg tgtccatttg tgctgatgtt aaattggcat tgcggggtat
gaataagatt 1260ctggagtcta gaatagggaa gctgaatttg gatttctcca
agtggagaga agaattaggt 1320gagcagaaga aggaattccc actgagtttt
aagacatttg gggatgcaat tcctccacaa 1380tatgccattc aggtgcttga
tgagttgacc aatggtaatg ctattataag tactggtgtt 1440gggcagcacc
aaatgtgggc tgcgcagcat tacaagtaca gaaaccctcg ccaatggctg
1500acctctggtg ggttgggggc tatggggttt gggctaccag ccgccattgg
agctgcagtt 1560gctcgaccag atgcagtggt tgtcgatatt gatggggatg
gcagttttat tatgaatgtt 1620caagagttgg ctacaattag ggtggaaaat
ctcccagtta agataatgct gctaaacaat 1680caacatttag gtatggttgt
ccaatgggaa gataggttct ataaagctaa ccgggcacat 1740acataccttg
gaaacccttc caaatctgct gatatcttcc ctgatatgct caaattcgct
1800gaggcatgtg atattccttc tgcccgtgtt agcaacgtgg ctgatttgag
ggccgccatt 1860caaacaatgt tggatactcc agggccgtac ctgctcgatg
tgattgtacc gcatcaagag 1920catgtgttgc ctatgattcc aagtggtgcc
ggtttcaagg ataccattac agagggtgat 1980ggaagaacct cttattga
19982665PRTBeta vulgaris 2Met Ala Ala Thr Phe Thr Asn Pro Thr Phe
Ser Pro Ser Ser Thr Pro1 5 10 15Leu Thr Lys Thr Leu Lys Ser Gln Ser
Ser Ile Ser Ser Thr Leu Pro 20 25 30Phe Ser Thr Pro Pro Lys Thr Pro
Thr Pro Leu Phe His Arg Pro Leu 35 40 45Gln Ile Ser Ser Ser Gln Ser
His Lys Ser Ser Ala Ile Lys Thr Gln 50 55 60Thr Gln Ala Pro Ser Ser
Pro Ala Ile Glu Asp Ser Ser Phe Val Ser65 70 75 80Arg Phe Gly Pro
Asp Glu Pro Arg Lys Gly Ser Asp Val Leu Val Glu 85 90 95Ala Leu Glu
Arg Glu Gly Val Thr Asn Val Phe Ala Tyr Pro Gly Gly 100 105 110Ala
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Lys Thr Ile Arg 115 120
125Asn Val Leu Pro Arg His Glu Gln Gly Gly Val Phe Ala Ala Glu Gly
130 135 140Tyr Ala Arg Ala Thr Gly Lys Val Gly Val Cys Ile Ala Thr
Ser Gly145 150 155 160Pro Gly Ala Thr Asn Leu Val Ser Gly Leu Ala
Asp Ala Leu Leu Asp 165 170 175Ser Val Pro Leu Val Ala Ile Thr Gly
Gln Val Pro Arg Arg Met Ile 180 185 190Gly Thr Asp Ala Phe Gln Glu
Thr Pro Ile Val Glu Val Thr Arg Ser 195 200 205Ile Thr Lys His Asn
Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg 210 215 220Ile Val Lys
Glu Ala Phe Phe Leu Ala Asn Ser Gly Arg Pro Gly Pro225 230 235
240Val Leu Ile Asp Leu Pro Lys Asp Ile Gln Gln Gln Leu Val Val Pro
245 250 255Asp Trp Asp Arg Pro Phe Lys Leu Gly Gly Tyr Met Ser Arg
Leu Pro 260 265 270Lys Ser Lys Phe Ser Thr Asn Glu Val Gly Leu Leu
Glu Gln Ile Val 275 280 285Arg Leu Met Ser Glu Ser Lys Lys Pro Val
Leu Tyr Val Gly Gly Gly 290 295 300Cys Leu Asn Ser Ser Glu Glu Leu
Arg Arg Phe Val Glu Leu Thr Gly305 310 315 320Ile Pro Val Ala Ser
Thr Leu Met Gly Leu Gly Ser Tyr Pro Cys Asn 325 330 335Asp Glu Leu
Ser Leu His Met Leu Gly Met His Gly Thr Val Tyr Ala 340 345 350Asn
Tyr Ala Val Asp Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg 355 360
365Phe Asp Asp Arg Val Thr Gly Lys Leu Glu Ala Phe Ala Ser Arg Ala
370 375 380Lys Ile Val His Ile Asp Ile Asp Ser Ala Glu Ile Gly Lys
Asn Lys385 390 395 400Gln Pro His Val Ser Ile Cys Ala Asp Val Lys
Leu Ala Leu Arg Gly 405 410 415Met Asn Lys Ile Leu Glu Ser Arg Ile
Gly Lys Leu Asn Leu Asp Phe 420 425 430Ser Lys Trp Arg Glu Glu Leu
Gly Glu Gln Lys Lys Glu Phe Pro Leu 435 440 445Ser Phe Lys Thr Phe
Gly Asp Ala Ile Pro Pro Gln Tyr Ala Ile Gln 450 455 460Val Leu Asp
Glu Leu Thr Asn Gly Asn Ala Ile Ile Ser Thr Gly Val465 470 475
480Gly Gln His Gln Met Trp Ala Ala Gln His Tyr Lys Tyr Arg Asn Pro
485 490 495Arg Gln Trp Leu Thr Ser Gly Gly Leu Gly Ala Met Gly Phe
Gly Leu 500 505 510Pro Ala Ala Ile Gly Ala Ala Val Ala Arg Pro Asp
Ala Val Val Val 515 520 525Asp Ile Asp Gly Asp Gly Ser Phe Ile Met
Asn Val Gln Glu Leu Ala 530 535 540Thr Ile Arg Val Glu Asn Leu Pro
Val Lys Ile Met Leu Leu Asn Asn545 550 555 560Gln His Leu Gly Met
Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala 565 570 575Asn Arg Ala
His Thr Tyr Leu Gly Asn Pro Ser Lys Ser Ala Asp Ile 580 585 590Phe
Pro Asp Met Leu Lys Phe Ala Glu Ala Cys Asp Ile Pro Ser Ala 595 600
605Arg Val Ser Asn Val Ala Asp Leu Arg Ala Ala Ile Gln Thr Met Leu
610 615 620Asp Thr Pro Gly Pro Tyr Leu Leu Asp Val Ile Val Pro His
Gln Glu625 630 635 640His Val Leu Pro Met Ile Pro Ser Gly Ala Gly
Phe Lys Asp Thr Ile 645 650 655Thr Glu Gly Asp Gly Arg Thr Ser Tyr
660 66531998DNABeta vulgarismutation(1706)..(1706)Substitution of a
Guanosine by a Thymidine 3atggcggcta ccttcacaaa cccaacattt
tccccttcct caactccatt aaccaaaacc 60ctaaaatccc aatcttccat ctcttcaacc
ctcccctttt ccacccctcc caaaacccca 120actccactct ttcaccgtcc
cctccaaatc tcatcctccc aatcccacaa atcatccgcc 180attaaaacac
aaactcaagc accttcttct ccagctattg aagattcatc tttcgtttct
240cgatttggcc ctgatgaacc cagaaaaggg tccgatgtcc tcgttgaagc
tcttgagcgt 300gaaggtgtta ccaatgtgtt tgcttaccct ggtggtgcat
ctatggaaat ccaccaagct 360ctcacacgct ctaaaaccat ccgcaatgtc
ctccctcgcc atgaacaagg cggggttttc 420gccgccgagg gatatgctag
agctactgga aaggttggtg tctgcattgc gacttctggt 480cctggtgcta
ccaacctcgt atcaggtctt gctgacgctc tccttgattc tgtccctctt
540gttgccatca ctggccaagt tccacgccgt atgattggca ctgatgcttt
tcaggagact 600ccaattgttg aggtgacaag gtctattact aagcataatt
atttagtttt ggatgtagag 660gatattccta gaattgttaa ggaagccttt
tttttagcta attctggtag gcctggacct 720gttttgattg atcttcctaa
agatattcag cagcaattgg ttgttcctga ttgggatagg 780ccttttaagt
tgggtgggta tatgtctagg ctgccaaagt ccaagttttc gacgaatgag
840gttggacttc ttgagcagat tgtgaggttg atgagtgagt cgaagaagcc
tgtcttgtat 900gtgggaggtg ggtgtttgaa ttctagtgag gagttgagga
gatttgttga gttgacaggg 960attccggtgg ctagtacttt gatggggttg
gggtcttacc cttgtaatga tgaactgtct 1020cttcatatgt tggggatgca
cgggactgtt tatgccaatt atgcggtgga taaggcggat 1080ttgttgcttg
ctttcggggt taggtttgat gatcgtgtga ccgggaagct cgaggcgttt
1140gctagccgtg ctaagattgt gcatattgat attgactctg ctgagattgg
gaagaacaag 1200cagccccatg tgtccatttg tgctgatgtt aaattggcat
tgcggggtat gaataagatt 1260ctggagtcta gaatagggaa gctgaatttg
gatttctcca agtggagaga agaattaggt 1320gagcagaaga aggaattccc
actgagtttt aagacatttg gggatgcaat tcctccacaa 1380tatgccattc
aggtgcttga tgagttgacc aatggtaatg ctattataag tactggtgtt
1440gggcagcacc aaatgtgggc tgcgcagcat tacaagtaca gaaaccctcg
ccaatggctg 1500acctctggtg ggttgggggc tatggggttt gggctaccag
ccgccattgg agctgcagtt 1560gctcgaccag atgcagtggt tgtcgatatt
gatggggatg gcagttttat tatgaatgtt 1620caagagttgg ctacaattag
ggtggaaaat ctcccagtta agataatgct gctaaacaat 1680caacatttag
gtatggttgt ccaattggaa gataggttct ataaagctaa ccgggcacat
1740acataccttg gaaacccttc caaatctgct gatatcttcc ctgatatgct
caaattcgct 1800gaggcatgtg atattccttc tgcccgtgtt agcaacgtgg
ctgatttgag ggccgccatt 1860caaacaatgt tggatactcc agggccgtac
ctgctcgatg tgattgtacc gcatcaagag 1920catgtgttgc ctatgattcc
aagtggtgcc ggtttcaagg ataccattac agagggtgat 1980ggaagaacct cttattga
19984665PRTBeta vulgarisMISC_FEATURE(569)..(569)Substitution of a
Tryptophan by a Leucine 4Met Ala Ala Thr Phe Thr Asn Pro Thr Phe
Ser Pro Ser Ser Thr Pro1 5 10 15Leu Thr Lys Thr Leu Lys Ser Gln Ser
Ser Ile Ser Ser Thr Leu Pro 20 25 30Phe Ser Thr Pro Pro Lys Thr Pro
Thr Pro Leu Phe His Arg Pro Leu 35 40 45Gln Ile Ser Ser Ser Gln Ser
His Lys Ser Ser Ala Ile Lys Thr Gln 50 55 60Thr Gln Ala Pro Ser Ser
Pro Ala Ile Glu Asp Ser Ser Phe Val Ser65 70 75 80Arg Phe Gly Pro
Asp Glu Pro Arg Lys Gly Ser Asp Val Leu Val Glu 85 90 95Ala Leu Glu
Arg Glu Gly Val Thr Asn Val Phe Ala Tyr Pro Gly Gly 100 105 110Ala
Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser Lys Thr Ile Arg 115 120
125Asn Val Leu Pro Arg His Glu Gln Gly Gly Val Phe Ala Ala Glu Gly
130 135 140Tyr Ala Arg Ala Thr Gly Lys Val Gly Val Cys Ile Ala Thr
Ser Gly145 150 155 160Pro Gly Ala Thr Asn Leu Val Ser Gly Leu Ala
Asp Ala Leu Leu Asp 165 170 175Ser Val Pro Leu Val Ala Ile Thr Gly
Gln Val Pro Arg Arg Met Ile 180 185 190Gly Thr Asp Ala Phe Gln Glu
Thr Pro Ile Val Glu Val Thr Arg Ser 195 200 205Ile Thr Lys His Asn
Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg 210 215 220Ile Val Lys
Glu Ala Phe Phe Leu Ala Asn Ser Gly Arg Pro Gly Pro225 230 235
240Val Leu Ile Asp Leu Pro Lys Asp Ile Gln Gln Gln Leu Val Val Pro
245 250 255Asp Trp Asp Arg Pro Phe Lys Leu Gly Gly Tyr Met Ser Arg
Leu Pro 260 265 270Lys Ser Lys Phe Ser Thr Asn Glu Val Gly Leu Leu
Glu Gln Ile Val 275 280 285Arg Leu Met Ser Glu Ser Lys Lys Pro Val
Leu Tyr Val Gly Gly Gly 290 295 300Cys Leu Asn Ser Ser Glu Glu Leu
Arg Arg Phe Val Glu Leu Thr Gly305 310 315 320Ile Pro Val Ala Ser
Thr Leu Met Gly Leu Gly Ser Tyr Pro Cys Asn 325 330 335Asp Glu Leu
Ser Leu His Met Leu Gly Met His Gly Thr Val Tyr Ala 340 345 350Asn
Tyr Ala Val Asp Lys Ala Asp Leu Leu Leu Ala Phe Gly Val Arg 355 360
365Phe Asp Asp Arg Val Thr Gly Lys Leu Glu Ala Phe Ala Ser Arg Ala
370 375 380Lys Ile Val His Ile Asp Ile Asp Ser Ala Glu Ile Gly Lys
Asn Lys385 390 395 400Gln Pro His Val Ser Ile Cys Ala Asp Val Lys
Leu Ala Leu Arg Gly 405 410 415Met Asn Lys Ile Leu Glu Ser Arg Ile
Gly Lys Leu Asn Leu Asp Phe 420 425 430Ser Lys Trp Arg Glu Glu Leu
Gly Glu Gln Lys Lys Glu Phe Pro Leu 435 440 445Ser Phe Lys Thr Phe
Gly Asp Ala Ile Pro Pro Gln Tyr Ala Ile Gln 450 455 460Val Leu Asp
Glu Leu Thr Asn Gly Asn Ala Ile Ile Ser Thr Gly Val465 470 475
480Gly Gln His Gln Met Trp Ala Ala Gln His Tyr Lys Tyr Arg Asn Pro
485 490 495Arg Gln Trp Leu Thr Ser Gly Gly Leu Gly Ala Met Gly Phe
Gly Leu 500 505 510Pro Ala Ala Ile Gly Ala Ala Val Ala Arg Pro Asp
Ala Val Val Val 515 520 525Asp Ile Asp Gly Asp Gly Ser Phe Ile Met
Asn Val Gln Glu Leu Ala 530 535 540Thr Ile Arg Val Glu Asn Leu Pro
Val Lys Ile Met Leu Leu Asn Asn545 550 555 560Gln His Leu Gly Met
Val Val Gln Leu Glu Asp Arg Phe Tyr Lys Ala 565 570 575Asn Arg Ala
His Thr Tyr Leu Gly Asn Pro Ser Lys Ser Ala Asp Ile 580 585 590Phe
Pro Asp Met Leu Lys Phe Ala Glu Ala Cys Asp Ile Pro Ser Ala 595 600
605Arg Val Ser Asn Val Ala Asp Leu Arg Ala Ala Ile Gln Thr Met Leu
610 615 620Asp Thr Pro Gly Pro Tyr Leu Leu Asp Val Ile Val Pro His
Gln Glu625 630 635 640His Val Leu Pro Met Ile Pro Ser Gly Ala Gly
Phe Lys Asp Thr Ile 645 650 655Thr Glu Gly Asp Gly Arg Thr Ser Tyr
660 66552013DNAArabidopsis thaliana 5atggcggcgg caacaacaac
aacaacaaca tcttcttcga tctccttctc caccaaacca 60tctccttcct cctccaaatc
accattacca atctccagat tctccctccc attctcccta 120aaccccaaca
aatcatcctc ctcctcccgc cgccgcggta tcaaatccag ctctccctcc
180tccatctccg ccgtgctcaa cacaaccacc aatgtcacaa ccactccctc
tccaaccaaa 240cctaccaaac ccgaaacatt catctcccga ttcgctccag
atcaaccccg caaaggcgct 300gatatcctcg tcgaagcttt agaacgtcaa
ggcgtagaaa ccgtattcgc ttaccctgga 360ggtgcatcaa tggagattca
ccaagcctta acccgctctt cctcaatccg taacgtcctt 420cctcgtcacg
aacaaggagg tgtattcgca gcagaaggat acgctcgatc ctcaggtaaa
480ccaggtatct gtatagccac ttcaggtccc ggagctacaa atctcgttag
cggattagcc 540gatgcgttgt tagatagtgt tcctcttgta gcaatcacag
gacaagtccc tcgtcgtatg 600attggtacag atgcgtttca agagactccg
attgttgagg taacgcgttc gattacgaag 660cataactatc ttgtgatgga
tgttgaagat atccctagga ttattgagga agctttcttt 720ttagctactt
ctggtagacc tggacctgtt ttggttgatg ttcctaaaga tattcaacaa
780cagcttgcga ttcctaattg ggaacaggct atgagattac ctggttatat
gtctaggatg 840cctaaacctc cggaagattc tcatttggag cagattgtta
ggttgatttc tgagtctaag 900aagcctgtgt tgtatgttgg tggtggttgt
ttgaattcta gcgatgaatt gggtaggttt 960gttgagctta cggggatccc
tgttgcgagt acgttgatgg ggctgggatc ttatccttgt 1020gatgatgagt
tgtcgttaca tatgcttgga atgcatggga cggtgtatgc gaattacgct
1080gtggagcata gtgatttgtt gttggcgttt ggggtgaggt ttgatgatcg
cgtcacgggt 1140aagcttgagg cttttgctag tagggctaag attgttcata
ttgatattga ctctgctgag 1200attgggaaga ataagactcc tcatgtgtct
gtgtgtggtg atgtcaagct ggctttgcaa 1260gggatgaata aggttcttga
gaaccgagct gaggagctta agcttgattt tggagtttgg 1320aggaatgagt
tgaacgtaca gaaacagaag tttccgttga gctttaagac gtttggggaa
1380gctattcctc cacagtatgc gattaaggtc cttgatgagt tgactgatgg
aaaagccata 1440ataagtactg gtgtcgggca acatcaaatg tgggcggcgc
agttctacaa ttacaagaag 1500ccaaggcagt ggctatcatc aggaggcctt
ggagctatgg gttttggact tcctgctgcc 1560attggagcgt ctgttgctaa
ccctgatgca atagttgtgg atattgacgg agatggaagc 1620tttataatga
atgtgcaaga gctggccaca atccgtgtag agcaacttcc agtgaagata
1680ctcttattaa acaaccagca tcttggcatg gttatgcaat gggaagatcg
gttctacaag 1740gctaaccgag ctcacacatt tctcggggat ccggctcagg
aggacgagat attcccgaac 1800atgttgctgt ttgcagcagc ttgcgggatt
ccagcggcga gggtgacaaa gaaagcagat 1860ctccgagaag ctattcagac
aatgctggat acaccaggac cttacctgtt ggatgtgatt 1920tgtccgcacc
aagaacatgt gttgccgatg atcccgagtg gtggcacttt caacgatgtc
1980ataacggaag gagatggccg gattaaatac tga 20136670PRTArabidopsis
thaliana 6Met Ala Ala Ala Thr Thr Thr Thr Thr Thr Ser Ser Ser Ile
Ser Phe1 5 10 15Ser Thr Lys Pro Ser Pro Ser Ser Ser Lys Ser Pro Leu
Pro Ile Ser 20 25 30Arg Phe Ser Leu Pro Phe Ser Leu Asn Pro Asn Lys
Ser Ser Ser Ser 35 40 45Ser Arg Arg Arg Gly Ile Lys Ser Ser Ser Pro
Ser Ser Ile Ser Ala 50 55 60Val Leu Asn Thr Thr Thr Asn Val Thr Thr
Thr Pro Ser Pro Thr Lys65 70 75 80Pro Thr Lys Pro Glu Thr Phe Ile
Ser Arg Phe Ala Pro Asp Gln Pro
85 90 95Arg Lys Gly Ala Asp Ile Leu Val Glu Ala Leu Glu Arg Gln Gly
Val 100 105 110Glu Thr Val Phe Ala Tyr Pro Gly Gly Ala Ser Met Glu
Ile His Gln 115 120 125Ala Leu Thr Arg Ser Ser Ser Ile Arg Asn Val
Leu Pro Arg His Glu 130 135 140Gln Gly Gly Val Phe Ala Ala Glu Gly
Tyr Ala Arg Ser Ser Gly Lys145 150 155 160Pro Gly Ile Cys Ile Ala
Thr Ser Gly Pro Gly Ala Thr Asn Leu Val 165 170 175Ser Gly Leu Ala
Asp Ala Leu Leu Asp Ser Val Pro Leu Val Ala Ile 180 185 190Thr Gly
Gln Val Pro Arg Arg Met Ile Gly Thr Asp Ala Phe Gln Glu 195 200
205Thr Pro Ile Val Glu Val Thr Arg Ser Ile Thr Lys His Asn Tyr Leu
210 215 220Val Met Asp Val Glu Asp Ile Pro Arg Ile Ile Glu Glu Ala
Phe Phe225 230 235 240Leu Ala Thr Ser Gly Arg Pro Gly Pro Val Leu
Val Asp Val Pro Lys 245 250 255Asp Ile Gln Gln Gln Leu Ala Ile Pro
Asn Trp Glu Gln Ala Met Arg 260 265 270Leu Pro Gly Tyr Met Ser Arg
Met Pro Lys Pro Pro Glu Asp Ser His 275 280 285Leu Glu Gln Ile Val
Arg Leu Ile Ser Glu Ser Lys Lys Pro Val Leu 290 295 300Tyr Val Gly
Gly Gly Cys Leu Asn Ser Ser Asp Glu Leu Gly Arg Phe305 310 315
320Val Glu Leu Thr Gly Ile Pro Val Ala Ser Thr Leu Met Gly Leu Gly
325 330 335Ser Tyr Pro Cys Asp Asp Glu Leu Ser Leu His Met Leu Gly
Met His 340 345 350Gly Thr Val Tyr Ala Asn Tyr Ala Val Glu His Ser
Asp Leu Leu Leu 355 360 365Ala Phe Gly Val Arg Phe Asp Asp Arg Val
Thr Gly Lys Leu Glu Ala 370 375 380Phe Ala Ser Arg Ala Lys Ile Val
His Ile Asp Ile Asp Ser Ala Glu385 390 395 400Ile Gly Lys Asn Lys
Thr Pro His Val Ser Val Cys Gly Asp Val Lys 405 410 415Leu Ala Leu
Gln Gly Met Asn Lys Val Leu Glu Asn Arg Ala Glu Glu 420 425 430Leu
Lys Leu Asp Phe Gly Val Trp Arg Asn Glu Leu Asn Val Gln Lys 435 440
445Gln Lys Phe Pro Leu Ser Phe Lys Thr Phe Gly Glu Ala Ile Pro Pro
450 455 460Gln Tyr Ala Ile Lys Val Leu Asp Glu Leu Thr Asp Gly Lys
Ala Ile465 470 475 480Ile Ser Thr Gly Val Gly Gln His Gln Met Trp
Ala Ala Gln Phe Tyr 485 490 495Asn Tyr Lys Lys Pro Arg Gln Trp Leu
Ser Ser Gly Gly Leu Gly Ala 500 505 510Met Gly Phe Gly Leu Pro Ala
Ala Ile Gly Ala Ser Val Ala Asn Pro 515 520 525Asp Ala Ile Val Val
Asp Ile Asp Gly Asp Gly Ser Phe Ile Met Asn 530 535 540Val Gln Glu
Leu Ala Thr Ile Arg Val Glu Asn Leu Pro Val Lys Val545 550 555
560Leu Leu Leu Asn Asn Gln His Leu Gly Met Val Met Gln Trp Glu Asp
565 570 575Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Phe Leu Gly Asp
Pro Ala 580 585 590Gln Glu Asp Glu Ile Phe Pro Asn Met Leu Leu Phe
Ala Ala Ala Cys 595 600 605Gly Ile Pro Ala Ala Arg Val Thr Lys Lys
Ala Asp Leu Arg Glu Ala 610 615 620Ile Gln Thr Met Leu Asp Thr Pro
Gly Pro Tyr Leu Leu Asp Val Ile625 630 635 640Cys Pro His Gln Glu
His Val Leu Pro Met Ile Pro Asn Gly Gly Thr 645 650 655Phe Asn Asp
Val Ile Thr Glu Gly Asp Gly Arg Ile Lys Tyr 660 665 670
* * * * *