U.S. patent application number 11/984821 was filed with the patent office on 2008-08-07 for methods for modification of plant inflorescence architecture.
Invention is credited to Vivijan Babic, Raju Datla, Tim Dumonceaux, Wilf Keller, Gopalan Selvaraj, Prakash Venglat.
Application Number | 20080189809 11/984821 |
Document ID | / |
Family ID | 23079244 |
Filed Date | 2008-08-07 |
United States Patent
Application |
20080189809 |
Kind Code |
A1 |
Datla; Raju ; et
al. |
August 7, 2008 |
Methods for modification of plant inflorescence architecture
Abstract
The present invention relates to methods for the use of the
Arabidopsis "BREVIPEDICELLUS" (BP) gene for alteration of plant
architecture, in particular alteration of the morphology of the
inflorescence of a flowering plant. The methods of the present
invention provide a means to alter the development of the peduncle,
notably the inflorescence branches, and the pedicels that subtend
the individual flowers as well as aspects of flower structure such
as the style, and subsequent seed pods, of a flowering plant. The
invention also relates to methods to identify and isolate
polynucleotides encoding genes with BP-related functions from other
plant species and methods for utilizing said polynucleotides to
alter the inflorescence of said plant species. Furthermore, the
invention encompasses transgenic plants generated by the methods
disclosed, and nucleotide sequences for use in generating the
transgenic plants.
Inventors: |
Datla; Raju; (Saskatoon,
CA) ; Babic; Vivijan; (Saskatoon, CA) ;
Dumonceaux; Tim; (Winnipeg, CA) ; Venglat;
Prakash; (Saskatoon, CA) ; Keller; Wilf;
(Saskatoon, CA) ; Selvaraj; Gopalan; (Saskatoon,
CA) |
Correspondence
Address: |
NATIONAL RESEARCH COUNCIL OF CANADA;1200 MONTREAL ROAD
BLDG M-58, ROOM EG12
OTTAWA, ONTARIO
K1A 0R6
CA
|
Family ID: |
23079244 |
Appl. No.: |
11/984821 |
Filed: |
November 21, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10471756 |
Mar 26, 2004 |
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PCT/CA02/00434 |
Mar 28, 2002 |
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11984821 |
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60281901 |
Mar 29, 2001 |
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Current U.S.
Class: |
800/287 ;
536/23.6 |
Current CPC
Class: |
C12N 15/827 20130101;
C07K 14/415 20130101 |
Class at
Publication: |
800/287 ;
536/23.6 |
International
Class: |
C12N 15/29 20060101
C12N015/29 |
Claims
1. A method of producing a transgenic plant comprising: (a)
introducing into a plant cell capable of being transformed and
regenerated into a whole plant a construct comprising, in addition
to DNA sequences required for transformation and selection in
plants, a nucleotide sequence having at least 90% sequence identity
to the nucleotide sequence as set forth in nucleotides 74 to 1231
of SEQ ID NO: 11 operably linked to a promoter; and (b) recovering
a plant which contains said nucleotide sequence and has shortened
pedicel length as a result of expression of said nucleotide
sequence compared to an unmodified plant.
2. The method of claim 1, wherein the nucleotide sequence has at
least 95% sequence identity to the nucleotide sequence as set forth
in nucleotides 74 to 1231 of SEQ ID NO: 11.
3. The method of claim 1, wherein the nucleotide sequence has at
least 99% sequence identity to the nucleotide sequence as set forth
in nucleotides 74 to 1231 of SEQ ID NO: 11.
4. The method of claim 1, wherein the nucleotide sequence comprises
nucleotides 74 to 1231 of SEQ ID NO: 11.
5. The method of claim 1, wherein the plant further has shortened
internode length compared to an unmodified plant.
6. The method of claim 1, wherein the plant further comprises
downwardly pointing pedicels and siliques compared to an unmodified
plant.
7. The method of claim 1, wherein the plant further comprises
downwardly pointing flowers compared to an unmodified plant.
8. The method of claim 1, wherein the plant is of genus
Arabidopsis.
9. The method of claim 1, wherein the plant is of genus
Brassica.
10. The method of claim 1, wherein the plant is a dicot, a monocot
or a member of Cruciferae.
11. The method of claim 1, wherein the promoter comprises a
transcriptional regulatory region normally in operable association
with an endogenous brevipedicellus gene or homologue thereof.
12. The method of claim 1, wherein the promoter comprises a
transcriptional regulatory region that is not normally in operable
association with an endogenous brevipedicellus gene or homologue
thereof.
13. The method of claim 1, wherein the promoter is a constitutive
promoter, an inducible promoter, an organ specific promoter, a
strong promoter, a weak promoter, or an endogenous promoter from
Arabidopsis as set forth in SEQ ID NO: 24.
14. The method of claim 1, wherein the nucleotide sequence
comprises nucleotides 74 to 1231 of SEQ ID NO: 11, the plant is of
genus Brassica, and the plant further has shortened internode
length and downwardly pointing flowers compared to an unmodified
plant.
15. The method of claim 14, wherein the promoter is a constitutive
promoter, an inducible promoter, an organ specific promoter, a
strong promoter, a weak promoter, or an endogenous promoter from
Arabidopsis as set forth in SEQ ID NO: 24.
16. An isolated nucleic acid molecule comprising a nucleotide
sequence as set forth in nucleotides 74 to 1231 of SEQ ID NO: 11
for generating a transgenic plant with decreased pedicel length
compared with an unmodified plant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 10/471,756 filed Mar. 26, 2004, which is the National
Stage of International Application No. PCT/CA02/00434 filed Mar.
28, 2002, which claims the benefit under 35 U.S.C. 119(e) of U.S.
provisional application Ser. No. 60/281,901 filed Mar. 29, 2001 now
abandoned, the entire contents of all of which are incorporated
herein by this reference.
FIELD OF THE INVENTION
[0002] The present invention relates to methods for altering plant
architecture, and in particular the morphology of the inflorescence
of a flowering plant, involving the use of the Arabidopsis
"BREVIPEDICELLUS" (BP) gene and homologues thereof.
BACKGROUND OF THE INVENTION
[0003] Plant architecture plays a very important role in overall
crop performance. The characteristics of the inflorescence, flower,
silique/fruit, and stem internodes have broad agronomic
implications in the overall productivity of any crop plant. Compact
architecture can contribute to productivity. For example, flowering
stalks or inflorescences that are compact in nature and do not
shade lower photosynthetic tissue can allow for greater
productivity. Similarly, a flowering stalk or inflorescence that is
spread out may allow for more photosynthesis to take place during
seed development within the flowering stalk. Thus, different
inflorescence architectures may be desired for different crops.
[0004] Since most crop varieties have been derived directly or
indirectly through breeding from wild species, productivity of
crops may be affected by characteristics that are evolutionarily
beneficial to wild species but impair performance in an
agricultural setting. For example, well spread-out flowers and
siliques with long pedicels on the inflorescence (along with genes
controlling seed dispersal mechanisms such as shattering) may be
evolutionarily beneficial to wild species, while in a crop setting
this confers significant disadvantages in terms of overall
productivity as measured by harvested seed.
[0005] An example of this is canola species, in which the shoot
architecture, especially involving inflorescence and siliques, is
not ideal for optimal productivity and recovery of seed. Though
there have been concerted efforts to produce crop plants with ideal
architecture, it has not been achieved in many crop species.
[0006] It widely known that the growth and developmental programs
of a plant species control pedicel development and determine its
length, attachment angle of the flowers and seed pods, and
contribute significantly towards the overall architecture of the
flower and/or inflorescence. Despite significant advances in the
understanding of flower development, very little is known about the
genetic and molecular control of pedicel development.
[0007] Plant architecture or morphology is a major determining
factor in plant productivity under agricultural settings. Plant
varieties that have well-defined morphology of a uniform nature and
pattern are preferred since they are amenable to mechanical
cultivation. In particular, plant species that produce seed are
selected for the uniformity of the placement of seed forming
structures (typically seed pods or cobs) to allow efficient
mechanical harvesting of seed. Plant varieties are also selected on
the basis of other seed forming characteristics, such as strong
pods to ensure no seed is lost or dispersed prior to harvesting, or
compact nature of the raceme of the plant that contains the
seedpods. Not all plants have these ideal characteristics. Thus,
there is a strong interest in modifying the placement of seed pods
and overall physical characteristics of many seed plants to produce
plants with desirable plant architecture and overall morphology.
Compact plants, with clustered seed pods can provide many benefits
for mechanical production of the crop, as well as lead to increased
productivity. Accordingly, control of plant form and plant
architecture is a desirable goal for the industry.
[0008] The building blocks of the plant architecture (body plan)
are composed of reiterative units referred to as phytomers and
these are elaborated during different phases of development
(Sussex, I. M. & Kerk, N. M. (2001) Curr. Opin. Plant Biol. 4,
33-37). In Arabidopsis thaliana, three types of phytomers have been
described (Schultz, E. A. & Haughn, G. W. (1991) Plant Cell 3,
771-781). The variations in the number of units and their size
among these three main types of phytomers in different plant
species contribute to the tremendous architectural diversity
observed in flowering plants (Steeves, T. A. & Sussex, I. M.
(1989)) Patterns in plant development (Cambridge University Press,
Cambridge). The activity of the shoot apical meristem (SAM),
together with additional meristems, regulates the growth and
development of all three types of phytomers (Medford, J. I.,
Behringer, F. J., Callos, J. D. & Feldmann, K. A. (1992) Plant
Cell 4, 631-643 & Simon, R. (2001) Semin. Cell Dev. Biol. 12,
357-362). The SAM contains three major domains defined by
cytoplasmic densities and cell division rates: the central zone
(CZ), which is responsible for maintaining the pluripotent stem
cells; the peripheral zone (PZ), which is involved in the
production of lateral organs; and the rib zone (RZ), from which the
bulk of the stem is derived (Bowman, J. L. & Eshed, Y. (2000)
Trends Plant Sci. 5, 110-115). Recent studies in Arabidopsis have
shown that several genes, including SHOOTMERISTEMLESS (STM),
WUSCHEL and CLAVATA-family receptor kinases and their putative
ligands define key functions in the SAM (Brand, U., Hobe, M. &
Simon, R. (2001) BioEssays 23, 134-141., Long, J. A., Moan, E. I,
Medford, J. I. & Barton, M. K. (1996) Nature 379, 66-69.,
Mayer, K. F., Schoof, H., Haecker, A., Lenhard, A., Jurgens, G.
& Laux, T. (1998) Cell 95, 805-815., & Clark, S. E. (2001)
Nat. Mol. Cell Biol. 2, 276-284.)
[0009] In Arabidopsis the inflorescence constitutes the major part
of the shoot and thus contributes significantly to the overall
shoot architecture. Several genes have been identified in
Arabidopsis that play key roles in defining the architecture of the
shoot/inflorescence. For example, dwarf plants with uniform effects
on all phytomers have been associated with altered levels of or
defects in the signaling pathways of certain plant hormones
(gibberellins or brassinosteriods--Hedden, N. P. & Kamiya, Y.
(1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 431-460.,
& Richards, D. E., King, K. E., Ait-ali, T. & Harberd, N.
P. (2001) Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 67-88.,
and references therein). The supershoot (Tantikanjana, T., Yong, J.
W., Letham, D. S., Griffith, M., Hussain, M., Ljung, K., Sandberg,
G. & Sundaresan, V. (2001) Genes Dev 15, 1577-1588.) and
altered meristem program (Chaudhury, A. M., Letham, S., Craig, S.
& Dennis, E. S. (1993) Plant J. 4, 907-916.) mutants display
abnormally high levels of cytokinins and produce extensive
branching and altered shoot and inflorescence architecture. Auxin
polar transport mutants, such as pinformed (Okada, K., Ueda, J.,
Komaki, M. K., Bell, C. J. & Shimura, Y. (1991) Plant Cell 3,
677-684.) and pinoid (Bennett, S. R. M., Alvarez, J., Bossinger, G.
& Smyth, D. R. (1995) Plant J. 8, 505-520.), form
inflorescences that are reduced to pin-like structures that do not
produce any lateral organs or meristems. A compact inflorescence is
caused by the erecta mutation, which involves a putative receptor
kinase (Torii, K. U., Mitsukawa, N., Oosumi, T., Matsuura, Y.,
Yokoyama, R., Whittier, R. F. & Komeda, Y. (1996) Plant Cell 8,
735-746).
[0010] An even stronger effect on inflorescence architecture is
conferred in a Landsberg erecta (Ler) background by the
brevipedicellus (BP) mutation, which is defined by a recessive
mutant with compact internodes and short, downward-pointing
pedicels (Koornneef, M., Eden, J. v., Hanhart, C. J., Stam, P.,
Braaksma, F. J. & Feenstra, W. J. (1983) J. Hered. 74,
265-272). Thus, mutants that exhibit altered architecture provide
an indication that architecture can be altered, but there is no
indication as to the molecular nature of the gene or the mechanisms
by which these changes are manifested.
[0011] The role of homeobox genes in defining body plan and their
evolutionary relationships in animals is well documented (Gehring,
W. J., Affoler, M. & Burglin, T. (1994) Annu. Rev. Biochem. 63,
487-526., Kappen, C. (2000) Proc. Natl. Acad. Sci. USA 97,
4481-4486.) More recently, several plant knotted-like homeobox
(KNOX) genes have been identified, which form two classes based
upon sequence similarities and expression domains (Bharathan, G.,
Janssen, B., Kellogg, E. & Sinha, N. (1999) Mol. Biol. Evol.
16, 553-563., Reiser, L., Sanchez, B. P. & Hake, S. (2000)
Plant Mol. Biol. 42, 151-166., Serikawa, K. A., Martinez-Laborda,
A. & Zambryski, P. (1996) Plant Mol. Biol. 32, 673-693).
[0012] In Arabidopsis, there are four different class I KNOX genes,
STM, KNAT1, KNAT2, and KNAT6 (Long, ibid., Lincoln, C., Long, J.,
Yamaguchi, J., Serikawa, K. & Hake, S. (1994) Plant Cell 6,
1859-1876. & Semiarti, E., Ueno, Y., Tsukaya, H., Iwakawa, H.,
Machida, C. & Machida, Y. (2001) Development 128, 1771-1783).
STM is expressed in the SAM, whereas KNAT1 and KNAT2 expression
observed in the PZ of the SAM. KNAT1 is also expressed in the
cortical cell layers of the peduncle and pedicel. STM, KNAT1 and
KNAT2 expression is excluded from the leaf primordia and developing
leaves by ASYMMETRICLEAVES 1 and 2 genes (Ori, N., Eshed, Y.,
Chuck, G., Bowman, J. L. & Hake, S. (2000) Development 127,
5523-5532., & Byrne, M., Barley, R., Curtis, M., Arroyo, J.,
Dunham, M., Hudson, A. & Martienssen, R. (2000) Nature 408,
967-971). Ectopic expression of KNAT1 and KNAT2 in leaves induces
altered symmetry and cell fate, and ectopic meristem/shoot
formation from the adaxial surface (Chuck, G., Lincoln, C. &
Hake, S. (1996) Plant Cell 8, 1277-1289). To date, loss-of-function
mutations in class I KNOX genes are known only for S.TM. and these
suggest a critical role in SAM maintenance and function.
Significantly, however, no such mutations have previously been
described for KNAT1, hampering study of the role of this homeobox
gene in plant development.
[0013] The future prospects of engineering optimal plant
architectures in plant species will depend on the availability of
critical morphology controlling genes and knowledge of their
functional regulatory properties. For example in canola, the
occurrence of an inflorescence and silique with long pedicels may
offer some unique challenges and opportunities to develop an ideal
architecture for improving productivity.
[0014] In summary, there remains a continuing need to develop novel
and efficient techniques for modifying the morphology and
architecture of plants, such as for example Brassica and other
plant types, to improve photosynthetic efficiency, overall yield,
and harvestability. This need extends to both crops and to
horticulturally grown species to improve aesthetic appeal.
SUMMARY OF THE INVENTION
[0015] The inventors of the present application have successfully
identified the gene responsible for the brevipedicellus (bp) mutant
in Arabidopsis. This mutation is known to give rise to plants
having a very compact architecture with shortened siliques pointing
downwards. Importantly, the inventors have realized that the
successful identification of this gene has important implications
on the generation of new crops and other plant species that exhibit
advantageously modified morphological features.
[0016] In this regard, the inventors have discovered that the bp
mutation has several productivity advantages if introduced for
example, into canola crop species. In Arabidopsis, the mutation
results in reduced pedicel length, and siliques pointing downward
with compact architecture. These features can improve exposure of
upper leaves to sunlight and thereby enhance their photosynthetic
efficiency: a well recognized problem in canola, especially during
the pod setting and maturation stages. In addition, during
harvesting the altered pod dynamics can reduce shattering losses,
an important problem facing canola farmers. Further, the
downward-pointing flowers may help in reducing disease
incidence.
[0017] Therefore, the present invention relates, in one embodiment,
to nucleic acid sequences derived from Arabidopsis encoding a
homeobox gene involved in the control of inflorescence
architecture, for use in modifying plant inflorescence
architecture. In addition, the present invention relates in other
embodiments to methods for modifying the morphological phenotype of
plants, by introducing the nucleotide sequences encompassed by the
present invention into a plant, and expressing the nucleotide
sequences as appropriate.
[0018] In another embodiment, the present invention relates to
nucleic acid sequences derived from Arabidopsis encoding a homeobox
gene involved in the control of inflorescence architecture, said
homeobox gene differing from wild type by at least a change in an
amino acid codon to produce a truncated protein.
[0019] The invention further relates to the proteins encoded by the
nucleic acids encompassed by the invention, and their use.
[0020] The present invention also relates to methods for alteration
of the expression of a native gene related to inflorescence
structure, in particular the reduction in the expression of said
gene.
[0021] In one aspect of the invention, nucleic acid sequences are
provided that encode an altered protein that when expressed confers
an altered inflorescence architecture phenotype in Arabidopsis,
particularly an inflorescence with an altered pedicel, peduncle or
style.
[0022] In one aspect of the invention, nucleic acid sequences are
provided that encode an altered protein that when expressed confers
an altered inflorescence architecture phenotype in Brassica,
particularly an inflorescence with an altered pedicel, peduncle or
style.
[0023] In another aspect of the present invention methods are
described that enable the heterologous expression of the nucleic
acid or portions or homologues thereof, described in SEQ ID NO: 5
in a host cell to obtain a plant with an altered inflorescence,
more particularly an inflorescence with an altered pedicel,
peduncle or style.
[0024] In yet another aspect of the present invention, methods are
described wherein the nucleic acid sequence or regions thereof as
described in SEQ ID NO: 6 and nucleic acids homologous to same are
used to alter the architecture of a flowering plant, in particular
the inflorescence, more particularly the pedicel, peduncle or
style.
[0025] In yet another aspect of the present invention, methods are
described wherein nucleic acid sequence or regions thereof as
described in SEQ ID. NO: 6 and nucleic acids homologous to same are
used to alter the architecture of the inflorescence of a plant from
the Crucifer (Cruciferae) family, particularly the pedicel,
peduncle or style of said plant.
[0026] In yet another aspect of the present invention, methods are
described wherein nucleic acid sequence or regions thereof as
described in SEQ ID. NO: 6 and nucleic acids homologous to same are
used to alter the architecture of the inflorescence of a plant from
the Crucifer (Cruciferae) family, particularly the pedicel of said
plant, said plant exhibiting an altered inflorescence, with compact
internodes, downward pointing pedicels and siliques that point
downward relative to the normal presentation of siliques.
[0027] In one embodiment, the present invention provides a method
of producing a transgenic plant with a modified inflorescence
architecture characterised in that the method comprises the steps
of: (a) introducing into a plant cell capable of being transformed
and regenerated into a whole plant a construct comprising, in
addition to the DNA sequences required for transformation and
selection in plants, a nucleotide sequence derived from a BP
(KNAT1) gene and encoding at least part of a BP (KNAT1) gene
product operably linked to a promoter; and (b) recovery of a plant
which contains said nucleotide sequence and has a modified
inflorescence architecture compared to an unmodified plant.
Preferably, the method involves nucleotide sequences encoding a
peptide having at least 50%, preferably 70%, more preferably 90%,
more preferably 95%, most preferably 99% homology to the peptide
encoded by SEQ ID NO: 5 or 6, or a part thereof, or a complement
thereof. Preferably, the method involves nucleotide sequences that
are able to bind under stringent conditions to SEQ ID NO: 5 or 6,
or a part thereof, or a complement thereof.
[0028] Preferably, the modification of inflorescence architecture
comprises an altered pedicel, peduncle or style, and more
preferably the altered pedicel has an altered length compared to an
unmodified plant. Moreover, the modified inflorescence architecture
preferably comprises downwardly pointing flowers.
[0029] In alternative embodiments, the invention provides methods
characterised in that the nucleotide sequences are derived from a
plant of the genus Arabidopsis or Brassica and/or the transformed
plants are of the genus Arabidopsis or Brassica or are selected
from the group consisting of: a dicot, a monocot, and a member of
Cruciferae.
[0030] Preferably, the methods of the invention can generate a
plant having either a compact or an open inflorescence compared to
an unmodified plant. The nucleotide sequences may be expressed in a
sense direction for complementary inhibition of an endogenous BP
(KNAT1) gene in the transgenic plant, such that the plant has a
compact inflorescence architecture compared to an unmodified
plant.
[0031] Preferably, the BP (KNAT1) gene may be in a mutated form. In
an alternative embodiment, the nucleotide sequence may be expressed
in an antisense direction for antisense inhibition of an endogenous
BP (KNAT1) gene such that the plant has a compact inflorescence
architecture and/or decreased pedicel length compared to an
unmodified plant. In an further alternative embodiment, the
nucleotide sequence may be overexpressed in a sense direction, such
that the plant has an open inflorescence architecture and/or
increased pedicel length compared to an unmodified plant.
[0032] In one aspect, the plant may harbour a bp mutation such that
expression of said nucleotide sequence is complementary to said
mutation, inducing the plant to exhibit a wild-type phenotype.
[0033] The promoters for use in accordance with the methods of the
present invention may take various forms. For example, the promoter
may comprise, in one embodiment a transcriptional regulatory region
normally in operable association with an endogenous BP (KNAT1) gene
or homologue thereof. Alternatively, the promoter may comprise a
transcriptional regulatory region that is not normally in operable
association with an endogenous BP (KNAT1) gene or homologue
thereof.
[0034] Further, the promoter may be selected from the group
consisting of: a constitutive promoter, an inducible promoter, an
organ specific promoter, a strong promoter, a weak promoter, and an
endogenous BP (KNAT1) promoter from Arabidopsis. Alternatively, the
promoter may be derived from a functional portion of SEQ ID NO: 23
or SEQ ID NO: 24.
[0035] The present invention further encompasses methods for
modifying the inflorescence architecture of a plant involving the
use of sequences homologous to SEQ ID NO: 5 or 6, such as, for
example, SEQ ID NOS: 11,14,15, and 20.
[0036] In another embodiment, the present invention provides a
method of identifying a plant that has been successfully
transformed with a construct, characterised in that the method
comprises the steps of: (a) introducing into plant cells capable of
being transformed and regenerated into whole plants a construct
comprising, in addition to the DNA sequences required for
transformation and selection in plants, a nucleotide sequence
derived from a BP (KNAT1) gene and encoding at least part of a BP
(KNAT1) gene product, operably linked to a promoter; (b)
regenerating said plant cells into whole plants; and (c) inspecting
the inflorescences of said plants to determine those plants
successfully transformed with said construct, and expressing said
nucleotide sequence. In a preferred embodiment, the plant cells and
the regenerated whole plants harbour a bp mutation, and successful
transformation and expression of said nucleotide sequence
complements said mutation, thereby generating a plant exhibiting a
wild-type phenotype. More preferably, the construct is bicistronic
and further comprises a second DNA expression cassette for
generating a transcript unrelated to said nucleotide sequence
derived from a BP (KNAT1) gene. In this way, the BP (KNAT1)-related
portion of the construct can complement a known mutation in a plant
and positively confirm transformation, and simultaneously a second
transcript can be produced from a second region of the bicistronic
construct, conferring desirable or otherwise properties to the
transgenic plant.
[0037] The present invention further encompasses transgenic plants
generated by any of the methods of the present invention. In this
regard, the transgenic plants are preferably of the genus
Arabidopsis or Brassica or plants selected from the group
consisting of: a dicot, a monocot, and a member of Cruciferae.
Moreover, the exogenous DNA or construct introduced into the plant
may preferably be derived from plants of the genus Arabidopsis or
Brassica.
[0038] The transgenic plants of the present invention preferably
comprise a modified inflorescence (e.g. compact or open) compared
to an unmodified plant. Preferably the modified inflorescence
architecture comprises an altered pedicel, peduncle or style, more
preferably a plant with altered pedicel length or downwardly
pointing flowers compared to an unmodified plant.
[0039] The present invention further encompasses, in other
embodiments, isolated nucleotide sequences for generating a
transgenic plant with modified inflorescence architecture,
characterised in that the isolated nucleotide sequences are derived
from a BP (KNAT1) gene and encode at least part of a BP (KNAT1)
gene product. The isolated nucleotide sequences preferably comprise
a sequence selected from: (a) SEQ ID NO: 5 or 6, or a part thereof,
or a complement thereof; and (b) a nucleotide sequence encoding a
peptide having at least 50%, preferably 70%, more preferably 90%,
more preferably 95%, and most preferably 99% homology to the
peptide encoded by the nucleotide sequence defined in (a).
[0040] Preferably the isolated nucleotide sequences of the present
invention are characterised in that the nucleotide sequences
hybridise under stringent conditions to the nucleotide sequence of
SEQ ID NO: 5 or 6, or a part thereof or a complement thereof. The
isolated nucleotide sequences for generating a transgenic plant
with a modified inflorescence architecture compared to an
unmodified plant, include sequences derived from a construct
selected from the group consisting of: pRD400-951/955,
pRD400-951/956, pRD400-35S::AtBPS, pRD400-35S::AtBPA/S,
pRD400-35S::Atbp-2, pRD400-951/952::Atbp-2, pRD400-951/952::BnBPS,
pRD400-35S::BnBPS, and pRD400-35S::BnBPA/S.
[0041] The present invention further encompasses, in further
embodiments, the use of isolated nucleotide sequences related to
the BP (KNAT1) gene, for generating a transgenic plant with a
modified inflorescence architecture.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0042] FIG. 1. Illustration of the bp BP phenotype in Arabidopsis.
In this figure the phenotypes of 6 week-old Ler wt, bp-1 Ler and
bp-2 Ler plants. (A) Whole plant. Close-up of floral nodes with
siliques of Ler wt (B) and bp-1 Ler (C). Close-up of inflorescence
apex in Ler wt (D) and bp-1 Ler (E).
[0043] FIG. 2. A comparison of internode and pedicel lengths
between Ler wt, bp-1 Ler and bp-2 Ler. The histograms represent the
percentage reduction in pedicel length for bp-1 and bp-2; the
actual measurements in mm (mean values standard deviation of 30
data points) are shown above the corresponding bars. The average
pedicel lengths represent the values for the floral nodes 1-5.
IN-1, IN-2, IN-3: coflorescence nodes 1, 2,3; FN1-5: floral nodes
1-5; FN6-10: floral nodes 6-10.
[0044] FIG. 3. SEM micrographs of inflorescences from Ler (A, B)
and bp-1 Ler (C-E). (A) Ler wt floral nodes. (B) Ler wt peduncle
internode magnified to show differentiated epidermal cells. (C)
bp-1 floral nodes. (D, E) bp-1 peduncle internode showing stripes
of less differentiated epidermal cells (arrows) that originate
below the node. Anatomy of the peduncle of Ler wt (F, H) and bp-1
(G, I). Cross sections through the internodal region of the
peduncle of Ler wt (F) and bp-1 (G). Longitudinal sections through
the nodal region of Ler wt (H) and bp-1 (I). Arrows in G demarcate
a band of less differentiated cells that originate below the node.
co, cortical cell layer; ad, adaxial; ab, abaxial. Bar=0.1 mm (A,
B, D, E-I); 1 mm (C).
[0045] FIG. 4. Pedicel development in Ler wt (A-E, K, L) and bp-1
Ler (F-J, M, N). SEM of pedicel of stage 12 flower of Ler wt (A)
showing complete epidermal cell differentiation on both the adaxial
(B) and abaxial (C) sides. Pedicel of stage 12 flower of bp-1 (F)
with narrow distal end (.rarw.), differentiated adaxial (G) and
less differentiated abaxial (H) sides. SEM of stage 13 flower of
Ler wt (D) and its pedicel (E). Stage 13 flower of bp-1 (I) and its
pedicel (J) showing less differentiated abaxial side. Cross section
through the mid-region of the pedicel of Ler wt (K) and bp-1 (L)
and the distal end of the pedicel of Ler wt (M) and bp-1 (N). Bar=1
mm (A, D, F, I); 0.1 mm (B, C, E, G, H, J-N). ad, adaxial; ab,
abaxial.
[0046] FIG. 5. SEM of the style of a stage 17 flower of Ler wt (A),
and bp-1 Ler (D). Longitudinal sections through the style of Ler
(B) and bp-1 (E). Cross sections through the style of Ler wt (C)
and bp-1 (F). Arrows in D-F indicate the lateral axis. Bar=0.1 mm.
sp, stigmatic papillae; st, style.
[0047] FIG. 6. Southern blot and RT-PCR of KNAT1. (A) Southern
blot. Genomic DNA from Col wt (lanes 1 and 2), Ler wt (lanes 3 and
4), RLD wt (lanes 5 and 6), bp-1 Ler (lanes 7 and 8), and bp-2 RLD
(lanes 9 and 10) was digested with BamHI (lanes 1,3,5,7,9) or EcoRI
(lanes 2,4,6,8,10) and probed with the KNAT1 cDNA. Sizes of the MW
standards (kb) are indicated. (B) RT-PCR using KNAT1 primers 954
and 955. Lane 1, Col wt; lane 2, RLD wt; lane 3, Ler wt; lane 4,
bp-1 Ler; lane 5, bp-2 Ler; lane 6, bp-2 RLD; lane 7, bp-2 Col. The
same cDNA pools were amplified with primers specific for gapC.
[0048] FIG. 7. Sequences of the polymorphic regions of the
BP-encoding cDNAs from Col wt, RLD wt, Ler wt, and bp-2. Numbering
is shown for the Col wt sequence (GenBank U14174). Stop codons are
indicated by an asterisk. (*), nucleotide and 25 amino acid
deletions relative to Col wt are indicated by a dash (-), and
nucleotide and amino acid insertions relative to Col wt are
indicated in parentheses ( ). The C-T transition that causes a stop
codon at position 535 in bp-2 is shown in bold. Nucleotides
downstream of position 540 were identical among all of the
BP-encoding genes analyzed and are not shown.
[0049] FIG. 8. Vector map of the plant transformation vector
referred to as pRD400-951/955, comprising of the KNAT1 cDNA cloned
downstream of the putative BP (KNAT1) promoter.
[0050] FIG. 9. Vector map of the plant transformation vector
referred to as pRD400-951/956, consisting of the putative BP
(KNAT1) promoter and the BP (KNAT1)-encoding ORF amplified from
genomic DNA.
[0051] FIG. 10. Vector map of the plant transformation vector
referred to as pRD400-35S::AtBPS, consisting of the A. thaliana BP
ORF under the control of the 35S promoter.
[0052] FIG. 11. Vector map of the plant transformation vector
referred to as pRD400-35S::AtBPA/S, consisting of the A. thaliana
BP ORF in an antisense orientation under the control of the 35S
promoter.
[0053] FIG. 12. Vector map of the plant transformation vector
referred to as pRD400-35S::Atbp-2, consisting of the altered BP
gene coding sequence (SEQ ID. NO: 6) under the control of the 35S
promoter. The asterisk denotes the approximate location of the stop
codon that results in a truncated predicted protein.
[0054] FIG. 13. The vector pRD400-951/952::Atbp-2, consisting of
the A. thaliana bp-2 cDNA under the control of the A. thaliana BP
(KNAT1) promoter. The asterisk denotes the approximate location of
the stop codon that results in a truncated predicted protein.
[0055] FIG. 14. The map of the vector of pRD400-951/952::BnBPS,
consisting of the B. trapus BP ORF (SEQ ID. NO: 11) under the
control of the A. thaliana BP (KNAT1) promoter.
[0056] FIG. 15. The vector map of pRD400-35S::BnBPS, consisting of
the B. napus BP ORF (SEQ ID NO: 11) under the control of an
optimized cauliflower mosaic virus (CaMV) 35S promoter.
[0057] FIG. 16. The vector map pRD400-35S::BnBPA/S, consisting of
the B. napus BP ORF (SEQ ID NO: 1) in an antisense orientation
under the control of the 35S promoter.
DETAILED DESCRIPTION OF THE INVENTION
[0058] Definitions The singular forms "a", "an", and "the" include
plural reference unless the context clearly dictates otherwise.
[0059] A "coding sequence" or "coding region" is the part of a gene
that codes for the amino acid sequence of a protein, or for a
functional RNA such as a tRNA or rRNA. A coding sequence typically
represents the final amino acid sequence of a protein or the final
sequence of a structural nucleic acid. Coding sequences may be
interrupted in the gene by intervening sequences, typically
intervening sequences are not found in the mature coding
sequence.
[0060] A "polynucleotide encoding an amino acid sequence" refers to
a nucleic acid sequence that encodes the genetic code of at least a
portion of a mature protein sequence, typically a contiguous string
of amino acids typically linked through a peptide bond. An "amino
acid sequence" is typically two or more amino acid residues, more
typically 10 or more amino acids in a specific defined order.
[0061] A "complement" or "complementary sequence" is a sequence of
nucleotides which forms a hydrogen-bonded duplex with another
sequence of nucleotides according to Watson-Crick base-pairing
rules. For example, the complementary base sequence for 5'-AGCT-3'
is 3'-TCGA-5'.
[0062] "Expression" refers to the transcription of a gene into
structural RNA (rRNA, tRNA) or messenger RNA (mRNA) with subsequent
translation into a protein in the case of the mRNA.
[0063] Polynucleotides are "functionally equivalent" if they
perform substantially the same biological function. By
substantially the same biological function it is meant that similar
protein activities or protein function are encoded by a mRNA
polynucleotide, or a structural polynucleotide has a similar
structure and biological activity.
[0064] Polynucleotides are "heterologous" to one another if they do
not naturally occur together in the same arrangement in the same
organism. A polynucleotide is heterologous to an organism if it
does not naturally occur in its particular form and arrangement in
that organism.
[0065] Polynucleotides or polypeptides have "homologous" or
"identical" sequences if the sequence of nucleotides or amino acid
residues, respectively, in the two sequences is the same when
aligned for maximum correspondence as described herein. Sequence
comparisons between two or more polynucleotides or polypeptides are
generally performed by comparing portions of the two sequences over
a portion of the sequence to identify and compare local regions.
The comparison portion is generally from about 20 to about 200
contiguous nucleotides or contiguous amino acid residues or more.
The "percentage of sequence identity" or "percentage of sequence
homology" for polynucleotides and polypeptides, such as 50, 60, 70,
80, 90, 95, 98, 99 or 100 percent sequence identity may be
determined by comparing two optimally aligned sequences which may
or may not include gaps for optimal alignment over a comparison
region, wherein the portion of the polynucleotide or polypeptide
sequence in the comparison may include additions or deletions
(i.e., gaps) as compared to the reference sequence (which does not
comprise additions or deletions) for optimal alignment of the two
sequences.
[0066] The percentage of homology or similarity is calculated by:
(a) determining the number of positions at which the identical
nucleic acid base or amino acid residue occurs in both sequences to
yield the number of matched positions; (b) dividing the number of
matched positions by the total number of positions in the window of
comparison; and, (c) multiplying the result by 100 to yield the
percentage of sequence identity.
[0067] Optimal alignment of sequences for comparison may be
conducted by computerized implementations of known algorithms, or
by inspection. Readily available sequence comparison and multiple
sequence alignment algorithms are, respectively, the Basic Local
Alignment Search Tool (BLAST) (Altschul, S. F. et al. 1990. J. Mol.
Biol. 215:403; Altschul, S. F. et al. 1997. Nucleic Acids Res. 25:
3389-3402) and ClustalW programs. BLAST is available on the
Internet at http://www.ncbi.nlm.nih.gov and a version of ClustalW
is available at www2.ebi.ac.uk. Other suitable programs include
GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package (Genetics Computer Group (GCG), 575 Science Dr., Madison,
Wis.). For greater certainty, as used herein and in the claims,
"percentage of sequence identity" or "percentage of sequence
homology" of amino acid sequences is determined based on optimal
sequence alignments determined in accordance with the default
values of the BLASTX program, available as described above.
[0068] Sequence, identity typically refers to sequences that have
identical residues in order, whereas sequence similarity refers to
sequences that have similar or functionally related residues in
order. For example an identical polynucleotide sequence would have
the same nucleotide bases in a specific nucleotide sequence as
found in a different polynucleotide sequence. Sequence similarity
would include sequences that are similar in character for example
purines and pyrimidines arranged in a specific fashion. In the case
of amino acid sequences, sequence identity means the same amino
acid residues in a specific order, where as sequence similarity
would allow for amino acids with similar chemical characteristics
(for instance basic amino acids, or hydrophobic amino acids) to
reside within a specific order.
[0069] The terms "stringent conditions" or "stringent hybridization
conditions" includes reference to conditions under which a probe
will hybridize to its target sequence, to a detectably greater
degree than other sequences (e.g. at least 2-fold over background).
Stringent conditions are sequence-dependent and will be different
in different circumstances. Longer sequences hybridize specifically
at higher temperatures. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (Tm) for the specific sequence at a defined ionic strength
and pH. The Tm is the temperature (under defined ionic strength and
pH at which 50% of a complementary target sequence hybridizes to a
perfectly matched probe. Typically, stringent conditions will be
those in which the salt concentration is less than about 1.0 M Na
ion, typically about 0.01 to 1.0 M Na ion concentration (or other
salts) at pH 7.0 to 8.3 and the temperature is at least about
30.degree. C. for short probes (e.g. 10 to 50 nucleotides) and at
least about 60.degree. C. for long probes (e.g. greater than 50
nucleotides). Stringent conditions may also be achieved with the
addition of destabilizing agents such as formamide. Exemplary low
stringency conditions include hybridization with a buffer solution
of 30% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
2.times.SSC at 50.degree. C. Exemplary high stringency conditions
include hybridization in 50% formamide, 1 M NaCl, 1% SDS at
37.degree. C., and a wash in 0.1.times.SSC at 60.degree. C.
Hybridization procedures are well-known in the art and are
described in Ausubel et al., (Ausubel F. M., et al., 1994, Current
Protocols in Molecular Biology, John Wiley & Sons Inc.).
[0070] "Isolated" refers to material that is: (1) substantially or
essentially free from components which normally accompany or
interact with it as found in its naturally occurring environment;
or (2) if in its natural environment, the material has been
non-naturally altered to a composition and/or placed at a locus in
the cell not native to a material found in that environment. The
isolated material optionally comprises material not found with the
material in its natural environment. For example, a naturally
occurring nucleic acid becomes an isolated nucleic acid if it is
altered, or if it is transcribed from DNA which is altered, by
non-natural, synthetic methods performed within the cell from which
it originates.
[0071] Two DNA sequences are "operably linked" if the linkage
allows the two sequences to carry out their normal functions
relative to each other. For instance, a promoter region would be
operably linked to a coding sequence if the promoter were capable
of effecting transcription of that coding sequence and said coding
sequence encoded a product intended to be expressed in response to
the activity of the promoter.
[0072] A "polynucleotide" is a sequence of two or more
deoxyribonucleotides (in DNA) or ribonucleotides (in RNA).
[0073] A "DNA construct" is a nucleic acid molecule that is
isolated from a naturally occurring gene or which has been modified
to contain segments of nucleic acid which are combined and
juxtaposed in a manner which would not normally otherwise exist in
nature.
[0074] A "polypeptide" is a sequence of two or more amino
acids.
[0075] A "homeobox" gene is a gene that is typically involved the
developmental process of an organism, and usually contains one or
more specific regions within the encoded protein that include a DNA
binding region and a second region that is distinct from the
binding region. Homeobox genes typically contain a homoedomain that
is homologous or has similarity to other homeodomain found in other
homeobox genes.
[0076] A "promoter" or transcriptional regulatory region is a
cis-acting DNA sequence, generally located upstream of the
initiation site of a gene, to which RNA polymerase may bind and
initiate correct transcription.
[0077] A "recombinant" polynucleotide, for instance a recombinant
DNA molecule, is a novel nucleic acid sequence formed through the
ligation of two or more nonhomologous DNA molecules (for example a
recombinant plasmid containing one or more inserts of foreign DNA
cloned into it).
[0078] "Transformation" means the directed modification of the
genome of a cell by the external application of recombinant DNA
from another cell of different genotype, leading to its uptake and
integration into the subject cell's genome.
[0079] A "transgenic plant" encompasses all descendants, hybrids,
and crosses thereof, whether reproduced sexually or asexually, and
which continue to harbour the foreign DNA.
[0080] An inflorescence is a portion of a flowering plant that
produces and supports flower development and typically seed
formation. An inflorescence is usually formed from a meristem
structure. The terms "inflorescence" and "flowering stalk" are used
interchangeably herein.
[0081] Unless defined otherwise all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs.
[0082] The present application describes nucleic acids encoding a
gene from Arabidopsis referred to as the BP gene, or the gene
encoding the brevipedicellus phenotype, and the role of said gene
in determining inflorescence morphology. Said gene is a member of
the KNOX gene family in Arabidopsis that is involved in control of
certain aspects of flower development. The gene identified in the
present invention represents an altered form of the wild-type gene.
As a result of the present discovery, it was found that the
wild-type gene is normally involved in the control of the
architecture of the inflorescence or at least the pedicel, peduncle
and style structures within the inflorescence.
[0083] The gene sequences responsible for the bp phenotype have not
been previously identified and hence the molecular nature of the bp
mutation was not known prior to this disclosure. Accordingly, the
utility of the bp mutation for practical purposes has not been
described. The present invention identifies the molecular basis of
the bp mutation and methods for using the gene encoded by the BP
locus in alteration of plant inflorescence structure.
[0084] The present application further describes the discovery that
the alteration in the expression levels of said gene, in particular
the reduction in expression of said gene, or expression of an
altered protein form of said gene results in changes in the
inflorescence structure. Loss of function of said gene results in a
compact inflorescence structure with changes in the length of the
internodes, pedicle length and angle of seed pod attachment. The
invention also provides evidence that gain of function can restore
a wild-type phenotype, hence providing direction on alteration of
inflorescence structure towards a compact structure or a structure
that exhibits a less compact, more spread out structure.
[0085] The present application further describes the molecular
basis of two loss of function alterations, providing a basis for
the engineering of altered inflorescence architecture. In the
present invention, the methods for the alteration of inflorescence
architecture were shown to be: loss of the expression of the gene
itself (inhibition of gene expression); and loss of function by
expression of an altered form of the protein (expression of altered
protein).
[0086] Accordingly, it is anticipated that the engineering of
similar loss of function phenotypes in numerous flowering plant
species can be easily and routinely accomplished by the use of
methods described herein to identify, modify and alter the
expression of the normally encoded gene or genes related to said
nucleic acid sequences described herein. Thus, the present
invention encompasses plants with altered inflorescence structures,
in particular plants with an altered pedicel, peduncle or style can
be obtained, alone or in combination, to produce an altered
inflorescence structure.
[0087] Portions of the gene sequence representing the native
wild-type protein coding sequence described in the present
invention were found to be identical to the previously identified
homeobox gene called KNAT1, but the involvement of the KNAT1 gene
in the control of the inflorescence architecture or its association
with the bp phenotype have not previously been described nor
anticipated. Indeed, the previous studies (Lincoln et al, Plant
Cell, 6: 1859-1876, 1994) on the KNAT1 gene expression failed to
identify a primary role for the gene in inflorescence architecture,
suggesting that the expression of the KNAT1 gene was restricted in
its expression in the inflorescence. No indication of the role of
KNAT1 in peduncle, pedicle or style formation was suggested.
Efforts to determine the function of the KNAT1 gene in this study
were restricted to ectopic constitutive expression of the KNAT1
native coding sequence. No loss of function information for the
KNAT1 was provided hence no definition of the nature of the
activity of KNAT1 could be inferred from these studies. Accordingly
the art did not describe a function for the KNAT1 gene, nor for
that matter link the expression of the KNAT1 gene with the bp
mutant.
[0088] The present invention has thus assigned function to the
KNAT1 gene, identified altered forms of the KNAT1 gene as the basis
of the BP phenotype and provides methods for the alteration of
wild-type gene expression to produce altered inflorescence, in
particular inflorescence structures with alterations in the
peduncle, pedicel or style or combinations thereof.
[0089] The present invention encompasses the use of the BP (KNAT1)
gene, and parts thereof, complements thereof, and homologues
thereof, for generating transgenic plants with altered
inflorescence structures. The present invention also encompasses
the use of nucleic acid sequences encoding peptides having at least
50% homology, preferably 70% homology, preferably 90%, more
preferably 95%, most preferably 99% to the peptides encoded by the
BP (KNAT1) gene or SEQ ID NOS: 5 and 6. In this regard, homologous
proteins with at least 50% or 70% predicted amino acid sequence
homology are expected to encompass proteins with activity as those
defined by the present invention, wherein disruption of expression
or overexpression of the homologous proteins is expected to
generate plants with altered structure as described in the present
application. Such proteins may be derived from similar or unrelated
species of plant.
[0090] The present invention also encompasses polynucleotide
sequences encoding peptides comprising at least 90%, 95% or 99%
sequence homology to the peptides encoded by the BP (KNAT1) gene or
SEQ ID NOS: 5 and 6. This class of related proteins is intended to
include close gene family members with very similar or identical
catalytic activity. In addition, peptides with 90% to 99% amino
acid sequence homology may be derived from functional homologues of
similar species of plant, or from directed mutations to the
sequences disclosed in the present application.
[0091] The present invention demonstrates the utility of said
nucleic acid sequences and altered forms of the protein encoded by
said nucleic acid sequences in controlling inflorescence
development and hence assigns a novel utility for the use of the BP
(KNAT1) gene, and homologues thereof, to alter floral structure in
flowering plants.
[0092] The nucleic acid sequences provided in the present invention
can be used to alter plant morphology by heterologous expression,
for example, of the nucleic acid sequences shown in SEQ ID. NOS: 5
and 6 and other homologous sequences as described herein.
[0093] The nucleic acid sequence of SEQ ID. NO: 5 encodes a KNAT1
protein that has been shown in the present invention to be involved
in maintaining the normal development of an inflorescence of a
flowering plant, wherein expression of the protein confers the
normal architecture of the inflorescence of a flowering plant. The
protein represents a member of the homeodomain proteins involved in
the control of plant development.
[0094] The nucleic acid sequence of SEQ ID. NO: 6 encodes an
altered form of the BP (KNAT1) protein, herein referred to as the
BP related protein that is preferentially expressed in the
inflorescence of a flowering plant, wherein expression of the
protein influences the architecture of the inflorescence of a
flowering plant. This protein represents an altered member of the
homeodomain proteins involved in the control of plant
development.
[0095] The present invention encompasses the expression of
nucleotide sequences derived from the BP (KNAT1) gene, including
SEQ ID NOS: 5 and 6 or homologues thereof to alter the
inflorescence of a flowering plant by using said polynucleotides to
alter the expression of the protein normally expressed by BP
(KNAT1) and related genes using methods familiar to those of skill
in the art.
[0096] In one aspect of the present invention, a gene sequence is
used to modify the architecture of a inflorescence in a flowering
plant by heterologous expression of the coding sequence of SEQ ID.
NO: 6 or parts thereof, or complements thereof, or homologues
thereof.
[0097] In another aspect of the present invention, one or more
portions, of at least 50 amino acids, but less than 400 amino
acids, most preferably about 179 amino acids of the protein encoded
by the nucleic acid sequence of SEQ ID. NO: 6 are expressed in a
host plant, said expression causing the alteration of inflorescence
architecture as illustrated herein.
[0098] In another aspect of the present invention, the nucleic acid
sequence, or coding region thereof described in the BP (KNAT1) gene
or in SEQ ID NO: 5 or 6 can used to modify the inflorescence of a
flowering plant by using said sequence to isolate a homologous
nucleic acid that encodes a protein that is at least 50% homologous
to the protein encoded by SEQ ID. NO: 6 and expressing said
homologous nucleic acid as part of a recombinant DNA construct in a
host plant species. The recombinant DNA construct so expressed is
engineered to express an altered form of the wild-type protein, or
engineered to reduce the expression of the wild-type gene. Method
for the identification and isolation of homologous DNA sequences
are very well known in the art and are included, for example in
Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989).
[0099] It will also be understood to a person of skill in the art
that site-directed mutagenesis techniques are readily applicable to
the polynucleotide sequences of the present invention, to make the
sequences better suited for use in generated morphologically
modified transgenic plants. Related techniques are well understood
in the art, for example as provided in Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring
Harbor, N.Y. (1989). In this regard, the present invention teaches
the use of nucleotide sequences derived from the BP (KNAT1) gene,
including, for example SEQ ID NOS: 5 and 6. However, the present
invention is not intended to be limited to these specific
sequences. Numerous directed mutagenesis techniques would permit
the non-informed technician to alter one or more residues in the
nucleotide sequences, thus changing the subsequently expressed
polypeptide sequences. Moreover, commercial `kits` are available
from numerous companies that permit directed mutagenesis to be
carried out (available for example from Promega and Biorad). These
include the use of plasmids with altered antibiotic resistance,
uracil incorporation and PCR techniques to generate the desired
mutation. The mutations generated may include point mutations,
deletions and truncations as required. The present invention is
therefore intended to encompass corresponding mutants of the BP
(KNAT1) gene, both cDNA and genomic DNA sequences in accordance
with the teachings of the present application.
[0100] The polynucleotide sequences of the present invention must
be ligated into suitable vectors before transfer of the genetic
material into plants. For this purpose, standard ligation
techniques that are well known in the art may be used. Such
techniques are readily obtainable from any standard textbook
relating to protocols in molecular biology, and suitable ligase
enzymes are commercially available.
[0101] In another aspect of the present invention, the BP (KNAT1)
gene sequence, and parts, complements, and homologues therefore
used to modify a plant inflorescence by the transformation of plant
cells with a plant transformation vector comprising a coding, for
example, a region of said nucleic acid described in SEQ ID. NO: 6
under the control of a heterologous or native/homologous
promoter.
[0102] In another aspect of the present invention, the nucleic acid
sequence described in SEQ ID. NO: 6 is used to modify plant
inflorescence architecture by the transformation of plant cells
with a plant transformation vector comprising a coding region of
said polynucleotide under the control of the promoter normally
associated with the nucleic acid sequence found in SEQ ID NO:
6.
[0103] In one aspect of the present invention, the nucleic acid
described in SEQ ID NO: 6 is used to alter the phenotype of an
Arabidopsis plant by introduction of said nucleic acid or portion
thereof into an Arabidopsis plant and recovering a plant wherein
the inflorescence architecture of the plant has changed as a result
of the introduction of the nucleic acid sequence, or portion
thereof into the plant.
[0104] In one aspect of the invention these nucleic acid sequences
may be used for identification of related homologous sequences
deposited in public databases through comparative techniques
well-known in the art, or as a hybridization probe for the
identification of related cDNA or genomic sequences from various
species, including plant species where the DNA sequence information
is not known. In particular it is contemplated that these sequences
so described can be used for the isolation of plant genes encoding
similar activities.
[0105] In another aspect of the present invention, nucleic acids
encoding a protein at least 50% homologous to the protein encoded
by SEQ ID. NO: 6 are isolated and said nucleic acids are used to
alter the phenotype of the inflorescence of the plant species from
which they were derived by introduction of said nucleic acid or
portion thereof into said plant species and recovering a plant
wherein the inflorescence architecture of the plant has changed as
a result of the introduction of the nucleic acid sequence, or
portion thereof into the plant species.
[0106] In another aspect of the present invention, said nucleic
acids that encode a protein at least 50% homologous to the protein
encoded by SEQ ID NO: 6 are used to alter the inflorescence
architecture of a flowering plant by introduction of said nucleic
acid into a plant species heterologous to the plant species from
which said nucleic acid sequence was derived.
[0107] In yet another aspect of the present invention, the nucleic
acid sequence described in SEQ ID NO: 6 is used as a visible marker
for plant transformation, said marker producing plants with an
altered inflorescence architecture relative to plants not
transformed with the same.
[0108] In order to isolate nucleic acid sequences involved in
inflorescence architecture, mutant plant lines with altered
inflorescence architecture were analyzed. A mutant in Arabidopsis
designated as bp has been described that exhibits a significant
reduction in pedicel length (.about.80-90%) along with shortening
in the internodal regions (40-60%). The bp mutant was first
described by Koornneef et al in 1983 (ibid.) and has been used
extensively in mapping studies as a classical chromosome 4 marker.
However, no studies explaining the developmental or molecular basis
of this mutation have been published to date.
[0109] In the present invention, mutant alleles of this gene were
isolated by screening T-DNA insertional lines for bp phenotypes. A
line was found that showed a bp mutant phenotype. As described
herein, this isolated line was designated as bp-2 and the Koornneef
isolate as bp-1. Thus, a new bp mutation was discovered by the
present inventors.
[0110] In order to establish the basis of the new mutation, pure
lines with single recessive alleles of bp-1 and bp-2 were
established in Arabidopsis ecotypes Landsberg erecta (Ler) and
Columbia (col).
[0111] These lines were analyzed for architectural changes by
Scanning Electron Microscopy (SEM) and the results indicated that
epidermal cell differentiation is affected in both pedicel and
internodes. Detailed SEM analysis of the pedicel showed that in the
abaxial region (lower side), epidermal cell differentiation is more
affected compared to the adaxial region (upper side) in addition to
an overall reduction in cell divisions along the whole pedicel.
Thus, the more pronounced abaxial changes in differentiation
coupled with reduced cell division contribute to the change in the
pedicel attachment angle and as a result produced shortened
siliques (seed pods) pointing downwards in the BP mutant. This
provides an architectural change in the morphology of the pedicel,
leading to a plant with an altered inflorescence.
[0112] Cross sections through the internodal regions showed that in
addition to alterations in epidermal cell differentiation, the
sub-epidermal cortical region was changed in bp lines. In these
lines, this region showed more intercellular spaces with larger
cortical cells. Analysis of pedicel cross sections also revealed
similar changes. Analysis of longitudinal sections through the
nodes showed there were fewer cells (between floral nodes) in the
bp lines compared to wild-type lines. The presence of fewer cells
in the internodes is indicative of reduced cell divisions in this
region, consistent with the significantly reduced internodal length
in the bp lines.
[0113] The anatomical analysis clearly demonstrates that changes in
cell differentiation coupled with reduced cell division contributes
to the altered, compact architectural phenotype in the bp lines.
Accordingly, the changes in the architecture of the plant as a
result of the BP mutation (or loss of its function) provide a new
and valuable phenotype for flowering plants with a compact
inflorescence and downward pointing seed pods.
[0114] Genetic analysis established that bp-2 is allelic to bp-1
previously mapped on chromosome 4. The bp-2 mutant phenotype is not
physically linked to the T-DNA. The present inventors used a novel
strategy of positional cloning to isolate the gene sequences
associated with the bp phenotype.
[0115] The available genetic and recombination data suggest that
the bp locus is located in between the marker DET2 and the
centromere on chromosome 4. The genomic sequence corresponding to
this region (.about.1.5 Mb) has been determined. To clone the BP
gene, a region between DET1 and the centromere on chromosome 4 was
chosen, based on genetic maps compiled from several data sets
(www.Arabidopsis.org; (Pepper, A., Delaney, T., Washburn, T.,
Poole, D. & Chory, J. (1994) Cell 78). As the loss-of-function
BP mutation mainly affects the pedicel and internodal regions but
not the leaves, the BP transcripts are also likely differentially
expressed. Probes corresponding to differentially expressed
transcripts were prepared from the pedicel and internodal region
and were used for subtraction hybridization with leaf-expressed
transcripts to identify potential BP candidate genes from this
.about.1.5 Mb genomic region.
[0116] Radioactively labeled probes representing the transcripts
preferentially expressed in the pedicel and internodal region were
generated and hybridized the probes to restriction-digested
overlapping BAC DNAs completely covering this region of chromosome
4. The results showed a single hybridizing band representing a
.about.20-kb BamHI fragment from BAC clone F9M13 (Mayer K F X,
Schiller C M E, et al. (1999) Sequence and analysis of chromosome 4
of the plant Arabidopsis thaliana. Nature 402: 769-777.) The
annotation and BLAST analysis of this .about.20 kb sequence showed
only one potential gene, with 100% identity to the previously
reported homeodomain containing protein KNAT1 (Lincoln, C., Long,
J., Yamaguchi, J., Serikawa, K. & Hake, S. (1994) Plant Cell 6,
1859-1876.).
[0117] Previous reports in the art have mapped the KNAT1 gene to
chromosome 5, however, the assignment of the chromosomal location
of the KNAT1 gene has now been found to be in error. Utilizing the
sequence comparison available based on screening the whole
Arabidopsis genome demonstrated that the KNAT1 gene as well as the
sequence of the BAC clone F9M13 containing the BP gene to be
located on chromosome 4. Thus, it was established that the KNAT1
gene resides on chromosome 4, not 5 as previously reported.
[0118] This discovery shows that the previously described KNAT1
gene, formerly thought to be on chromosome 5 and encoding a protein
previously thought to be involved in various facets of plant
development, is the gene affected in the bp mutation and is in fact
intimately involved in the control of inflorescence
architecture.
[0119] Whereas the previous study with the KNAT1 gene demonstrated
that overexpression of the coding region of the gene (cDNA) under
CaMV 35S promoter produced several abnormal phenotypes including
the ectopic production of meristems from adaxial (upper) surface of
the leaves and altered leaf shape, the involvement of KNAT1 in
pedicel architecture and control of inflorescence was not reported.
The art failed to provide correlation between KNAT1 and the bp
mutation herein described. Hence function of the KNAT1 gene was not
assigned nor was the utility of the gene for controlling
inflorescence architecture known or suggested. In addition, the
chromosomal location of KNAT1 was also incorrectly reported further
confusing the nature and utility of the KNAT1 gene.
[0120] However, as described herein, the second bp phenotype, bp-2
was unequivocally established as residing on chromosome 4 within
the BAC F9M13 clone. Since the brevipedicellus (bp) mutation was
described before the report of KNAT1, the inventors adopted the BP
designation for this locus, according to conventional practice.
[0121] To determine if sequence differences existed between bp-1,
bp-2 and wild type plants at the BP locus, a Southern blot with
restriction digested genomic DNAs as target and the BP (KNAT1) cDNA
as probe was carried out. It was demonstrated that the bp-1 (Ler)
lacks the BP (KNAT1) gene entirely, indicating that a deletion of
this gene had occurred in this mutant. In contrast, bp-2 showed
hybridizing bands similar to wild type. Thus bp-1 represents a
deletion mutation of the BP gene, (or the KNAT1 gene) whereas bp-2
represents an alteration of the gene (and encoded protein)
itself.
[0122] The expression of the BP transcripts in mutant and wt plants
was analyzed. RT-PCR results confirm that bp-1 produces no BP
transcript, while bp-2 produces an apparently full-length
transcript comparable to the wild type. To identify the molecular
basis for the bp-2 mutant phenotype, BP-encoding RT-PCR products
from duplicate reverse transcription reactions using Ler (wt), RLD
(wt), bp-2 (col), bp-2 (Ler), and bp-2 (RLD) were then cloned and
their sequences determined.
[0123] In wt Ler and RLD the BP ORFs encoded predicted proteins of
400 amino acids, compared with a predicted protein of 398 amino
acids for col wt. Minor sequence polymorphisms among the three
wild-type BP cDNAs were detected, some of which resulted in
differences in the predicted proteins. The BP gene, or KNAT1 gene
contains two domains, a homeodomain, and an ELK region as typically
found in plant and animal homeobox genes.
[0124] Changes were noted between wild-type and mutant BP proteins
(bp-2 protein from Ler, col and RLD bp lines). In particular, the
third and fourth asparagine/histidine-rich regions contained
differing numbers of N residues among the three predicted proteins,
which accounted for the differences in the total number of amino
acids. The predicted BP proteins from bp-2 (col), bp-2 (Ler), and
bp-2 (RLD) were identical and contained several unique
polymorphisms compared with the wt sequences, hence the altered
protein structure of the protein encoded by the bp-2 gene,
conferring altered functionality. This similarity between the
different bp-2 proteins is expected since the original bp-2
mutation was introgressed into these three backgrounds.
Interestingly, within the wt BP protein, minor polymorphisms were
identified. Thus, protein polymorphisms are found in both wt and
bp-2 proteins. For example, the third N-rich region contained only
three N residues in the bp-2 lines, compared with five in col (wt)
and six each in Ler (wt) and RLD (wt). Most importantly, bp-2
contained a C-T transition corresponding to position 535 of the col
(wt) ORF. This point mutation changed codon 179 from cag to tag,
thereby introducing a stop codon and resulting in a truncated
predicted protein. The predicted BP protein of bp-2 is truncated
upstream of both the important homeodomain and ELK regions, and as
result this protein would not be expected to have normal
function.
[0125] Further supporting evidence was obtained by transforming the
bp-1 and bp-2 mutant lines with wild type BP genomic and cDNA
constructs, which showed complementation of the mutant phenotype in
transgenic plants and restoration of wild-type plant
architecture.
[0126] In addition to simple complementation, control of
inflorescence architecture can be regulated by expression levels of
wt BP protein. The pedicels in col wt are much longer than Ler wt
pedicels. Based on expression analysis, it was found that there is
a 24 times higher transcript level of wt BP mRNA in col wt ecotype
when compared to Ler wt, indicating that transcriptional regulation
of BP contributes to the observed differences between these
ecotypes. Thus, reducing the BP transcript levels can lead to a
significant reduction in pedicel and internodal length. It is also
desirable to increase the length of pedicel and/or internodes by
up-regulating the expression of BP functional homologues. Thus, the
results presented herein provide obvious strategies for the
manipulation of inflorescence architecture.
[0127] Accordingly, the present invention ascribes a function to a
previously identified homeobox gene, KNAT1, demonstrating that
KNAT1 encodes a protein normally involved in the control of
inflorescence development. This invention also demonstrates that
KNAT1 is located on chromosome 4, not 5 as previously reported. In
addition, this invention demonstrates the function of the KNAT1
gene in pedicel architecture and demonstrates alterations in the
coding sequence of the KNAT1 gene can lead to a bp phenotype, thus
establishing KNAT1 as the BP gene.
[0128] For the purposes of the present invention, nucleic acid
sequences encoding a protein with substantial homology of 50% or
more to the protein encoded by SEQ ID NO: 5, said proteins at least
differentially expressed in the inflorescence of a flowering plant,
and having a role in regulating inflorescence architecture, are
herein referred to as "BP" coding sequences, encoding a "BP"
protein. Hence a "BP gene" from a flowering plant represents a
coding sequence substantially similar to the SEQ ID NO: 5 in both
protein sequence and protein function.
[0129] A "BP" gene may or may not include the 5' and 3' regions
normally associated with said coding sequence, as a native "BP"
gene will include at least functional portion of these regulatory
regions, whereas a recombinant "BP" gene will have at least one
portion of the 3' or 5' regions altered by the addition of new DNA
sequences. The alteration of the 5' or 3' regions of said BP gene
will be at least expected to cause altered expression in the native
plant species from which the BP gene was derived when compared to
the expression of the wt BP gene normally found in said plant
species.
[0130] In one embodiment of the present invention, the expression
of the BP gene in a plant species is altered by the inhibition of
expression of the native BP gene coding sequence. Accordingly, it
is one object of the present invention to alter the expression
levels of the protein encoded by the BP gene normally found in a
plant species by introduction of a recombinant BP gene that alters
the expression of the wt BP gene by reduction of the native BP gene
expression and reduction of the levels of the protein encoded by
the wt BP gene in said plant species.
[0131] It is a further embodiment of the present invention to alter
the expression of a wt BP gene in a plant species by introduction
of a recombinant version of said BP gene, said recombinant version
altered by the addition of one of more DNA sequences that lead to
the increased expression of said gene relative to the expression of
the wt BP gene in said plant species, leading to the increased
expression levels of the protein encoded by a wt BP gene coding
sequence in said plant species.
[0132] It is still another embodiment of the present invention to
express a non-native BP coding sequence in a plant species. Said
non-native BP coding sequence can be an altered form of the BP
coding region normally found in said plant species, or a BP
functional homologue from a different plant species. Expression of
the non-native BP protein can be expected to alter the activity of
the native BP protein by competition for DNA binding regions, or
the non-native BP protein can encode an activity that provides a
phenotypic distinction.
[0133] Accordingly, it is one embodiment of the present invention
to alter the activity of the protein encoded by the BP gene
normally found in a plant species is altered by introduction of a
recombinant version of a non-native BP gene, said recombinant
version altered by the addition of one of more DNA sequences that
lead to expression of said gene in said plant species, leading to
altered activity of the native BP protein. In the present case,
altered activity of the BP protein is defined as changes in the
inflorescence structure in plants that comprise the non-native BP
gene.
[0134] Similarly, in a further embodiment of the present invention
to alter the expression of a wt BP gene in a plant species by
introduction of a recombinant non-native BP gene that alters the
activity of the wt BP gene by reduction of the native BP gene
expression and reduction of the expression of the protein encoded
by the wt BP gene in said plant species.
[0135] The identification of this unique genetic activity and
specific function allows for novel strategies to manipulate plant
morphology or architecture. The sequence can also be used to
isolate corresponding related similar or identical sequences from
other plant species. Related techniques are well understood in the
art, for example as provided in Sambrook et al., Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor,
N.Y. (1989).
[0136] The applications of this gene for engineering useful
flower/inflorescence architectures in crop and economically
important plant species include both the production of more compact
flowering structures and conversely methods for the genetic
reprogramming of inflorescence structure to produce less compact
and more spreading floral structures useful for horticultural
applications.
[0137] One preferred application is to develop a bp phenotype in
canola crop species (e.g. Brassica napus, B. rapa). A compact
inflorescence architecture in canola will offer several advantages
to this crop that may include reduced shattering and improved
overall performance. As one aspect of the present invention,
BP-related genes from canola have been isolated and are used to
engineer bp-phenotypes.
[0138] Similar strategies can be applied to other crop plants by
using BP functional homologues from the respective species.
Engineering novel and useful architectures using BP or functional
homologues is not limited to crop species; potential applications
could be extended to horticultural plants to create aesthetically
appealing flowers or inflorescences.
[0139] Accordingly, in one embodiment of the invention the subject
method includes the steps of expressing a BP gene in a plant
species comprising the steps of:
[0140] a) introducing into a plant cell capable of being
transformed a genetic construct comprising a first DNA expression
cassette that comprises, in addition to the DNA sequences required
for transformation and selection in said cells, a DNA sequence
derived from a BP (KNAT1) gene, for example, that encodes a peptide
having at least 50% homology to the peptide encoded by SEQ ID NO:
5, operably linked to a suitable transcriptional regulatory region
and,
[0141] b) recovery of a plant which contains said recombinant DNA,
said plant exhibiting altered inflorescence architecture.
[0142] The suitable transcriptional regulatory region can be the
regulatory region normally associated with the BP (KNAT1) gene or
BP coding sequence or a heterologous transcriptional regulatory
region capable of expression in the inflorescence.
[0143] In another preferred embodiment of the invention the subject
method includes a method for modifying the inflorescence
architecture of a plant comprising:
[0144] (a) introducing into a plant cell capable of being
transformed and regenerated to a whole plant a genetic construct
comprising a first DNA expression cassette that comprises, in
addition to the DNA sequences required for transformation and
selection in plant cells, a DNA sequence that comprises a
polynucleotide region derived from SEQ ID NO: 5 or 6 encoding a BP
gene sequence or part thereof, operably linked to a suitable
transcriptional regulatory region and,
[0145] (b) recovery of a plant which contains said recombinant DNA
and has altered inflorescence architecture.
[0146] The chimeric gene is introduced into a plant cell and a
plant cell recovered wherein said gene is integrated into the plant
chromosome. The plant cell is induced to regenerate and a whole
plant is recovered with altered inflorescence architecture.
[0147] The method further relies on the use of transformation to
introduce the gene encoding the enzyme into plant cells.
Transformation of the plant cell can be accomplished by a variety
of different means. Methods that have general utility include
Agrobacterium-based systems, using either binary and cointegrate
plasmids of both A. tumifaciens and S. rhyzogenies (e.g., U.S. Pat.
No. 4,940,838, U.S. Pat. No. 5,464,763), the biolistic approach
(e.g., U.S. Pat. No. 4,945,050, U.S. Pat. No. 5,015,580, U.S. Pat.
No. 5,149,655), microinjection, (e.g., U.S. Pat. No. 4,743,548),
direct DNA uptake by protoplasts, (e.g., U.S. Pat. No. 5,231,019,
U.S. Pat. No. 5,453,367) or needle-like whiskers (e.g., U.S. Pat.
No. 5,302,523). Any method for the introduction of foreign DNA
and/or genetic transformation of a plant cell may be used within
the context of the present invention.
[0148] It is also apparent to one skilled in the art that the
polynucleotide and deduced amino acid sequence of SEQ ID NO: 5 or 6
can be used to isolate related genes from various other plant
species. The similarity or identity of two polypeptide or
polynucleotide sequences is determined by comparing sequences. In
the art, this is typically accomplished by alignment of the amino
acid or nucleotide sequences and observing the strings of residues
that match. The identity or similarity of sequences can be
calculated by known means including, but not limited to, those
described in Computational Molecular Biology, Lesk A. M., ed.,
Oxford University Press, New York, 1988, Biocomputing: Informatics
and Genome Projects, Smith, D. W., ed., Academic Press, New York,
1993., Computer Analysis of Sequence Data, Part I, Griffin, A. M.
and Griffin, H. G., eds., Humana Press, New Jersey, 1994 and other
protocols known to those skilled in the art. Moreover, programs to
determine relatedness or identity are codified in publicly
available programs. One of the most popular programs comprises a
suite of BLAST programs, three designed for nucleic acid sequences
(BLASTN, BLASTX and TBLASTX), and two designed for protein
sequences (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology,
12:76-80, 1994). The BLASTX program is publicly available from NCBI
and other sources such as the BLAST Manual, Altschul, S., et al.,
NCBI NLM NIH Bethesda Md. 20984, also
www.ncbi.nlm.nih.gov/BLAST/blast_help.html) provides online help
and further literature references for BLAST and related protein
analysis methods, and Altschul, S., et al., J. Mol. Biol
215:403-410, 1990.
[0149] Within the BP gene two regions are found, the homeodomain
and the ELK region. Within the homeodomain region BP shares
significant homology with number of other homeodomain proteins
(approximately 50 in Arabidopsis), and also other plant and animal
homeodomain proteins, thus the BP protein represents one of the
many homeobox genes.
[0150] The isolated polynucleotide can be sequenced and the DNA
sequence used to further screen DNA sequence collections to
identify related sequences from other species. The DNA sequence
collections can comprise EST sequences, genomic sequences or
complete cDNA sequences.
[0151] In Arabidopsis the entire BP coding sequence shares the
highest homology with STM which is implicated in meristem
maintenance and function (41%), whereas outside of Arabidopsis, it
shares 53% homology with maize RS1 and 52% with rice OSH15. These
genes have been identified by utilizing the conserved domains of
plant homeobox genes. Similarly, hybridization can be used to
isolate BP functional homologues genes from other species. In the
present invention, we have used probes derived from SEQ ID NO: 5 to
isolate cDNA sequences homologous to the BP gene of
Arabidopsis.
[0152] The present inventors have isolated BP genes from Brassica
napus, B. oleracea and B. rapa using hybridization. These Brassica
BP genes have been incorporated into plant transformation vectors
and have been used to transform plants to obtain plants with
altered inflorescence structures.
[0153] Accordingly, in one embodiment of the invention the subject
method for modifying the inflorescence of a plant comprising the
steps of:
[0154] a) introducing into a plant cell capable of being
transformed a genetic construct comprising a first DNA expression
cassette that comprises, in addition to the DNA sequences required
for transformation and selection in said cells, a DNA sequence that
encodes a BP coding sequence encoding a peptide having of at least
50% sequence identity to the peptide encoded by SEQ ID NO: 5,
operably linked to a suitable transcriptional regulatory region
and,
[0155] b) recovery of a plant which contains said recombinant
DNA.
[0156] In another embodiment of the present invention, alteration
of Brassica inflorescence structure is contemplated. Accordingly,
the present invention encompasses a method for modifying the
inflorescence of a Brassica plant comprising the steps of:
[0157] a) introducing into a Brassica plant cell capable of being
transformed a genetic construct comprising a first DNA expression
cassette that comprises, in addition to the DNA sequences required
for transformation and selection in said cells, a DNA sequence that
encodes a Brassica BP coding sequence encoding a protein of at
least 50% sequence identity to the protein sequence encoded by SEQ
ID NO: 5, operably linked to a suitable transcriptional regulatory
region and,
[0158] b) recovery of a Brassica plant which contains said
recombinant DNA and exhibits an altered inflorescence.
[0159] The use of gene inhibition technologies such as antisense
RNA or co-suppression or double stranded RNA interference is within
the scope of the present invention. In these approaches, the
isolated gene sequence is operably linked to a suitable regulatory
element.
[0160] Accordingly, in one embodiment of the invention the subject
method includes a method to modify the inflorescence of a plant
comprising the steps of:
[0161] a) introducing into a plant cell capable of being
transformed a genetic construct comprising a first DNA expression
cassette that comprises, in addition to the DNA sequences required
for transformation and selection in said cells, a DNA sequence that
encodes a BP coding sequence encoding a protein of at least 50%
sequence identity to the protein encoded by SEQ ID NO: 5, at least
a portion of said DNA sequence in an antisense orientation relative
to the normal presentation to the transcriptional regulatory
region, operably linked to a suitable transcriptional regulatory
region such that said recombinant DNA construct expresses an
antisense RNA or portion thereof of an antisense RNA and,
[0162] b) recovery of a plant which contains said recombinant
DNA.
[0163] It is apparent to the skilled artisan that the
polynucleotide encoding the sequence can be in the antisense (for
inhibition by antisense RNA) or sense (for inhibition by
co-suppression) orientation, relative to the transcriptional
regulatory region, or a combination of sense and antisense RNA, to
induce double stranded RNA interference (Chuang and Meyerowitz,
PNAS 97:4985-4990, 2000, Smith et al., Nature 407:319-320,
2000).
[0164] A transcriptional regulatory region is often referred to as
a promoter region and there are numerous promoters that can be used
within the scope of the present invention. In addition, the skilled
artisan will readily recognize that the sequence of the inserted
recombinant gene must contain regions of sufficient homology to
allow for sequence-specific inhibition of gene expression.
[0165] Another application for the BP gene is as a visible marker
for plant transformation. The advantages of using selection systems
that do not include antibiotic/herbicide resistance marker genes
for producing transgenic plants are well recognized. Since the bp-1
null mutant represents a phenotype that is clearly visible and
easily distinguishable from wild type plants, it is possible to
develop transformation vectors based on the BP gene that are devoid
of any antibiotic or herbicide selection markers to provide a novel
and very efficient alternative to the currently available selection
systems. As evidenced by the present invention, the use of the BP
gene for complementation of the bp phenotype in Arabidopsis
demonstrates that it is possible to select for plants that have
received a BP gene as a result of transformation with said
gene.
[0166] It is apparent to the skilled practitioner that any number
of methods for the construction of a heterologous genetic construct
encoding the protein or portion thereof encoded by SEQ ID NO: 5 or
homologues thereof can be used to alter the architecture of plant
wherein said DNA construct has been introduced.
[0167] The following examples serve to illustrate the method and in
no way limit the utility of the invention.
EXAMPLE 1
Construction and Analysis of Arabidopsis bp Mutant Lines
[0168] Plant material and genetic analysis. Plants were grown at
22.degree. C. (90% relative humidity) under fluorescent and
incandescent light at .about.60 .mu.E/m.sup.2/s with 16 h days. The
bp mutant seeds were obtained from the Arabidopsis Biological
Resources Center (ABRC), Ohio State University (stock number CS30;
(Koornneef, M., Eden, J. v., Hanhart, C. J., Stam, P., Braaksma, F.
J. & Feenstra, W. J. (1983) J. Hered. 74, 265-272)). This
allele was designated bp-1. A second bp allele (bp-2) was isolated
from promoter-tagged Arabidopsis lines in RLD background. This
allele was introgressed into Ler and backcrossed five times with
wild type (wt). bp-2 was introduced into Columbia (Col) wt
background from Ler and backcrossed three times.
[0169] Histology. Plant samples were fixed for 24 h at room
temperature in FAA and paraffin embedded as described (Johansen, D.
A. (1940) Plant microtechnique (McGraw-Hill Book Co., New York).).
Serial sections were taken at 8 .mu.m on a rotary microtome,
attached to glass slides with Mayer's egg albumin (Sigma) solution,
and dried on a warming tray (42.degree. C.). Sections were stained
after removal of the embedding medium in toluidine blue O. The
sections were observed under a Leitz (Wetzlar) microscope and
images were captured using Optronics DEI 750 digital microscope
camera.
[0170] Scanning electron microscopy. For scanning electron
microscopy (SEM) the samples were fixed in 3% glutaraldehyde and
processed as described (Venglat, S. P. & Sawhney, V. K. (1996)
Planta 1968, 480-487.). Samples were mounted on aluminum stubs and
coated with gold in an Edwards S150B sputter-coater. Observations
were made with a Phillips SEM 505 scanning electron microscope at
30 kV and recorded using Polaroid type 665 P/N. Images were scanned
and enhanced using Adobe Photoshop 4.0.
[0171] Architectural Changes in the inflorescence of bp mutants. In
all bp plants the earliest signs of alteration of the inflorescence
are evident at the time of bolting, with more compactly arranged
floral buds at the apex; the effects were more pronounced when the
first few co-florescence internodes from the rosette leaves started
elongating. At maturity, bp plants display a marked reduction in
overall height, primarily as a result of shortened internodes;
moreover, the floral internodes were affected to a greater extent
than the co-florescence internodes (FIGS. 1,2). Additionally,
bending at nodes was observed and this phenotype was more severe in
bp-1 than bp-2 plants. bp-2 in RLD (the original isolate) and Col
backgrounds showed similar patterns, although the reduction in
internodal lengths was less than observed in Ler background. bp
affects cell division and cell differentiation in the internodes of
the inflorescence. SEM analysis showed that the floral buds began
pointing downwards quite early in their development and that the
internodal elongation is significantly reduced. The peduncle
surface showed stripes consisting of cell files (.about.15 cells in
width) with changes in epidermal cell differentiation (defined by
alterations in bp lines in cell size, shape, and/or cell type
(stomata) in relation to similar regions in wt) associated with
regions below the nodes (FIG. 3). Cross sections through internodes
in bp indicated that the overall radial pattern, in terms of tissue
types, was very similar to the wt (FIG. 3). However, small sectors
with changes in epidermal cell differentiation are observed, and
these corresponded to the stripes of differentiation-altered cells
observed by SEM. Furthermore, the cortical cells below these
sectors were had changes in differentiation (indicated by a lack of
chloroplasts), and the cells were relatively larger with less
intercellular space. Longitudinal sections through the nodes showed
sectors of epidermal and sub-epidermal changes. As the cell number
per unit area along the main axis of the peduncle in BP was
comparable to the wt, the reduced internodal length was interpreted
to be a result of fewer cell divisions.
[0172] BP causes chanaes in inflorescence development. Pedicels in
bp plants at all the floral nodes showed a drastic reduction in
length compared with wt (FIG. 2), in addition to downward-pointing
siliques (FIG. 1). The degree of the latter phenotype conferred by
bp-2 varied in different backgrounds from downward-pointing (Ler)
to less acute bending in RLD and Col backgrounds. Since very little
is known about pedicel development in any plant species, including
Arabidopsis, we determined its ontogeny in Ler wt compared with bp.
Pedicel initiation was first observed around stage 3 flowers,
followed by elaboration of the pedicel with coordinated development
on both the abaxial and adaxial sides, and along the proximo-distal
axis. The first signs of epidermal differentiation (defined by
characteristic changes in cell shape and the appearance of stomata)
were observed on the abaxial side at stage 9, and this was closely
followed by differentiation on the adaxial side in subsequent
stages. By stage 12 epidermal cell differentiation was completed
with no apparent differences observed between the abaxial and
adaxial sides in the wt (FIG. 4). In bp, no detectable differences
from wt were observed up to stage 3. However, the pedicel
differentiation and elaboration processes lagged behind the wt and
the first sign of epidermal cell differentiation was observed only
at stage 12, and this was restricted to the adaxial surface; no
corresponding differentiation was observed on the abaxial side,
even by the mature stage (FIG. 4). Anatomical analysis showed that
while the major part of the pedicel in bp contained defects in the
differentiation of abaxial-side epidermal cells and cortical cells
(FIG. 4), the distal region including the receptacle was more
strongly affected with a significantly reduced pith region, cell
size and differentiation, and radial growth (FIG. 4). Longitudinal
sections through the pedicels also showed that the cells in the
epidermal layer and cortical tissues on the abaxial side were less
elongated (FIG. 3). Furthermore, there were fewer cells in the
proximo-distal axis of the pedicel, indicative of fewer cell
divisions. Although there were no apparent defects observed in the
sepals, petals, and stamens, the carpels showed detectable
differences in bp. Notably, there was reduced radial growth of the
style (FIG. 5), although there was variability observed between
plants regarding this phenotype. The epidermal and cortical cells
of the style, especially in the lateral axis, were defective in
differentiation and elongation, and as a consequence the
arrangement of stigmatic papillae was significantly altered (FIG.
5). These observations support a functional role for BP in
maintaining the normal growth and radial symmetry of the style. The
developmental and anatomical studies suggested that the defects in
bp were only associated with the peduncle and parts of the flower
but not with the leaves.
EXAMPLE 2
Isolation of the BP (KNAT1) Coding Sequence
[0173] To isolate the BP (KNAT1) coding sequence, cDNA cloning was
used. Reverse transcription was carried out using 3-5 .mu.g of
total RNA from stem tissue of wt (Col, Ler, RLD) and bp plants and
Superscript II RT (Life Technologies). To amplify the BP (KNAT1)
open reading frame (ORF), 1 .mu.l of cDNA was used for PCR with
primers
TABLE-US-00001 SEQ ID NO: 1 954 DNA SEQ 5'
cgggatccatggaagaataccagcatgac 3' and SEQ ID NO: 2 955 DNA SEQ 5'
cgggatccggtacctggatgtcttatggaccgag 3'
and Pfu polymerase (1 U). Amplification of the cytosolic
glyceraldehyde-3-phosphate dehydrogenase (gapC) cDNA (Shih, M.-C.,
Heinrich, P. C. & Goodman, H. M. (1991) Gene 104, 133-138) from
the same cDNA pools was performed under the same conditions
TABLE-US-00002 SEQ ID NO: 3 DNA SEQ gapC-UP 5' accactaactgccttgctc
3' and SEQ ID NO: 4 DNA SEQ gapC-DN 5' caatttcacaaacttgtcgctc
3'
BP (KNAT1)-encoding PCR products were cloned and sequenced by
primer walking using an ABI 377 DNA sequencer. The sequence of the
wt BP (KNAT1) gene is shown in SEQ ID NO: 5.
EXAMPLE 3
Expression of BP Genes
[0174] Based on the discovery that BP represents the previously
described KNAT1 gene, probes for BP (KNAT1) were generated as
described above and used to analyzed BP transcript levels in col wt
and Ler wt by northern blots and by the more sensitive RT-PCR.
Results from these experiments showed 24 times higher transcript
levels in col wt ecotype (data not shown).
EXAMPLE 4
Isolation of BP Genomic Regions
[0175] The BP appears to be expressed predominantly in stem and
pedicel tissues in wt plants. To clone BP, a region between DET1
and the centromere on chromosome 4 was chosen, based on genetic
maps compiled from several data sets (www.Arabidopsis.org; (Pepper,
A., Delaney, T., Washburn, T., Poole, D. & Chory, J. (1994)
Cell 78)). To produce probes reflecting the anticipated expression
pattern of BP, polyA+RNA was isolated from both stem/pedicel and
leaf tissues in Col wt plants and a Suppression subtractive
hybridization (SSH) was performed using leaf cDNA as driver. Total
RNA was harvested from stem/pedicel and leaf tissues of Col wt
using Trizol Reagent (Life Technologies). Poly A+RNA was isolated
using mRNA spin columns (Clontech). cDNA synthesis was carried out
using a cDNA synthesis kit (Life Technologies). A total of 2 .mu.g
each of leaf cDNA (driver) and stem/pedicel cDNA (tester) was
digested with HaeIII (New England Biolabs) and used for suppression
subtractive hybridization as described (Diatchenko, L., et al.
(1996) Proc. Natl. Acad. Sci. USA 93, 6025-6030). The subtracted
mix was 32 P-labeled using a RediPrime kit (AP Biotech) and used to
screen Bacterial Artificial Chromosome (BAC) DNA preparations as
described below.
[0176] BAC clones from chromosome 4 were obtained from the ABRC.
DNA was prepared from 10-ml cultures of BACs T17A2, T13D4, F9M13,
T12G3, T28D5, T15F16, T3F12, T32A17, T3H13, F23J3, T8A17, T30A10,
T15G18, T25P22, and T24H23 using an alkaline lysis miniprep method
(Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman,
J. G., Smit, J. A. & Struhl, K. (1995), (John Wiley and Sons,
Inc., New York).). BAC DNA was digested with BamHI, EcoRI, or
HindIII (Life Technologies), fractionated on a 0.8% agarose gel,
then blotted to a Zeta Probe membrane (BioRad) using standard
procedures as described (Ausubel, et al, ibid.) The blot was probed
with the pooled subtracted mix representing cDNAs expressed in
stem/pedicel tissue, prepared as described above. Genomic DNA (5
.mu.g) isolated from leaves (Dellaporta, S. (1994) in The Maize
Handbook, eds. Freeling, M. & Walbot, V. (Springer-Verlag, New
York), pp. 522-525.) of wt and bp plants was digested using 30 U
BamHI or EcoRI (Life Technologies) at 37.degree. C. for 8 hours,
processed as above, and probed with the 32 P-labeled KNAT1 RT-PCR
product from Col wt. Hybridization proceeded for 3 h (BAC screen)
or overnight (genomic Southern blot) at 65.degree. C. in QuickHyb
hybridization solution (Stratagene); the most stringent wash was in
0.1.times.SSC/0.1% SDS at 65.degree. C. The blots were exposed to
X-OMAT AR film (Kodak) overnight at -70.degree. C. PCR, RT-PCR, and
DNA sequencing.
[0177] The pooled subtracted products were then used as a probe in
a Southern blot with 15 BACs as targets spanning a region of
approximately 1.5 Mb on chromosome 4 between DET1 and the
centromere. A BamHI fragment of about 20 kb from BAC F9M13 was the
only band that showed any hybridization to the subtracted probe.
BAC F9M13 (GenBank AC006267) contains a single gene on this 20-kb
BamHI fragment within a region rich in repeats. Subsequent
fingerprinting of F9M13 with this probe confirmed that the probe
detected the previously reported homeobox gene KNAT1.
EXAMPLE 5
Determination of BP Gene Coding Sequence in bp Mutant and Wild Type
Lines
[0178] It was found that the bp-1 mutant represented a deletion of
the BP gene. To identify the molecular basis for the bp-2 mutant
phenotype, BP-encoding RT-PCR products from duplicate reverse
transcription reactions using L. er (wt), RLD (wt), bp-2 (col),
bp-2 (Ler), and bp-2 (RLD) were then cloned and their sequences
determined. It was found that the bp-2 lines contained an altered
protein coding sequence which has a C-T transition corresponding to
position 535 of the col (wt) ORF. This is shown in SEQ ID NO: 6,
the bp-2 coding region. This point mutation changed codon 179 from
cag to tag, thereby introducing a stop codon and resulting in a
truncated predicted protein sequence shown in SEQ ID NO: 7.
[0179] This sequence analysis further demonstrated that in wt Ler
and RLD the BP ORFs encoded predicted proteins of 400 aa, compared
with a predicted protein of 398 aa for col wt.
EXAMPLE 6
Complementation of bp Mutant Lines
[0180] In order to demonstrate the function of the BP (KNAT1) gene
in the bp phenotype, plants exhibiting a bp phenotype were
transformed with a wild-type BP (KNAT1) gene under the control of
the native BP (KNAT1) promoter. Two different complementation
constructs were prepared. The structure of the vectors used for
complementation of the bp phenotype in Arabidopsis thaliana is as
follows:
[0181] The backbone for both vectors was pRD400 (Datla, R. S. S.,
J. K. Hammerlindl, B. Panchuk, L. E. Pelcher, and W. Keller. 1992.
Modified binary plant transformation vectors with the wild-type
gene encoding NPTII. Gene 122: 383-384). This vector was used to
derive two plant transformation vectors. In both constructs, the
BamHI sites at the junction of the promoter and ORF were introduced
to facilitate assembly of the constructs. B, BamHI; Bg, BglII, K,
KpnI. Parentheses indicate sites destroyed by ligation.
[0182] Construct A. Referred to as pRD400-951/955, consisting of
the BP (KNAT1) cDNA (SEQ ID NO: 5) cloned downstream of the
putative BP (KNAT1) promoter as shown in FIG. 8. The BP (KNAT1)
promoter was isolated by PCR using the following primers:
TABLE-US-00003 SEQ ID NO: 8 DNA SEQ 951 5'
cccaagcttagatctttcggtctagtgcagtgatg 3' and SEQ ID NO: 9 DNA SEQ 952
5' ccggatcccagatgagtaaagatttg 3'
for amplification of the putative BP (KNAT1) promoter; 1536 bp
product corresponding to the region immediately upstream of the BP
(KNAT1) start codon. Amplification conditions for all primers using
genomic DNA as template were as follows: 94.degree. C., 2 min
followed by 30 cycles of 94.degree. C., 15 sec; 55.degree. C., 30
sec; and 72.degree. C., 4-6 min. A final extension of 10 min at
72.degree. C. was performed. All amplifications from genomic DNA
used Pfu polymerase (Stratagene) (2.5 U) and a PTC-200 thermal
cycler (MJ Research).
[0183] Construct B. Referred to as pRD400-951/956, consisting of
the putative BP (KNAT1) promoter and the BP (KNAT1)-encoding ORF
amplified from genomic DNA. To amplify the BP (KNAT1) coding
region, two primers were used, SEQ ID NO: 1 (as described in
example 2), and
TABLE-US-00004 SEQ ID NO: 10 DNA SEQ 956 5'
gaagatctgtcgacgccttgtgcttgattgagactcca 3'
for amplification of the protein coding region and terminator from
genomic DNA; 3347-bp product from the BP (KNAT1) start codon to a
point 705 bp downstream of the stop codon, including the putative
transcriptional terminator.
[0184] Agrobacterium tumefaciens GV3101 containing these
recombinant constructs were used to transform bp-2 (Ler) plants by
vacuum infiltration (Bechtold, N., Ellis, J. & Pelletier, G.
(1993) C. R. Acad. Sci. Ser. III 316, 1194-1199). Transformation of
bp-2 (Ler) with the genomic clone of BP (KNAT1) resulted in 20
transformants; 4 were completely rescued to wt, while the others
were partially rescued. Southern analysis confirmed that these
complemented lines contained the BP (KNAT1) wt transgene. Further
analysis of two of these single transgene copy lines showed a 3:1
(wt:bp) segregation pattern in the T2 generation, providing genetic
confirmation of complementation. Complementation of BP with BP
(KNAT1) cDNA was also observed.
EXAMPLE 7
Overexpression of the Arabidopsis BP Gene
[0185] In this example, the native Arabidopsis BP gene (wt KNAT1)
was used for over expression. The BP gene coding region (SEQ ID NO:
5) was used to make an over expression construct with enhanced 35S
promoter referred to as pRD400-35S::AtbpS, consisting of the A.
thaliana BP ORF under the control of the 35S promoter assembled
using routine methods. This vector is shown in FIG. 10. The vector
was used to transform Arabidopsis as above.
EXAMPLE 8
Expression of the Arabidopsis BP Gene in Heterologous Species
[0186] The vector pRD400-35S::AtBPS was used to transform B. napus.
The vector was inserted into Agrobacterium stain MP90 by standard
triparental mating followed by Agrobacterium-mediated
transformation of Brassica. Transformation was essentially carried
out as described by Moloney et al., Plant Cell Reports 8:238-242,
1989.
EXAMPLE 9
Construction of Antisense Arabidopsis BP Genes
[0187] The BP coding region was used to construct an antisense
construct under its own promoter (1.5 kb) and also the 35S
promoter. For expression of antisense RNA under the 35S promoter,
the vector pRD400-35S::AtBPA/S, consisting of the A. thaliana bp
ORF in an antisense orientation under the control of the 35S
promoter was constructed and is shown in FIG. 11.
[0188] For expression of antisense RNA under the BP (KNAT1)
promoter, the vector pRD400-951/952::AtbpA/S, consisting of the A.
thaliana bp cDNA (nucleotides 481-1227) in an antisense orientation
under the control of the A. thaliana BP (KNAT1) promoter was
constructed and is shown in FIG. 12.
[0189] Terms used: Nos Ter, Nos terminator. B, BamHI; Bg, BglII, K,
KpnI; H, HindIII, S, SstI.
EXAMPLE 10
Expression of an Altered Arabidopsis BP Gene
[0190] In this example, the protein encoded by SEQ ID NO: 6 was
expressed under its own promoter (1.5 kb) and also the 35S
promoter.
[0191] For expression of the altered BP gene under the 35S
promoter, the vector pRD400-35S::Atbp-2, consisting of the A.
thaliana bp-2 ORF under the control of the 35S promoter is
constructed using the same procedures as for the wild-type coding
sequence and is shown in FIG. 12.
[0192] For expression of the altered BP gene under the BP (KNAT1)
promoter, the vector pRD400-951/952::Atbp-2, consisting of the A.
thaliana bp-2 cDNA (nucleotides 481-1227) in an antisense
orientation under the control of the A. thaliana BP (KNAT1)
promoter is constructed as above and is shown in FIG. 13.
EXAMPLE 11
Isolation of BP Related Coding Sequences from Other Species
B. napus
[0193] The A. thaliana BP (KNAT1) cDNA isolated in Example 2 was
used to screen a cDNA library prepared from stem tissues of B.
napus. A total of 200,000 pfu were initially screened under
moderate stringency hybridization conditions (hybridization
solution contained 30% formamide/5.times.SSC/5.times.Denhardt's
solution/0.5% SDS/50 .mu.g/ml salmon sperm DNA at 42.degree. C.,
with final washes in 0.1.times.SSC/0.1% SDS at 55.degree. C.). 13
plaques were purified from these primary screens; these were
excised from their phagemid hosts and sequenced.
[0194] BLAST analysis showed that the clones fell into 3 groups:
nonspecific clones (discarded); homeobox gene-like sequences that
were not likely orthologs of A. thaliana Knat1; and apparent Knat1
orthologs. The latter group was represented the most frequent and
consisted of both full-length and 5' truncated clones. The complete
sequence on both strands was determined for the longest cDNA
isolated from these screens (1515 bp), which was designated the
name Bnbp.
[0195] This cDNA contained an ORF of 1158 bp with 73 nucleotides of
5' untranslated region (UTR) and 284 nucleotides of 3'UTR and is
shown in SEQ ID NO: 11. The predicted protein encoded by this cDNA
was 385 amino acids in length and showed 86.3% similarity (PAM250
residue weight table) to A. thaliana bp.
TABLE-US-00005 PCR primers SEQ ID NO: 12:
5'-cgggatccatggaagaatatcaacatgaa-3' SEQ ID NO: 13:
5'-cgggatccggtaccttatggtccaagacgat-3'
were designed to amplify the ORF from this cDNA with ends modified
with BamHI/NcoI (5'end) and BamHI/KpnI (3' end) to facilitate its
insertion into the expression constructs described above. The Bnbp
ORF was amplified from the cDNA isolated from the library screens
with Pfu polymerase (Stratagene) and cloned into a standard PCR
product cloning vector (pCR2.1; Invitrogen).
EXAMPLE 12
Isolation of Bp Related Coding Sequences from Other Species
B. rapa, B. oleracea
[0196] The PCR primers that were designed to amplify the Bnbp ORF
were used to isolate bp orthologs from species closely related to
B. napus. Total RNA was extracted from B. rapa and B. oleracea
(kale) hypocotyls harvested 6 days after germination and used as a
template for first strand cDNA synthesis with Superscript II
reverse transcriptase (Life Technologies). This cDNA was then used
as a PCR template to amplify bp-like cDNAs from these species. PCR
products were cloned into a standard vector (pCR2.1) and their
sequences determined by primer walking. The sequences are shown in
SEQ ID NOS: 14 and 15.
EXAMPLE 13
Isolation of Bp Related Genes from Other Species
B. rapa, B. oleracea
[0197] Isolation of genomic clones encoding BP from B. napus, B.
rapa and B. oleracea. Using the sequences of the bp cDNAs
determined as described above, PCR primers were designed to amplify
the genomic copies (including introns) of the BP genes from each
species (B. napus, B. rapa, B. oleracea). The primers used
were:
TABLE-US-00006 SEQ ID NO: 16: PCR Primer used to amplify B. napus
BP from genomic DNA 5'-ataacaccaccaccaacaac-3' SEQ ID NO: 17: PCR
Primer used to amplify B. napus BP from genomic DNA
5'-actaggaagtctcaaacccc-3' SEQ ID NO: 18: PCR Primer used to
amplify B. rapa, B. oleracea BP from genomic DNA
5'-tcaacatgaaagcagatccac-3' SEQ ID NO: 19: PCR Primer used to
amplify B. rapa, B. oleracea BP from genomic DNA
5-aacgagagaggcaacaaaag-3'
[0198] The PCR products (approximately 3.8 kb for each species)
were cloned into pCR2.1 and their sequences were determined by
primer walking.
[0199] The sequence of the B. napus bp genomic region isolated
using primers as described in SEQ ID NOS: 16 and 17 is shown in SEQ
ID NO: 20.
[0200] The sequence analysis revealed that the B. napus BP coding
region, like that of A. thaliana BP, is interrupted by 4 introns.
The positions and relative lengths of the introns were all similar
to the A. thaliana BP gene.
EXAMPLE 14
Isolation of BP Promoter Regions from B. napus
[0201] Isolation of sequences upstream of B. napus BP, including
the probable promoter. The sequence of the BP-encoding cDNA from B.
napus was used to design primers to isolate 5' regions of the bp
gene. Primers used were:
TABLE-US-00007 SEQ ID NO: 21: PCR Primer used to amplify the region
upstream of the bp gene from B. napus
5'-catgatcggatcggaagcaattctcagtcg-3' SEQ ID NO: 22: PCR Primer used
to amplify the region upstream of the bp gene from B. napus
5'-aaaagttgagagagaaagagagagagagag-3'
to isolate the putative promoter-containing region from genomic
DNA. For this purpose, a Genome Walker kit (Clontech) was used.
Following the standard protocols in the kit, two fragments (840 and
950 bp) were isolated that represent the likely promoters of the
two BP genes of B. napus. The sequences are presented in SEQ ID
NOS: 23 and 24.
EXAMPLE 15
Construction of a Vector Comprising the Brassica BP Gene Under the
Control of an Arabidopsis Promoter
[0202] The map of the vector of pRD400-951/952::BnBPS, consisting
of the B. napus BP ORF (SEQ ID. NO: 11) under the control of the A.
thaliana KNAT1 promoter constructed using standard techniques is
shown in FIG. 14. The vector was used to transform Arabidopsis and
Brassica napus as described.
EXAMPLE 16
Construction of a Vector Comprising the Brassica BP Gene Under the
Control of a Constitutive Promoter
[0203] The vector map of pRD400-35S::BnBPS, consisting of the B.
napus BP ORF (SEQ ID NO: 11) under the control of an optimized
cauliflower mosaic virus (CaMV) 35S promoter (Datla, R. S. S., F.
Bekkaoui, J. K. Hammerlindl, G. Pilate, D. I. Dunstan, and W. L.
Crosby. 1993. Improved high-level constitutive foreign gene
expression in plants using an AMV RNA4 untranslated leader
sequence. Plant Sci. 94:139-149) is shown in FIG. 15. The vector
was assembled using well-known techniques as described. The vector
was used to transform Arabidopsis and Brassica napus as described
herein.
EXAMPLE 17
Construction of a Vector Comprising a Brassica Antisense BP Gene
Under the Control of a Constitutive Promoter
[0204] The vector map pRD400-35S::BnbpA/S, consisting of the B.
napus BP ORF in an antisense orientation under the control of the
35S promoter is shown in FIG. 16. The vector was assembled using
well-known techniques as described. The vector was used to
transform Arabidopsis and Brassica napus as described herein.
TABLE-US-00008 Sequence listing free text SEQ ID NO: 1 954 DNA
SEQ-PCR primer SEQ ID NO: 2 955 DNA SEQ-PCR primer SEQ ID NO: 3 DNA
SEQ gapC-UP-PCR primer SEQ ID NO: 4 DNA SEQ gapC-DN SEQ ID NO: 8
DNA SEQ 951-PCR primer SEQ ID NO: 9 DNA SEQ 952-PCR primer SEQ ID
NO: 10 DNA SEQ 956-PCR primer SEQ ID NO: 12 PCR primer SEQ ID NO:
13 PCR primer SEQ ID NO: 16 PCR primer SEQ ID NO: 17 PCR primer SEQ
ID NO: 18 PCR primer SEQ ID NO: 19 PCR primer SEQ ID NO: 21 PCR
primer SEQ ID NO: 22 PCR primer
TABLE-US-00009 Sequence ID listing SEQ ID NO: 1 954 DNA SEQ
(Synthetic DNA) 5'-cgggatccatggaagaataccagcatgac-3' SEQ ID NO: 2
955 DNA SEQ (Synthetic DNA)
5'-cgggatccggtacctggatgtcttatggaccgag-3' SEQ ID NO: 3 DNA SEQ
gapC-UP (Synthetic DNA) 5'-accactaactgccttgctc-3' SEQ ID NO: 4 DNA
SEQ gapC-DN (Synthetic DNA) 5'-caatttcacaaacttgtcgctc-3' SEQ ID NO:
5 KNAT1 gene (cDNA Sequence)
5'-cgggatccatggaagaataccagcatgacaacagcaccactcctcaa
agagtaagtttcttgtactctccaatctcttcttccaacaaaaacgataa
cacaagtgataccaacaacaacaacaacaataataatagtagcaattatg
gtcctggttacaataatactaacaacaacaatcatcaccaccaacacatg
ttgtttccacatatgagctctcttctccctcaaacaaccgagaattgctt
ccgatctgatcatgatcaacccaacaacaacaacaacccatctgttaaat
ctgaagctagctcctcaagaatcaatcattactccatgttaatgagagcc
atccacaatactcaagaagctaacaacaacaacaatgacaacgtaagcga
tgttgaagccatgaaggctaaaatcattgctcatcctcactactctaccc
tcctacaagcttacttggactgccaaaagattggagctccacctgatgtg
gttgatagaattacggcggcacggcaagactttgaggctcgacaacagcg
gtcaacaccgtctgtctctgcctcctctagagacccggagttagatcaat
tcatggaagcatactgtgacatgttggttaaatatcgtgaggagctaaca
aggcccattcaggaagcaatggagtttatacgtcgtattgaatctcagct
tagcatgttgtgtcagagtcccattcacatcctcaacaatcctgatggga
agagtgacaatatgggatcatcagacgaagaacaagagaataacagcgga
ggggaaacagaattaccggaaatagacccgagggccgaagatcgggaact
caagaaccatttgctgaagaagtatagtggatacttaagcagtttgaagc
aagaactatccaagaagaaaaagaaaggtaaacttcctaaagaagcacgg
cagaagcttctcacgtggtgggagttgcattacaagtggccatatccttc
tgagtcagagaaggtagcgttggcggaatcaacggggttagatcagaaac
aaatcaacaattggttcataaaccaaagaaagcgtcactggaaaccatct
gaagacatgcagttcatggtgatggatggtctgcagcacccgcaccacgc
agctctgtacatggatggtcattacatgggtgatggaccttatcgtctcg
gtccataagacatccaggtaccggatcccg-3' SEQ ID NO: 6 the bp-2 coding
region (cDNA Sequence)
5'-cgggatccatggaagaataccagcatgacaacagctccactcctcaa
agagtaagtttcttgtactctccaatctcttcttccaacaaaaacgataa
cacaagtgataccaacaacaacaacaacaataataatagtagcaattatg
gtcctggttacaataatactaacaacaacaatcatcaccaccaacacatg
ttgtttccacatatgagctctcttctccctcaaacaaccgagaattgctt
ccgatccgatcatgatcaacccaacaacaacccatctgttaaatctgaag
ctagctcctcaagaatcaatcattactccatgttaatgagagccatccac
aatactcaagaagctaacaacaacaacaatgataacgtaagcgatgttga
agccatgaaggctaaaatcattgctcatcctcactactctaccctcctac
aagcttacttggactgccaaaagattggagctccacctgacgtggttgat
agaattacggcggcacggcaagactttgaggctcgacaatagcggtcaac
accgtctgtctctgcctcctctagagacccggagttagatcaattcatgg
aagcatactgtgacatgttggttaaatatcgtgaggagctaacaaggccc
attcaggaagcaatggagtttatacgtcgtattgaatctcagcttagcat
gttgtgtcagagtcccattcacatcctcaacaatcctgatgggaagagtg
acaatatgggatcatcagacgaagaacaagagaataacagcggaggggaa
acagaattaccggaaatagacccgagggccgaagatcgggaactcaagaa
ccatttgctgaagaagtatagtggatacttaagcagtttgaagcaagaac
tatccaagaagaaaaagaaaggtaaacttcctaaagaagcacggcagaag
cttctcacgtggtgggagttgcattacaagtggccatatccttctgagtc
agagaaggtagcgttggcggaatcaacggggttagatcagaaacaaatca
acaattggttcataaaccaaagaaagcgtcactggaaaccatctgaagac
atgcagttcatggtgatggatggtctgcagcacccgcaccacgcagctct
gtacatggatggtcattacatgggtgatggaccttatcgtctcggtccat
aagacatccaggtaccggatcccg-3' SEQ ID NO: 7 predicted bp-2 protein
(Protein Sequence)
MEEYQHDNSSTPQRVSFLYSPISSSNKNDNTSDTNNNNNNNNSSNYGPGY
NNTNNNNHHHQHMLFPHMSSLLPQTTENCFRSDHDQPNNNPSVKSEASSS
RINHYSMLMRAIHNTQEANNNNNDNVSDVEAMKAKIIAHPHYSTLLQAYL
DCQKIGAPPDVVDRITAARQDFEARQ* SEQ ID NO: 8 DNA SEQ 951 (Synthetic
DNA) 5'-cccaagcttagatctttcggtctagtgcagtgatg-3' SEQ ID NO: 9 DNA SEQ
952 (Synthetic DNA) 5'-ccggatcccagatgagtaaagatttg-3' SEQ ID NO: 10
DNA SEQ 956 (Synthetic DNA)
5'-gaagatctgtcgacgccttgtgcttgattgagactcca-3' SEQ ID NO: 11 B. napus
bp gene (BnBP) (cDNA Sequence)
5'-ggcacgagcacattagttttttatattctctctctctctctctcttt
ctctctcaacttttattcatctgggtatggaagaatatcaacatgaaagc
agatccactcctcatagagtaagtttcttgtactctccaatctcttcttc
caacaaaaatgataacaccaccaccaacaacaataataccaattatggtt
ctggttacaataatactaataacaataatcatcaacaacacatgttgttc
ccacatatgagctctcttcttcctcaaacgactgagaattgcttccgatc
cgatcatgatcagcctaccaacgcatctgttaaatcagaagcaagctcct
caagaatcaatcactactctatgttgatgaaagccatccacaatactcaa
gaaactaacaacaacaacaatgatacggaatccatgaaagctaagatcat
cgctcatccccactactccaccctcctacacgcctacttggactgccaga
agattggagcaccacctgaggtggtcgataaaattacggcggcaagacaa
gagttcgaggcgaggcagcagcggccaacagcgtccgtaactgcgctgtc
tagagacccggaattggatcaattcatggaagcatactgtgatatgctgg
ttaaatatcgagaggagctaacacggcccattgaagaagcaatggagtat
atacgtcgtattgaatctcaaattagcatgttgtgtcagggtcccattca
catcctcaacaatcctgatgggaaaagtgaaggaatagaatcatcagacg
aagagcaagataataacaacagtggaggggaagcagaattaccggaaata
gacccgagggcggaagatcgggaactcaagaatcacttgctgaagaagta
cagtggatacttgagcagtctaaagcaagaactgtccaagaaaaaaaaga
aaggtaaacttcccaaagaagcaaggcagaagcttctcacgtggtgggaa
ttgcattacaagtggccgtatccttctgaatcagagaaggtggcgttggc
ggaatcaacggggttagatcagaaacagatcaacaattggttcataaacc
aaagaaaacgtcactggaaaccgtccgaggacatgcagttcatggtgatg
gatggtctacagcacccgcaccacgcagctctatacatggatggtcatta
catgggcgatggtccttatcgtcttggaccataagagaccacatgcagat
atccagaagggttagccatataataacaaccttttgttgcctctctcgtt
tacagttcatgatttcaactttccttcacaagtttgctacctatagcttt
attttcttacccgtatttaatgtcttatatcgttcaaggggtttgagact
tcctagtcattttcactttttattttgtatttttcataatgttttattta
taatatgtgttctaataatgtgtgaaaagagatgtttttatgaattttaa
aaaaaaaaaaaaaaaaaa-3' SEQ ID NO: 12: PCR Primer (Synthetic DNA)
5'-cgggatccatggaagaatatcaacatgaa-3' SEQ ID NO: 13: PCR Primer
(Synthetic DNA) 5'-cgggatccggtaccttatggtccaagacgat-3' SEQ ID NO:
14: B. rapa bp gene (cDNA Sequence)
5'-cgggatccatggaagaatatcaacatgaaagcagatccactcctcat
agagtaagtttcttgtactctccaatctcttcttccaacaaaaatgataa
caccaccaccaacaacaataataccaattatggttctggttacaataata
ctaataacaataatcatcaacaacacatgttgttcccacatatgagctct
cttcttcctcaaacgactgagaattgcttccgatccgatcatgatcagcc
aaccaacgcatctgttaaatcagaagcaagctcctcaagaatcaatcact
actctatgttgatgaaagccatccacaatactcaagaagctaacaacaac
aacaacaacaaygatatggaatccatgaaagctaagatcatcgctcatcc
tcactactccaccctcctacacgcctacttggactgccagaagattggag
caccacctgaagtggttgataaaattacggcggcaagacaagaattcgag
gcgaggcagcagcggccaacagcgtccgtaactgcgctgtctagagaccc
cgaattggatcaattcatggaagcatactgtgatatgctggttaaatatc
gagaggagctaacacggcccattgaagaagcaatggagtatatacgtcgt
attgaatctcagattagcatgttgtgtcagggtcccattcacatcctcaa
caatcctgatgggaaaagtgaaggaatggaatcatcagacgaagagcaag
ataataacaacagtggaggggaagcagaattaccggaaatagacccgagg
gcggaagatcgggaactcaagaatcacttgctgaagaaatacagtggata
cttgagcagtctaaagcaagaactgtccaagaaaaaaaagaaaggtaaac
ttcccaaagaagcaaggcagaagcttctcacgtggtgggaattgcattac
aagtggccgtatccttctgaatcagagaaggtggcgttggcggaatcaac
ggggttagatcagaaacagatcaacaattggttcataaaccaaagaaaac
gtcactggaaaccgtccgargacatgcagttcatggtgatggatggtcta
cagcacccgcaccacgcagctctatacatggatggtcattacatgggcga
tggcccttatcgtcttggaccataaggtaccggatcccg-3' SEQ ID NO: 15: B.
oleracea bp gene (cDNA Sequence)
5'-cgggatccatggaagaatatcaacatgaaagcagatccactcctcat
agagtaagtttcttgtactctccaatctcttcttccaacaaaaatgataa
caccaccaccaacaacaataataccaattatggttctggttacaataata
ctaataacaataatcatcaacaacacatgttgttcccacatatgagctct
cttcttcctcaaacgactgagaattgcttccgatccgatcatgatcagcc
taccaacgcatctgttaaatcagaagcaagctcctcaagaatcaatcact
actctatgttgatgaaagccatccacaatactcaagaaactaacaacaac
aacaatgatacggaatccatgaaagctaagatcatcgctcatccccacta
ctccaccctcctacacgcctacttggactgccagaagattggagcaccac
ctgaggtggtcgataaaattacggcggcaagacaagagttcgaggcgagg
cagcagcggccaacagcgtccgtaactgcgctgtctagagacccggaatt
ggatcaattcatggaagcatactgtgatatgctggttaaatatcgagagg
agctaacacggcccattgaagaagcaatggagtatatacgtcgtattgaa
tctcaaattagcatgttgtgtcagggtcccattcacatcctcaacaatcc
tgatgggaaaagtgaaggaatagaatcatcagacgaagagcaagataata
acaacagtggaggggaagcagaattaccggaaatagacccgagggcggaa
gatcgggaactcaagaatcacttgctgaagaagtacagtggatacttgag
cagtctaaagcaagaactgtccaagaaaaaaaagaaaggtaaacttccca
aagaagcaaggcagaagcttctcacgtggtgggaattgcattacaagtgg
ccgtatccttctgaatcagagaaggtggcgttggcggaatcaacggggtt
agatcaaaaacagatcaacaattggttcataaaccaaagaaaacgtcact
ggaaaccgtccgaggacatgcagttcatggngatggatggtctacagcac
ccgcaccacgcagctctatacatggatggtcattacatgggcgatggtcc
ttatcgtcttggaccataaggtaccggatcccg-3' SEQ ID NO: 16: PCR Primer
(Synthetic DNA) 5'-ataacaccaccaccaacaac-3' SEQ ID NO: 17: PCR
Primer (Synthetic DNA) 5'-actaggaagtctcaaacccc-3' SEQ ID NO: 18:
PCR Primer (Synthetic DNA) 5'-tcaacatgaaagcagatccac-3' SEQ ID NO:
19: PCR Primer (Synthetic DNA) 5'-aacgagagaggcaacaaaag-3' SEQ ID
NO: 20: B. napus BP genomic fragment (Genomic DNA)
5'-ataacaccaccaccaacaacaataataccaattatggttctggttac
aataatactaataacaataatcatcaacaacacatgttgttcccacatat
gagctctcttcttcctcaaacgactgagaattgcttccgatccgatcatg
atcagccaaccaacgcatctgttaaatcagaagcaagctcctcaagaatc
aatcactactctatgttgatgaaagccatccacaatactcaagaagctaa
caacaacaacaacaacaatgatatggaatccatgaaagctaagatcatcg
ctcatccgcactactccaccctcctacacgcctacttggactgccagaag
gttatatagatttagcactggatttcgttttatttttgttgtagtaatat
ataaaataccactcttgtttgtttaaattaacgagatgatatgcgtaaat
atgttcacgggttgcatatacagattggagcaccacctgaagtggttgat
aaaattacggcggcaacacaagagttcgaggcgaggcagcagcggccaac
agcatccgtaactgcgctgtctagagaccccgaattggatcaattcatgg
taaattaattatcaaactgaattatagtgggtcgtttcttcaagtgtata
tgttaagtctttatttttgtttgtatcgtaaattttatcaacaggaagca
tactgtgatatgctggttaaatatcgagaggagctaacacggcccattga
agaagcaatggagtatatacgtcgtattgaatctcagattagcatgttgt
gtcagggtcccattcacatcctcaacaatcctggtaaatgtcataaaact
cacaaatacatatacatgcatatacccacatgtaaccattgaatgtagaa
aagaaaatataatgccaaggtagggctcatgatgaatttcaagagcaaca
ttggcgcgtatttctttggttcccgggaaagttttgtaccaattagatta
tgataaggcgaccaaaaaataattatgattatatttggttaaaatttttc
atctaaacattcaagtgttaattaagatcataaaatataatagttaatat
gatagaaattcgtaggctgcagacagatgtgcacatttgctcttgttttc
cctattgtagaatccatccaaagagggtggggctttttttggtttcttac
ttttaacccggcccaaagtactactgtcacaaacacttttgttgttcact
atgaaaaaaaatacaaataggtattctcaattccagtatgcaaaatgttt
caaattttcataaaaaagtcagtacgactaaattgctcgtgaattatgaa
tcaaaatataagactgatgaaaagctaaaatttgaaacagatgggaaaag
tgaaggaatggaatcatcagacgaagagcaagataataacaacagtggag
gggaagcagaaattaccggaaatagacccggagggcggaagatcgggaac
tcaagaatcacttgctgaagaagtacagtggatacttgagcagtctaaag
caagaactgtccaagaaaaaaaagaaaggtaaacttcccaaagaagcaag
gcagaagcttctcacgtggtgggaattgcattacaagtggccgtatcctt
ctgtacgtataattttactctcatctctctatgctttcagtcttttaaaa
tatacactctatataaatactagaaccagtcttttggaaaacaatgtaga
tgctgggaatctccaatttgccctgattttctctaaagggccttccttag
gccgattaggctctttgcagggatcatttgtagatgctaggctctttgca
gagataatttgtgttcaaacctttatgcgtttccatatttcataacatat
gtatatatacatatatcaaacacgtttttatctatagttatctaaatttt
gaaataattttgaagtttaagtccgtggatctattgttatagtttatcag
cttcaggaaataaaacaaataaaaccgaatgtggtgatggcgaaggtctt
taatattgggtatacatatttaccacaaaaaaaatgatatattatataga
atggctgtttgttgttaaaaaatcctggtattttttttggtaaatatgat
accatttccaatgaacaccaaaaatgataccatcccaccaaatttgttgt
aatgtaaaaagtattacaccaaattaacaatattcattacaccaaatatt
aaaataatatattttattattttttatttaataatagatagattagtttt
ttacttagttataacttatagttaaaatgagtatatcataatatcttgta
tttttaatccatatttttacattactaaaacattaaactattattttatt
ttataatttaattaatagtatataattaaatgagtattataaattatatt
aaatggtaacaaaataaaaatgatcttcattttaaatgcaaaaagtttta
atttttacaaatattttaaataaaataaataataaagtatacacattmac
taaaagaaaaatagcttatataaaaataaaattaccaaatattaatatat
atatatatatatatataaactaaatgtgatacatatatataattagtcaa
ttataaacaattaatgtattaaattactaaaactaaaaagttgataatat
aaaatattattttggtgtagaatttggtgtgatggttggacatgaaaaat
aaagtttaacmcttaaacmccmmtyctggtgtaatttcarcactaatttt
agtgttatggttggagataccctaacagaaccatgcttcgtgctttgaaa
aaaaaatcagtcgtctaaagctacaataaaaaaattggagggaaatattt
tgtttcaaattaggttatgtatttacacagatatttgtttggattcttgt
ctgagaagtgcatggcattacattttgtgttacaaaagaagttgaatgat
ctgagtatcatatttattgaaagcgtgttggtatatgtgtgttgctaaaa
agttctataagaaaattggataaatttgctttaaaatttccatagtatat
cactattttgtatgttcggaaaccttgatatgtatacttttcccttataa
cgagggccttaatattctttagtcatctagattgttcgaagcagcagact
gtaatttataacttcgtctgactatcatctaccttttttatagaacatac
cttttcttttattgaaactaatatcgtctagcttttgtgattaaatctac
cgtttttaaacaatgaacaatactaaaaaagtgatgatatggatatggtt
ctgatttgtgttgtgtggcaggagtcagagaaggtggcgttggcggaatc
caacggggttagatcagaaacagatcaacaattggttcataaaccaaaga
aaacgtcactggaaaccgtccgaagacatgcagttcatggtgatggatgg
tctacagcacccgcaccacgcagctctatacatggatggtcattacatgg
gcgatggtccttatcgtcttggaccataagagaccgcatgcagatatcca
gaagggttagccatataataacaaccttttgttgcctctctcgtttacag
ttcatgatttcaactttccttcacaagtttgctacctatagctttatttt
cttacccgtatttaatgtcttatatcgttcaaggggtttgagacttccta gt-3' SEQ ID NO:
21: PCR Primer (Synthetic DNA) 5'-catgatcggatcggaagcaattctcagtcg-3'
SEQ ID NO: 22: PCR Primer (Synthetic DNA)
5'-aaaagttgagagagaaagagagagagagag-3' SEQ ID NO: 23: BnBP promoter
(Bnbppr 900) (Genomic DNA)
5'-aaaaaatgcttacaaatatctgcacatcaaccaatctgttacataaa
tagatcttcttgtgggggtagggttaacaaatattttcctctttttcttt
tctcaaaaatgtatcggtactgatatagccgcggagacctggttcattaa
aacattggcggtacatcttaataatcaaaacattgacggcacatcttaat
cctagagtttaaccacattatatatcatagagtaacaaacttagtttttg
acccaaaagaagaaaaaaaacttccaattttctagtacagaataagccta
cgagagggaaacagaagagaaaggaggaaagaagggaagcctttgcctta
tctcttgtccattctctcttacctttatttttaattttcaaatatttatt
attgccaccaaagcaaacgacgtcttgtcaatccactcaacccacccaac
ttcttaattattgttaacacatctctcctctttctctctcatctttttat
aatttcttctcttccatgtcactttttgacgaattctamacttagttcgt
tttttcttcctcaaaatatctcgttttcaatttatttgttttgttgggtg
caacttcacctcacaattttttttatgaagcacctttctgattcgtagat
atgagtcgtctagtcatgggatttgatttggttaaagtctaacatcgacc
tttgattgaaataaggacaaaaagaaagaatacatacatccccttcattt
tgcacccatccctttattttctagggttttatttttatcacattagtttt
ttatattctctctctctctctctctttctctctcaactttt-3' SEQ ID NO: 24: BnBP
promoter (Bnbppr 1000) (Genomic DNA)
5'-aaatctttatcttctctgtttcttgtgcaatcttctatccgaaaacg
agtacaatataatctctctccaccgatgtaatacgaatatcaaatcagaa
attaatcatttgatcatattctcaaaacatctaaatttattttacaaatt
gcttacaaatatctgcacatcaaccaatctgttacataaatagatcttct
tgtaggggtaaggttaacaaatatttttttctttttcttttctccaaaat
gtatcggtactgatatagccgcggagacctggttcatcaaaacattgacg
gtacatcttaattcgagagtttaaccaaattatatcatagagtaacaaac
ttagtttttgacccaaaataagagaaaaaactttcaattttctaatacgg
aataagctatgagagggagacagaagagaaagtaggaaagaagggaagcc
tttgccttatctcttgtccattctctcttacctttattttaattttcaaa
tatttattattgccaccaaagcaaacgacgtcttgtcaatccactcaacc
cacccaacttcttaattattgttaacacatctctcctctttctctctcat
ctttttataatttcttctcttccatgtcactttttgacgaattctattta
cttagttcgttttttcttcctcaaaatatctcgttttcaatttatttgtt
ttgttgggtgcaacttcacctcacaattttttttatgaagcacctttctg
attcgtagatatgagtcgtctagtcatgtggatttgatttggttaaagtc
taacatcgacctttgattgaaataagaacaaaagaaagaatacatacatc
cccttcattttgcacccatccctttattttctagggttttatttttatca
cattagttttttatattctctctctctctctctctctttctctctcaact ttt-3'
Sequence CWU 1
1
24129DNAArtificial954 DNA SEQ - PCR Primer 1cgggatccat ggaagaatac
cagcatgac 29234DNAArtificial955 DNA SEQ - PCR Primer 2cgggatccgg
tacctggatg tcttatggac cgag 34319DNAArtificialDNA SEQ gapC-UP - PCR
Primer 3accactaact gccttgctc 19422DNAArtificialDNA SEQ gapC-DN
4caatttcaca aacttgtcgc tc 2251227DNAArabidopsis sp. 5cgggatccat
ggaagaatac cagcatgaca acagcaccac tcctcaaaga gtaagtttct 60tgtactctcc
aatctcttct tccaacaaaa acgataacac aagtgatacc aacaacaaca
120acaacaataa taatagtagc aattatggtc ctggttacaa taatactaac
aacaacaatc 180atcaccacca acacatgttg tttccacata tgagctctct
tctccctcaa acaaccgaga 240attgcttccg atctgatcat gatcaaccca
acaacaacaa caacccatct gttaaatctg 300aagctagctc ctcaagaatc
aatcattact ccatgttaat gagagccatc cacaatactc 360aagaagctaa
caacaacaac aatgacaacg taagcgatgt tgaagccatg aaggctaaaa
420tcattgctca tcctcactac tctaccctcc tacaagctta cttggactgc
caaaagattg 480gagctccacc tgatgtggtt gatagaatta cggcggcacg
gcaagacttt gaggctcgac 540aacagcggtc aacaccgtct gtctctgcct
cctctagaga cccggagtta gatcaattca 600tggaagcata ctgtgacatg
ttggttaaat atcgtgagga gctaacaagg cccattcagg 660aagcaatgga
gtttatacgt cgtattgaat ctcagcttag catgttgtgt cagagtccca
720ttcacatcct caacaatcct gatgggaaga gtgacaatat gggatcatca
gacgaagaac 780aagagaataa cagcggaggg gaaacagaat taccggaaat
agacccgagg gccgaagatc 840gggaactcaa gaaccatttg ctgaagaagt
atagtggata cttaagcagt ttgaagcaag 900aactatccaa gaagaaaaag
aaaggtaaac ttcctaaaga agcacggcag aagcttctca 960cgtggtggga
gttgcattac aagtggccat atccttctga gtcagagaag gtagcgttgg
1020cggaatcaac ggggttagat cagaaacaaa tcaacaattg gttcataaac
caaagaaagc 1080gtcactggaa accatctgaa gacatgcagt tcatggtgat
ggatggtctg cagcacccgc 1140accacgcagc tctgtacatg gatggtcatt
acatgggtga tggaccttat cgtctcggtc 1200cataagacat ccaggtaccg gatcccg
122761221DNAArabidopsis sp. 6cgggatccat ggaagaatac cagcatgaca
acagctccac tcctcaaaga gtaagtttct 60tgtactctcc aatctcttct tccaacaaaa
acgataacac aagtgatacc aacaacaaca 120acaacaataa taatagtagc
aattatggtc ctggttacaa taatactaac aacaacaatc 180atcaccacca
acacatgttg tttccacata tgagctctct tctccctcaa acaaccgaga
240attgcttccg atccgatcat gatcaaccca acaacaaccc atctgttaaa
tctgaagcta 300gctcctcaag aatcaatcat tactccatgt taatgagagc
catccacaat actcaagaag 360ctaacaacaa caacaatgat aacgtaagcg
atgttgaagc catgaaggct aaaatcattg 420ctcatcctca ctactctacc
ctcctacaag cttacttgga ctgccaaaag attggagctc 480cacctgacgt
ggttgataga attacggcgg cacggcaaga ctttgaggct cgacaatagc
540ggtcaacacc gtctgtctct gcctcctcta gagacccgga gttagatcaa
ttcatggaag 600catactgtga catgttggtt aaatatcgtg aggagctaac
aaggcccatt caggaagcaa 660tggagtttat acgtcgtatt gaatctcagc
ttagcatgtt gtgtcagagt cccattcaca 720tcctcaacaa tcctgatggg
aagagtgaca atatgggatc atcagacgaa gaacaagaga 780ataacagcgg
aggggaaaca gaattaccgg aaatagaccc gagggccgaa gatcgggaac
840tcaagaacca tttgctgaag aagtatagtg gatacttaag cagtttgaag
caagaactat 900ccaagaagaa aaagaaaggt aaacttccta aagaagcacg
gcagaagctt ctcacgtggt 960gggagttgca ttacaagtgg ccatatcctt
ctgagtcaga gaaggtagcg ttggcggaat 1020caacggggtt agatcagaaa
caaatcaaca attggttcat aaaccaaaga aagcgtcact 1080ggaaaccatc
tgaagacatg cagttcatgg tgatggatgg tctgcagcac ccgcaccacg
1140cagctctgta catggatggt cattacatgg gtgatggacc ttatcgtctc
ggtccataag 1200acatccaggt accggatccc g 12217176PRTArabidopsis sp.
7Met Glu Glu Tyr Gln His Asp Asn Ser Ser Thr Pro Gln Arg Val Ser1 5
10 15Phe Leu Tyr Ser Pro Ile Ser Ser Ser Asn Lys Asn Asp Asn Thr
Ser20 25 30Asp Thr Asn Asn Asn Asn Asn Asn Asn Asn Ser Ser Asn Tyr
Gly Pro35 40 45Gly Tyr Asn Asn Thr Asn Asn Asn Asn His His His Gln
His Met Leu50 55 60Phe Pro His Met Ser Ser Leu Leu Pro Gln Thr Thr
Glu Asn Cys Phe65 70 75 80Arg Ser Asp His Asp Gln Pro Asn Asn Asn
Pro Ser Val Lys Ser Glu85 90 95Ala Ser Ser Ser Arg Ile Asn His Tyr
Ser Met Leu Met Arg Ala Ile100 105 110His Asn Thr Gln Glu Ala Asn
Asn Asn Asn Asn Asp Asn Val Ser Asp115 120 125Val Glu Ala Met Lys
Ala Lys Ile Ile Ala His Pro His Tyr Ser Thr130 135 140Leu Leu Gln
Ala Tyr Leu Asp Cys Gln Lys Ile Gly Ala Pro Pro Asp145 150 155
160Val Val Asp Arg Ile Thr Ala Ala Arg Gln Asp Phe Glu Ala Arg
Gln165 170 175835DNAArtificialDNA SEQ 951 - PCR Primer 8cccaagctta
gatctttcgg tctagtgcag tgatg 35926DNAArtificialDNA SEQ 952 - PCR
Primer 9ccggatccca gatgagtaaa gatttg 261038DNAArtificialDNA SEQ 956
- PCR Primer 10gaagatctgt cgacgccttg tgcttgattg agactcca
38111515DNABrassica napus 11ggcacgagca cattagtttt ttatattctc
tctctctctc tctctttctc tctcaacttt 60tattcatctg ggtatggaag aatatcaaca
tgaaagcaga tccactcctc atagagtaag 120tttcttgtac tctccaatct
cttcttccaa caaaaatgat aacaccacca ccaacaacaa 180taataccaat
tatggttctg gttacaataa tactaataac aataatcatc aacaacacat
240gttgttccca catatgagct ctcttcttcc tcaaacgact gagaattgct
tccgatccga 300tcatgatcag cctaccaacg catctgttaa atcagaagca
agctcctcaa gaatcaatca 360ctactctatg ttgatgaaag ccatccacaa
tactcaagaa actaacaaca acaacaatga 420tacggaatcc atgaaagcta
agatcatcgc tcatccccac tactccaccc tcctacacgc 480ctacttggac
tgccagaaga ttggagcacc acctgaggtg gtcgataaaa ttacggcggc
540aagacaagag ttcgaggcga ggcagcagcg gccaacagcg tccgtaactg
cgctgtctag 600agacccggaa ttggatcaat tcatggaagc atactgtgat
atgctggtta aatatcgaga 660ggagctaaca cggcccattg aagaagcaat
ggagtatata cgtcgtattg aatctcaaat 720tagcatgttg tgtcagggtc
ccattcacat cctcaacaat cctgatggga aaagtgaagg 780aatagaatca
tcagacgaag agcaagataa taacaacagt ggaggggaag cagaattacc
840ggaaatagac ccgagggcgg aagatcggga actcaagaat cacttgctga
agaagtacag 900tggatacttg agcagtctaa agcaagaact gtccaagaaa
aaaaagaaag gtaaacttcc 960caaagaagca aggcagaagc ttctcacgtg
gtgggaattg cattacaagt ggccgtatcc 1020ttctgaatca gagaaggtgg
cgttggcgga atcaacgggg ttagatcaga aacagatcaa 1080caattggttc
ataaaccaaa gaaaacgtca ctggaaaccg tccgaggaca tgcagttcat
1140ggtgatggat ggtctacagc acccgcacca cgcagctcta tacatggatg
gtcattacat 1200gggcgatggt ccttatcgtc ttggaccata agagaccaca
tgcagatatc cagaagggtt 1260agccatataa taacaacctt ttgttgcctc
tctcgtttac agttcatgat ttcaactttc 1320cttcacaagt ttgctaccta
tagctttatt ttcttacccg tatttaatgt cttatatcgt 1380tcaaggggtt
tgagacttcc tagtcatttt cactttttat tttgtatttt tcataatgtt
1440ttatttataa tatgtgttct aataatgtgt gaaaagagat gtttttatga
attttaaaaa 1500aaaaaaaaaa aaaaa 15151229DNAArtificialPCR Primer
12cgggatccat ggaagaatat caacatgaa 291331DNAArtificialPCR Primer
13cgggatccgg taccttatgg tccaagacga t 31141186DNABrassica rapa
14cgggatccat ggaagaatat caacatgaaa gcagatccac tcctcataga gtaagtttct
60tgtactctcc aatctcttct tccaacaaaa atgataacac caccaccaac aacaataata
120ccaattatgg ttctggttac aataatacta ataacaataa tcatcaacaa
cacatgttgt 180tcccacatat gagctctctt cttcctcaaa cgactgagaa
ttgcttccga tccgatcatg 240atcagccaac caacgcatct gttaaatcag
aagcaagctc ctcaagaatc aatcactact 300ctatgttgat gaaagccatc
cacaatactc aagaagctaa caacaacaac aacaacaayg 360atatggaatc
catgaaagct aagatcatcg ctcatcctca ctactccacc ctcctacacg
420cctacttgga ctgccagaag attggagcac cacctgaagt ggttgataaa
attacggcgg 480caagacaaga attcgaggcg aggcagcagc ggccaacagc
gtccgtaact gcgctgtcta 540gagaccccga attggatcaa ttcatggaag
catactgtga tatgctggtt aaatatcgag 600aggagctaac acggcccatt
gaagaagcaa tggagtatat acgtcgtatt gaatctcaga 660ttagcatgtt
gtgtcagggt cccattcaca tcctcaacaa tcctgatggg aaaagtgaag
720gaatggaatc atcagacgaa gagcaagata ataacaacag tggaggggaa
gcagaattac 780cggaaataga cccgagggcg gaagatcggg aactcaagaa
tcacttgctg aagaaataca 840gtggatactt gagcagtcta aagcaagaac
tgtccaagaa aaaaaagaaa ggtaaacttc 900ccaaagaagc aaggcagaag
cttctcacgt ggtgggaatt gcattacaag tggccgtatc 960cttctgaatc
agagaaggtg gcgttggcgg aatcaacggg gttagatcag aaacagatca
1020acaattggtt cataaaccaa agaaaacgtc actggaaacc gtccgargac
atgcagttca 1080tggtgatgga tggtctacag cacccgcacc acgcagctct
atacatggat ggtcattaca 1140tgggcgatgg cccttatcgt cttggaccat
aaggtaccgg atcccg 1186151180DNABrassica
oleraceamisc_feature(1078)..(1078)n = a, c, g, or t 15cgggatccat
ggaagaatat caacatgaaa gcagatccac tcctcataga gtaagtttct 60tgtactctcc
aatctcttct tccaacaaaa atgataacac caccaccaac aacaataata
120ccaattatgg ttctggttac aataatacta ataacaataa tcatcaacaa
cacatgttgt 180tcccacatat gagctctctt cttcctcaaa cgactgagaa
ttgcttccga tccgatcatg 240atcagcctac caacgcatct gttaaatcag
aagcaagctc ctcaagaatc aatcactact 300ctatgttgat gaaagccatc
cacaatactc aagaaactaa caacaacaac aatgatacgg 360aatccatgaa
agctaagatc atcgctcatc cccactactc caccctccta cacgcctact
420tggactgcca gaagattgga gcaccacctg aggtggtcga taaaattacg
gcggcaagac 480aagagttcga ggcgaggcag cagcggccaa cagcgtccgt
aactgcgctg tctagagacc 540cggaattgga tcaattcatg gaagcatact
gtgatatgct ggttaaatat cgagaggagc 600taacacggcc cattgaagaa
gcaatggagt atatacgtcg tattgaatct caaattagca 660tgttgtgtca
gggtcccatt cacatcctca acaatcctga tgggaaaagt gaaggaatag
720aatcatcaga cgaagagcaa gataataaca acagtggagg ggaagcagaa
ttaccggaaa 780tagacccgag ggcggaagat cgggaactca agaatcactt
gctgaagaag tacagtggat 840acttgagcag tctaaagcaa gaactgtcca
agaaaaaaaa gaaaggtaaa cttcccaaag 900aagcaaggca gaagcttctc
acgtggtggg aattgcatta caagtggccg tatccttctg 960aatcagagaa
ggtggcgttg gcggaatcaa cggggttaga tcaaaaacag atcaacaatt
1020ggttcataaa ccaaagaaaa cgtcactgga aaccgtccga ggacatgcag
ttcatggnga 1080tggatggtct acagcacccg caccacgcag ctctatacat
ggatggtcat tacatgggcg 1140atggtcctta tcgtcttgga ccataaggta
ccggatcccg 11801620DNAArtificialPCR Primer 16ataacaccac caccaacaac
201720DNAArtificialPCR Primer 17actaggaagt ctcaaacccc
201821DNAArtificialPCR Primee 18tcaacatgaa agcagatcca c
211920DNAArtificialPCR Primer 19aacgagagag gcaacaaaag
20203750DNABrassica napus 20ataacaccac caccaacaac aataatacca
attatggttc tggttacaat aatactaata 60acaataatca tcaacaacac atgttgttcc
cacatatgag ctctcttctt cctcaaacga 120ctgagaattg cttccgatcc
gatcatgatc agccaaccaa cgcatctgtt aaatcagaag 180caagctcctc
aagaatcaat cactactcta tgttgatgaa agccatccac aatactcaag
240aagctaacaa caacaacaac aacaatgata tggaatccat gaaagctaag
atcatcgctc 300atccgcacta ctccaccctc ctacacgcct acttggactg
ccagaaggtt atatagattt 360agcactggat ttcgttttat ttttgttgta
gtaatatata aaataccact cttgtttgtt 420taaattaacg agatgatatg
cgtaaatatg ttcacgggtt gcatatacag attggagcac 480cacctgaagt
ggttgataaa attacggcgg caacacaaga gttcgaggcg aggcagcagc
540ggccaacagc atccgtaact gcgctgtcta gagaccccga attggatcaa
ttcatggtaa 600attaattatc aaactgaatt atagtgggtc gtttcttcaa
gtgtatatgt taagtcttta 660tttttgtttg tatcgtaaat tttatcaaca
ggaagcatac tgtgatatgc tggttaaata 720tcgagaggag ctaacacggc
ccattgaaga agcaatggag tatatacgtc gtattgaatc 780tcagattagc
atgttgtgtc agggtcccat tcacatcctc aacaatcctg gtaaatgtca
840taaaactcac aaatacatat acatgcatat acccacatgt aaccattgaa
tgtagaaaag 900aaaatataat gccaaggtag ggctcatgat gaatttcaag
agcaacattg gcgcgtattt 960ctttggttcc cgggaaagtt ttgtaccaat
tagattatga taaggcgacc aaaaaataat 1020tatgattata tttggttaaa
atttttcatc taaacattca agtgttaatt aagatcataa 1080aatataatag
ttaatatgat agaaattcgt aggctgcaga cagatgtgca catttgctct
1140tgttttccct attgtagaat ccatccaaag agggtggggc tttttttggt
ttcttacttt 1200taacccggcc caaagtacta ctgtcacaaa cactttttgt
tgttcactat gaaaaaaaat 1260acaaataggt attctcaatt ccagtatgca
aaatgtttca aattttcata aaaaagtcag 1320tacgactaaa ttgctcgtga
attatgaatc aaaatataag actgatgaaa agctaaaatt 1380tgaaacagat
gggaaaagtg aaggaatgga atcatcagac gaagagcaag ataataacaa
1440cagtggaggg gaagcagaaa ttaccggaaa tagacccgga gggcggaaga
tcgggaactc 1500aagaatcact tgctgaagaa gtacagtgga tacttgagca
gtctaaagca agaactgtcc 1560aagaaaaaaa agaaaggtaa acttcccaaa
gaagcaaggc agaagcttct cacgtggtgg 1620gaattgcatt acaagtggcc
gtatccttct gtacgtataa ttttactctc atctctctat 1680gctttcagtc
ttttaaaata tacactctat ataaatacta gaaccagtct tttggaaaac
1740aatgtagatg ctgggaatct ccaatttgcc ctgattttct ctaaagggcc
ttccttaggc 1800cgattaggct ctttgcaggg atcatttgta gatgctaggc
tctttgcaga gataatttgt 1860gttcaaacct ttatgcgttt ccatatttca
taacatatgt atatatacat atatcaaaca 1920cgtttttatc tatagttatc
taaattttga aataattttg aagtttaagt ccgtggatct 1980attgttatag
tttatcagct tcaggaaata aaacaaataa aaccgaatgt ggtgatggcg
2040aaggtcttta atattgggta tacatattta ccacaaaaaa aatgatatat
tatatagaat 2100ggctgtttgt tgttaaaaaa tcctggtatt ttttttggta
aatatgatac catttccaat 2160gaacaccaaa aatgatacca tcccaccaaa
tttgttgtaa tgtaaaaagt attacaccaa 2220attaacaata ttcattacac
caaatattaa aataatatat tttattattt tttatttaat 2280aatagataga
ttagtttttt acttagttat aacttatagt taaaatgagt atatcataat
2340atcttgtatt tttaatccat atttttacat tactaaaaca ttaaactatt
attttatttt 2400ataatttaat taatagtata taattaaatg agtattataa
attatattaa atggtaacaa 2460aataaaaatg atcttcattt taaatgcaaa
aagttttaat ttttacaaat attttaaata 2520aaataaataa taaagtatac
acattmacta aaagaaaaat agcttatata aaaataaaat 2580taccaaatat
taatatatat atatatatat atataaacta aatgtgatac atatatataa
2640ttagtcaatt ataaacaatt aatgtattaa attactaaaa ctaaaaagtt
gataatataa 2700aatattattt tggtgtagaa tttggtgtga tggttggaca
tgaaaaataa agtttaacmc 2760ttaaacmccm mtyctggtgt aatttcarca
ctaattttag tgttatggtt ggagataccc 2820taacagaacc atgcttcgtg
ctttgaaaaa aaaatcagtc gtctaaagct acaataaaaa 2880aattggaggg
aaatattttg tttcaaatta ggttatgtat ttacacagat atttgtttgg
2940attcttgtct gagaagtgca tggcattaca ttttgtgtta caaaagaagt
tgaatgatct 3000gagtatcata tttattgaaa gcgtgttggt atatgtgtgt
tgctaaaaag ttctataaga 3060aaattggata aatttgcttt aaaatttcca
tagtatatca ctattttgta tgttcggaaa 3120ccttgatatg tatacttttc
ccttataacg agggccttaa tattctttag tcatctagat 3180tgttcgaagc
agcagactgt aatttataac ttcgtctgac tatcatctac cttttttata
3240gaacatacct tttcttttat tgaaactaat atcgtctagc ttttgtgatt
aaatctaccg 3300tttttaaaca atgaacaata ctaaaaaagt gatgatatgg
atatggttct gatttgtgtt 3360gtgtggcagg agtcagagaa ggtggcgttg
gcggaatcca acggggttag atcagaaaca 3420gatcaacaat tggttcataa
accaaagaaa acgtcactgg aaaccgtccg aagacatgca 3480gttcatggtg
atggatggtc tacagcaccc gcaccacgca gctctataca tggatggtca
3540ttacatgggc gatggtcctt atcgtcttgg accataagag accgcatgca
gatatccaga 3600agggttagcc atataataac aaccttttgt tgcctctctc
gtttacagtt catgatttca 3660actttccttc acaagtttgc tacctatagc
tttattttct tacccgtatt taatgtctta 3720tatcgttcaa ggggtttgag
acttcctagt 37502130DNAArtificialPCR Primer 21catgatcgga tcggaagcaa
ttctcagtcg 302230DNAArtificialPCR Primer 22aaaagttgag agagaaagag
agagagagag 3023840DNABrassica napus 23aaaaaatgct tacaaatatc
tgcacatcaa ccaatctgtt acataaatag atcttcttgt 60gggggtaggg ttaacaaata
ttttcctctt tttcttttct caaaaatgta tcggtactga 120tatagccgcg
gagacctggt tcattaaaac attggcggta catcttaata atcaaaacat
180tgacggcaca tcttaatcct agagtttaac cacattatat atcatagagt
aacaaactta 240gtttttgacc caaaagaaga aaaaaaactt ccaattttct
agtacagaat aagcctacga 300gagggaaaca gaagagaaag gaggaaagaa
gggaagcctt tgccttatct cttgtccatt 360ctctcttacc tttattttta
attttcaaat atttattatt gccaccaaag caaacgacgt 420cttgtcaatc
cactcaaccc acccaacttc ttaattattg ttaacacatc tctcctcttt
480ctctctcatc tttttataat ttcttctctt ccatgtcact ttttgacgaa
ttctatttac 540ttagttcgtt ttttcttcct caaaatatct cgttttcaat
ttatttgttt tgttgggtgc 600aacttcacct cacaattttt tttatgaagc
acctttctga ttcgtagata tgagtcgtct 660agtcatggga tttgatttgg
ttaaagtcta acatcgacct ttgattgaaa taaggacaaa 720aagaaagaat
acatacatcc ccttcatttt gcacccatcc ctttattttc tagggtttta
780tttttatcac attagttttt tatattctct ctctctctct ctctttctct
ctcaactttt 84024950DNABrassica napus 24aaatctttat cttctctgtt
tcttgtgcaa tcttctatcc gaaaacgagt acaatataat 60ctctctccac cgatgtaata
cgaatatcaa atcagaaatt aatcatttga tcatattctc 120aaaacatcta
aatttatttt acaaattgct tacaaatatc tgcacatcaa ccaatctgtt
180acataaatag atcttcttgt aggggtaagg ttaacaaata tttttttctt
tttcttttct 240ccaaaatgta tcggtactga tatagccgcg gagacctggt
tcatcaaaac attgacggta 300catcttaatt cgagagttta accaaattat
atcatagagt aacaaactta gtttttgacc 360caaaataaga gaaaaaactt
tcaattttct aatacggaat aagctatgag agggagacag 420aagagaaagt
aggaaagaag ggaagccttt gccttatctc ttgtccattc tctcttacct
480ttattttaat tttcaaatat ttattattgc caccaaagca aacgacgtct
tgtcaatcca 540ctcaacccac ccaacttctt aattattgtt aacacatctc
tcctctttct ctctcatctt 600tttataattt cttctcttcc atgtcacttt
ttgacgaatt ctatttactt agttcgtttt 660ttcttcctca aaatatctcg
ttttcaattt atttgttttg ttgggtgcaa cttcacctca 720caattttttt
tatgaagcac ctttctgatt cgtagatatg agtcgtctag tcatgtggat
780ttgatttggt taaagtctaa catcgacctt tgattgaaat aagaacaaaa
gaaagaatac 840atacatcccc ttcattttgc acccatccct ttattttcta
gggttttatt tttatcacat 900tagtttttta tattctctct ctctctctct
ctctttctct ctcaactttt 950
* * * * *
References