U.S. patent application number 14/163400 was filed with the patent office on 2014-06-26 for molecular markers of plant embryogenesis.
This patent application is currently assigned to MALAYSIAN PALM OIL BOARD. The applicant listed for this patent is MALAYSIAN PALM OIL BOARD. Invention is credited to Meilina Ong ABDULLAH, Harikrishna KULAVEERASINGAM.
Application Number | 20140182017 14/163400 |
Document ID | / |
Family ID | 3826247 |
Filed Date | 2014-06-26 |
United States Patent
Application |
20140182017 |
Kind Code |
A1 |
ABDULLAH; Meilina Ong ; et
al. |
June 26, 2014 |
MOLECULAR MARKERS OF PLANT EMBRYOGENESIS
Abstract
The present invention relates generally to a molecular marker
for a plant physiological process and more particularly for plant
embryogenesis. The molecular marker is, in one form, a genetic
sequence from a monocot plant such as but not limited to oil-palm
plants. In another form, the molecular marker is a polypeptide
encoded by said genetic sequence. More particularly, the molecular
marker of the present invention enables embryogenic tissue to be
detected in vitro. The early detection of embryogenic tissue
enables non-embryogenic tissue to be discarded. The ability to
detect embryogenesis facilitates maximization of embryogenic
potential. The present invention further contemplates a molecular
marker comprising in one form a sequence of nucleotides encoding an
antioxidant or in another form a sequence of amino acids defining a
polypeptide having antioxidant activity. The antioxidant according
to this aspect of the present invention is particularly useful in
tablet or cream form as an anti-aging agent. The molecular markers
of the present invention therefore also have uses in the inhibition
or retardation of apoptotic processes. Such an effect has benefits
in both plant and animal cells. The present invention further
contemplates a promoter sequence encoding the molecular marker and
its use in generating male sterile plants.
Inventors: |
ABDULLAH; Meilina Ong;
(Kajang Selangor, MY) ; KULAVEERASINGAM; Harikrishna;
(Kajan, MY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MALAYSIAN PALM OIL BOARD |
Kajang Selangor |
|
MY |
|
|
Assignee: |
MALAYSIAN PALM OIL BOARD
KAJANG SELANGOR
MY
|
Family ID: |
3826247 |
Appl. No.: |
14/163400 |
Filed: |
January 24, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12788725 |
May 27, 2010 |
8673549 |
|
|
14163400 |
|
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|
10028346 |
Dec 20, 2001 |
7745111 |
|
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12788725 |
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Current U.S.
Class: |
800/298 ;
435/320.1; 435/419; 435/468; 435/6.12; 435/7.92; 436/501; 436/94;
514/21.2; 530/370; 530/387.9; 536/23.6 |
Current CPC
Class: |
C12N 15/8287 20130101;
C12Q 1/6895 20130101; G01N 33/53 20130101; C12Q 2600/158 20130101;
C07K 14/415 20130101; C12N 9/0065 20130101; Y10T 436/143333
20150115; C07K 2299/00 20130101; A01K 2217/05 20130101; A61K 38/00
20130101; A61P 39/06 20180101 |
Class at
Publication: |
800/298 ;
536/23.6; 435/320.1; 435/419; 530/370; 435/468; 435/6.12;
530/387.9; 436/501; 514/21.2; 436/94; 435/7.92 |
International
Class: |
C07K 14/415 20060101
C07K014/415; G01N 33/53 20060101 G01N033/53; C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 20, 2000 |
AU |
PR 2213/00 |
Claims
1. An isolated nucleic acid molecule encoding a polypeptide
comprising an amino acid sequence substantially as set forth in SEQ
ID NO:2 or an amino acid sequence having at least about 71%
similarity to SEQ ID NO:2, wherein said polypeptide is present in
plant zygotic embryos or embryogenic callus and is substantially
not present in non-embryogenic tissue.
2. An isolated nucleic acid molecule of claim 1, wherein said
nucleic acid molecule comprises a sequence of nucleotides
substantially as set forth in SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form, or a nucleotide sequence having at least about
71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its complementary
form or a nucleotide sequence capable of hybridizing to SEQ ID NO:1
or SEQ ID NO:3 or its complementary form under low stringency
conditions.
3. An isolated nucleic acid molecule of claim 1, wherein the
nucleic acid molecule is developmentally regulated.
4. An isolated nucleic acid molecule of claim 1, 2 or 3, wherein
the nucleic acid molecule is expressed substantially in embryogenic
material of oil-palm plants or related plants but not in
non-embryogenic material.
5. An isolated nucleic acid molecule of claim 1, wherein the
nucleic acid molecule comprises the nucleotide sequence
substantially as set forth in SEQ ID NO:1 or SEQ ID NO:3.
6. An isolated nucleic acid molecule of claim 1, wherein the
nucleic acid molecule comprises the nucleotide sequence
substantially as set forth in SEQ ID NO:1.
7. An isolated nucleic acid molecule of claim 1, wherein the
nucleic acid molecule comprises the nucleotide sequence
substantially as set forth in SEQ ID NO:3.
8. A genetic construct comprising a nucleic acid molecule encoding
a polypeptide comprising an amino acid sequence substantially as
set forth in SEQ ID NO:2 or an amino acid sequence having at least
about 71% similarity to SEQ ID NO:2, wherein said polypeptide is
present in plant zygotic embryos or embryogenic callus and is
substantially not present in non-embryogenic tissue.
9. A genetic construct of claim 8, wherein the nucleic acid
molecule is substantially as set forth in SEQ ID NO:1 or SEQ ID
NO:3 or its complementary form, or a nucleotide sequence having at
least about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form or a nucleotide sequence capable of hybridizing
to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form under low
stringency conditions.
10. A genetic construct of claim 8, wherein the nucleic acid
molecule is developmentally regulated.
11. A genetic construct of claim 8, 9 or 10, wherein the nucleic
acid molecule is expressed substantially in embryogenic material of
oil-palm plants or related plants but not in non-embryogenic
material.
12. A genetic construct of claim 8, wherein the nucleic acid
molecule comprises a sequence of nucleotides substantially as set
forth in SEQ ID NO:1 or SEQ ID NO:3.
13. A genetic construct of claim 12, wherein the nucleic acid
molecule comprises a sequence of nucleotides substantially as set
forth in SEQ ID NO:1.
14. A genetic construct of claim 12, wherein the nucleic acid
molecule comprises a sequence of nucleotides substantially as set
forth in SEQ ID NO:3.
15. A genetic construct of claim 8 or 9, wherein said construct
further comprises one or more promoter sequences or transcription
termination sequences.
16. A genetic construct of claim 15, wherein said construct further
comprises one or more origins of replication and/or selectable
marker gene sequences.
17. A vector comprising a construct of any one of claims 8 to
16.
18. A host cell comprising a nucleic acid molecule encoding a
polypeptide comprising an amino acid sequence substantially as set
forth in SEQ ID NO:2 or an amino acid sequence having at least
about 71% similarity to SEQ ID NO:2, wherein said polypeptide is
present in plant zygotic embryos or embryogenic callus and is
substantially not present in non-embryogenic tissue.
19. A host cell of claim 18, wherein said nucleic acid molecule
comprises a sequence of nucleotides substantially as set forth in
SEQ ID NO:1 or SEQ ID NO:3 or its complementary form, or a
nucleotide sequence having at least about 71% similarity to SEQ ID
NO:1 or SEQ ID NO:3 or its complementary form or a nucleotide
sequence capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3 or
its complementary form under low stringency conditions.
20. A host cell of claim 18, wherein the nucleic acid molecule is
developmentally regulated.
21. A host cell of claim 18, 19 or 20 wherein the nucleic acid
molecule is expressed substantially in embryogenic material of
oil-palm plants or related plants but not in non-embryogenic
material.
22. A host cell of claim 18, wherein the nucleic acid molecule
comprises the nucleotide sequence substantially as set forth in SEQ
ID NO:1 or SEQ ID NO:3.
23. A host cell of claim 18, wherein the nucleic acid molecule
comprises the nucleotide sequence substantially as set forth in SEQ
ID NO:1.
24. A host cell of claim 18, wherein the nucleic acid molecule
comprises the nucleotide sequence substantially as set forth in SEQ
ID NO:3.
25. A host cell of claim 18, wherein the cell is a plant cell.
26. A plant cell of claim 25, wherein the cell is from an oil-palm
plant.
27. An isolated polypeptide or biologically-active fragment thereof
or a variant or derivative of these, said polypeptide comprising
the amino acid sequence set forth in SEQ ID NO:2 or an amino acid
sequence having at least about 71% similarity to SEQ ID NO:2,
wherein said polypeptide is present in plant zygotic embryos or
embryogenic callus and is substantially not present in
non-embryogenic tissue.
28. An isolated polypeptide of claim 27, wherein the amino acid
sequence is encoded by the nucleotide sequence substantially as set
forth in SEQ ID NO:1 or SEQ ID NO:3 or a nucleotide sequence having
at least about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form or a nucleotide sequence capable of hybridizing
to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form under low
stringency conditions.
29. An isolated polypeptide of claim 27, wherein the amino acid
sequence is expressed substantially in embryogenic material of
oil-palm plants or related plants but not in non-embryogenic
material.
30. An isolated polypeptide of claim 27 or 28, wherein the
nucleotide sequence is substantially as set forth in SEQ ID
NO:1.
31. An isolated polypeptide of claim 27 or 28, wherein the
nucleotide sequence is substantially as set forth in SEQ ID
NO:3.
32. An isolated polypeptide of any one of claims 27 to 31, wherein
the polypeptide has antioxidant properties.
33. An isolated polypeptide of claim 32, wherein the polypeptide is
peroxiredoxin.
34. A method for producing a recombinant polypeptide in a host cell
or tissue, said method comprising introducing into the said cell or
tissue an expression vector comprising a nucleic acid molecule
wherein said nucleic acid molecule comprises a sequence of
nucleotides substantially as set forth in SEQ ID NO:1 or SEQ ID
NO:3 or its complementary form, or a nucleotide sequence having at
least about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form or a nucleotide sequence capable of hybridizing
to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form under low
stringency conditions, wherein said nucleic acid molecule is
operably linked to one or more regulatory sequences such that the
nucleic acid molecule is capable of being expressed in said cell or
tissue.
35. A method of claim 34, wherein said expression vector comprises
a genetic construct comprising a nucleic acid molecule comprising a
sequence of nucleotides encoding an amino acid sequence
substantially as set forth in SEQ ID NO:2 or an amino acid sequence
having at least about 71% similarity to SEQ ID NO:2.
36. A method of claim 34, wherein said nucleic acid molecule
comprises a sequence of nucleotides substantially as set forth in
SEQ ID NO:1 or SEQ ID NO:3.
37. A method of claim 36, wherein said nucleic acid molecule
comprises a sequence of nucleotides substantially as set forth in
SEQ ID NO:1.
38. A method of claim 36, wherein said nucleic acid molecule
comprises a sequence of nucleotides substantially as set forth in
SEQ ID NO:3.
39. A method for modulating apoptotic processes in a cell or
tissue, said method comprising introducing into said cell or tissue
an expression vector comprising a nucleic acid molecule, said
nucleic acid molecule comprising a sequence of nucleotides
substantially as set forth in SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form, a nucleotide sequence having at least about 71%
similarity to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form
or a nucleotide sequence capable of hybridizing SEQ ID NO:1 or SEQ
ID NO:3 or its complementary form under low stringency conditions
wherein said nucleic acid molecule is operably linked to one or
more regulatory sequences such that the nucleic acid molecule is
capable of being expressed in said cell or tissue.
40. A method of claim 39, wherein said expression vector comprises
a genetic construct comprising a nucleic acid molecule comprising a
sequence of nucleotides encoding an amino acid sequence
substantially as set forth in SEQ ID NO:2 or an amino acid sequence
having at least about 71% similarity to SEQ ID NO:2.
41. A method of claim 39, wherein said nucleic acid molecule
comprises a sequence of nucleotides substantially as set forth in
SEQ ID NO:1 or SEQ ID NO:3.
42. A method of claim 41, wherein said nucleic acid molecule
comprises a sequence of nucleotides substantially as set forth in
SEQ ID NO:1.
43. A method of claim 41, wherein said nucleic acid molecule
comprises a sequence of nucleotides substantially as set forth in
SEQ ID NO:3.
44. A method for modulating apoptotic processes in a cell, said
method comprising administering to said cell an apoptotic
process-controlling effective amount of a recombinant polypeptide,
said polypeptide comprising the amino acid sequence set forth in
SEQ ID NO:2 or an amino acid sequence having at least about 71%
similarity to SEQ ID NO:2, said administration being for a time and
under conditions sufficient to modulate apoptosis.
45. A method for detecting embryogenic plant material, said method
comprising immobilizing a sample putatively containing RNA from the
material to be screened on a solid support and contacting said
immobilized RNA with a labelled nucleotide sequence capable of
hybridizing to all or part of an mRNA transcript corresponding to
the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or
their derivatives or homologues as defined herein and then
detecting the presence of said label.
46. A method of claim 45, wherein said nucleotide sequence is
substantially as set forth in SEQ ID NO:1 or SEQ ID NO:3.
47. A method of claim 45, wherein said nucleotide sequence is
substantially as set forth in SEQ ID NO:1.
48. A method of claim 45, wherein said nucleotide sequence is
substantially as set forth in SEQ ID NO:3.
49. An antibody to a polypeptide, said polypeptide comprising a
sequence of amino acids substantially as set forth in SEQ ID NO:2
or an amino acid sequence having at least 71% similarity to SEQ ID
NO:2, wherein said polypeptide is present in plant zygotic embryos
or embryogenic callus and is substantially not present in
non-embryogenic tissue.
50. An antibody of claim 49, wherein the amino acid sequence is
encoded by a nucleotide sequence substantially as set forth in SEQ
ID NO:1 or SEQ ID NO:3 or a nucleotide sequence having at least
about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form or a nucleotide sequence capable of hybridizing
to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form under low
stringency conditions.
51. An antibody of claim 49, wherein the amino acid sequence is
encoded by a nucleotide sequence substantially as set forth in SEQ
ID NO:1 or SEQ ID NO:3.
52. An antibody of claim 49, wherein the nucleotide sequence is
substantially as set forth in SEQ ID NO:1.
53. An antibody of claim 49, wherein the nucleotide sequence is
substantially as set forth in SEQ ID NO:3.
54. An antibody of claim 49, wherein the polypeptide has
antioxidant properties.
55. An antibody of claim 54, wherein the polypeptide is
peroxiredoxin.
56. A method for detecting a polypeptide which is indicative of the
presence of embryogenic tissue in oil-palm or related plants, said
method comprising contacting the tissue or an extract thereof with
an antibody specific for said polypeptide or its derivatives or
homologues for a time and under conditions sufficient for an
antibody-polypeptide complex to form, and then detecting said
complex.
57. A method of claim 56, wherein the polypeptide comprises a
sequence of amino acids substantially as set forth in SEQ ID NO:2
or an amino acid sequence having at least 71% similarity to SEQ ID
NO:2.
58. A method of claim 57, wherein the amino acid sequence is
encoded by a nucleotide sequence substantially as set forth in SEQ
ID NO:1 or SEQ ID NO:3 or a nucleotide sequence having at least
about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form or a nucleotide sequence capable of hybridizing
to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form under low
stringency conditions.
59. A method of claim 56, wherein the amino acid sequence is
encoded by a nucleotide sequence substantially as set forth in SEQ
ID NO:1 or SEQ ID NO:3.
60. A method of claim 56, wherein the amino acid sequence is
encoded by a nucleotide sequence substantially as set forth in SEQ
ID NO:1.
61. A method of claim 56, wherein the amino acid sequence is
encoded by a nucleotide sequence substantially as set forth in SEQ
ID NO:3.
62. A pharmaceutical composition comprising the polypeptide having
an amino acid sequence as set forth in SEQ ID NO:2 or a functional
homologue thereof or a molecule having at least 71% similarity to
SEQ ID NO:2 and one or more pharmaceutically-acceptable carriers
and/or diluents.
63. A pharmaceutical composition of claim 62, wherein the amino
acid sequence is as set forth in SEQ ID NO:2.
64. A regenerated differentiated plant comprising a nucleic acid
molecule encoding a polypeptide comprising an amino acid sequence
substantially as set forth in SEQ ID NO:2 or an amino acid sequence
having at least about 71% similarity to SEQ ID NO:2, wherein said
polypeptide is present in plant zygotic embryos or embryogenic
callus and is substantially not present in non-embryogenic
tissue.
65. A regenerated differentiated plant of claim 64, wherein nucleic
acid molecule comprises a sequence of nucleotides substantially as
set forth in SEQ ID NO:1 or SEQ ID NO:3 or its complementary form,
or a nucleotide sequence having at least about 71% similarity to
SEQ ID NO:1 or SEQ ID NO:3 or its complementary form or a
nucleotide sequence capable of hybridizing to SEQ ID NO:1 or SEQ ID
NO:3 or its complementary form under low stringency conditions.
66. A regenerated differentiated plant of claim 64, wherein the
amino acid sequence is encoded by a nucleotide sequence
substantially as set forth in SEQ ID NO:1 or SEQ ID NO:3.
67. A regenerated differentiated plant of claim 64, wherein the
amino acid sequence is encoded by a nucleotide sequence
substantially as set forth in SEQ ID NO:1.
68. A regenerated differentiated plant of claim 64, wherein the
amino acid sequence is encoded by a nucleotide sequence
substantially as set forth in SEQ ID NO:3.
69. A regenerated differentiated plant of claim 64, wherein the
plant is an oil palm plant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 12/788,725, filed on May 27, 2010, which is a
divisional application of Ser. No. 10/028,346, filed on Dec. 20,
2001, which benefits Australian Provisional Application No.
PR2213/00, filed Dec. 20, 2000.
FIELD OF THE INVENTION
[0002] The present invention relates generally to a molecular
marker for a plant physiological process and more particularly for
plant embryogenesis. The molecular marker is, in one form, a
genetic sequence from a monocot plant such as but not limited to
oil-palm plants. In another form, the molecular marker is a
polypeptide encoded by said genetic sequence. More particularly,
the molecular marker of the present invention enables embryogenic
tissue to be detected in vitro. The early detection of embryogenic
tissue enables non-embryogenic tissue to be discarded. The ability
to detect embryogenesis facilitates maximization of embryogenic
potential. The present invention further contemplates a molecular
marker comprising in one form a sequence of nucleotides encoding an
antioxidant or in another form a sequence of amino acids defining a
polypeptide having antioxidant activity. The antioxidant according
to this aspect of the present invention is particularly useful in
tablet or cream form as an anti-aging agent. The molecular markers
of the present invention therefore also have uses in the inhibition
or retardation of apoptotic processes. Such an effect has benefits
in both plant and animal cells. The present invention further
contemplates a promoter sequence encoding the molecular marker and
its use in generating male sterile plants.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0003] The Sequence Listing in the ASCII text file, named as
15179.seq.txt of 16 KB, created on Jan. 13, 2005, and submitted to
the United States Patent and Trademark Office via first class mail,
is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0004] Bibliographic details of the publications referred to by
author in this specification are collected at the end of the
description.
[0005] Reference to any prior art in this specification is not, and
should not be taken as, an acknowledgment or any form of suggestion
that this prior art forms part of the common general knowledge in
Australia or any other country.
[0006] The study of plant embryogenesis has been regarded as
fundamental to understanding plant development. It is during
embryogenesis that meristems and basic plant tissue systems are
established. Basically, embryogenesis involves two main processes:
the induction of embryogenic potential and the expression of the
embryogenesis programme. Because of the totipotency of the plant
cells, each cell has the capability to become embryogenic and to
develop into a whole plant.
[0007] Recent advances in micropropagation and manipulation of
tissue culture conditions has led to the possibility of multiplying
vegetatively many plant species efficiently and rapidly in vitro.
For many commercial production systems, conventional plant breeding
and seed production methods are slow and therefore limit the
ability to realize the maximum potential of selected genotypes.
However, the development of economically-viable propagation systems
necessitates the existence of efficient methods of embryo- or
organogenesis. Such methods have been generated for many, but not
all species.
[0008] There are very high demands for oil-palm. Accordingly, a
major area of study in the oil-palm industry seeks to find improved
ways to increase oil yield. With the ability to maintain uniformity
of planting materials in tissue culture, improvements in yield of
up to 20% may be able to be realized. In the case of oil-palm,
however, little is known about the biology of somatic embryogenesis
despite the economic importance of the crop and work to date has
resulted in average rates of in vitro embryogenesis of only 6%
(Wooi, 1995). Such low rates are inconsistent with an economically
viable system.
[0009] Most of the earlier studies concentrated on the development
of methodologies for the initiation and production of somatic
embryos (Jones, 1974; Ahee et al., 1981; Pannetier et al., 1981).
These groups worked mainly on the manipulation of phytohormones in
the media as well as on introducing tissues with better clonability
to further improve the process. Schwendiman and colleagues (1988)
carried out histological analysis of somatic embryogenesis from
leaf-derived callus, detailing the emergence of callus and the
subsequent formation of somatic embryos, with shoot and root
apices. Not long before that, Turnham and Northcote (1982)
investigated the occurrence of biochemical indicators that are
useful in the prediction of embryogenic potential.
[0010] More recently, the importance of understanding molecular
switches, that occur in somatic cells and induce them to become
embryogenic, has been highlighted (Dudits et al., 1995).
[0011] In this regard, the rapid introduction of and improvements
in recombinant DNA technologies has greatly facilitated the study
of plant development and provided researchers with sophisticated
precision tools for investigating underlying molecular
mechanisms.
[0012] There is a need to develop an effective and efficient method
for the production of somatic embryos and new approaches to be
brought to bear in attempts to realize that end.
[0013] In work leading up to the present invention, the inventors
sought to identify underlying factors involved in the induction of
embryogenesis. In so doing the inventors located and isolated a
polynucleotide sequence which was surprisingly found to be
expressed only in zygotic embryo and embryogenic callus. The
polynucleotide sequence or an amino acid encoded thereby of the
present invention is useful inter alia as a means of discriminating
embryogenic from non-embryogenic material. The molecular marker
represents a member of a new class of molecules from monocot plants
such as but not limited to oil-palm and related plants.
SUMMARY OF THE INVENTION
[0014] Throughout this specification, unless the context requires
otherwise, the words "comprise", "comprises" and "comprising" will
be understood to imply the inclusion of a stated step or element or
group of steps or elements but not the exclusion of any other step
or element or group of steps or elements.
[0015] Nucleotide and amino acid sequences are referred to by a
sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond
numerically to the sequence identifiers <400>1, <400>2,
etc. A sequence listing is provided after the claims.
[0016] The present invention provides a developmentally-regulated
nucleic acid molecule designated herein as OPEm1. The nucleic acid
molecule comprises a nucleotide coding sequence substantially as
set forth in SEQ ID NO:1. Additional 3' and 5' sequences are
provided in SEQ ID NO:3. The nucleic acid molecule is expressed
only in zygotic embryo and embroyonic callus to produce a
polypeptide comprising the amino acid sequence set forth in SEQ ID
NO:2 (corresponding to SEQ ID NO:1) or SEQ ID NO:4 (corresponding
to SEQ ID NO:3). The identification of the nucleic acid molecule
permits the discrimination of plant tissue at different
developmental stages. The nucleic acid molecule, therefore, permits
identification of a plant physiological process or tissue or other
plant material associated with a plant physiological process.
[0017] The nucleic acid molecule and/or the polypeptide encoded
thereby of the present invention may be used as a means of
discriminating embryogenic from non-embryogenic material in plants,
in particular monocot plants and even more particularly in oil-palm
and related plants. The present invention provides a nucleic acid
molecule, recombinant and purified naturally-occurring
polypeptides, antibodies to the polypeptides as well as transgenic
and genetically-modified plants. Furthermore, the polypeptides of
the present invention also have anti-apoptotic properties, making
them useful in the preparation of pharmaceutical compositions for
use as anti-ageing agents.
[0018] Accordingly, one aspect of the present invention provides an
isolated nucleic acid molecule encoding a polypeptide comprising an
amino acid sequence substantially as set forth in SEQ ID NO:2 or an
amino acid sequence having at least about 71% similarity to SEQ ID
NO:2, wherein said polypeptide is present in plant zygotic embryos
or embryogenic callus and is substantially not present in
non-embryogenic tissue.
[0019] Another aspect of the present invention provides an isolated
nucleic acid molecule comprising a sequence of nucleotides
substantially as set forth in SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form, or a nucleotide sequence having at least about
71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its complementary
form or a nucleotide sequence capable of hybridizing to SEQ ID NO:1
or SEQ ID NO:3 or its complementary form under low stringency
conditions.
[0020] A further aspect of the present invention provides an
isolated nucleic acid molecule capable of discriminating
embryogenic from non-embryogenic material, wherein said nucleic
acid molecule comprises a sequence of nucleotides substantially as
set forth in SEQ ID NO:1 or SEQ ID NO:3 or its complementary form,
or a nucleotide sequence having at least about 71% similarity to
SEQ ID NO:1 or SEQ ID NO:3 or its complementary form or a
nucleotide sequence capable of hybridizing to SEQ ID NO:1 or SEQ ID
NO:3 or its complementary form under low stringency conditions.
[0021] Yet another aspect of the present invention provides an
isolated nucleic acid molecule comprising a polynucleotide sequence
substantially as set forth in SEQ ID NO:1 or SEQ ID NO:3.
[0022] Still another aspect of the present invention provides a
genetic construct comprising a nucleic acid molecule encoding a
polypeptide comprising an amino acid sequence substantially as set
forth in SEQ ID NO:2 or an amino acid sequence having at least
about 71% similarity to SEQ ID NO:2, wherein said polypeptide is
present in plant zygotic embryos or embryogenic callus and is
substantially not present in non-embryogenic tissue.
[0023] Even still another spect of the present invention provides a
genetic construct comprising a nucleic acid molecule comprising a
sequence of nucleotides substantially as set forth in SEQ ID NO:1
or SEQ ID NO:3 or its complementary form, or a nucleotide sequence
having at least about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3
or its complementary form or a nucleotide sequence capable of
hybridizing to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form
under low stringency conditions.
[0024] Even yet another aspect of the present invention provides a
vector comprising a nucleic acid molecule capable of discriminating
embryogenic from non-embryogenic material, wherein said nucleic
acid molecule comprises a sequence of nucleotides substantially as
set forth in SEQ ID NO:1 or SEQ ID NO:3 or its complementary form,
or a nucleotide sequence having at least about 71% similarity to
SEQ ID NO:1 or SEQ ID NO:3 or a nucleotide sequence capable of
hybridizing to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form
under low stringency conditions.
[0025] Another aspect of the instant invention provides a host cell
comprising a nucleic acid molecule encoding a polypeptide
comprising an amino acid sequence substantially as set forth in SEQ
ID NO:2 or an amino acid sequence having at least about 71%
similarity to SEQ ID NO:2, wherein said polypeptide is present in
plant zygotic embryos or embryogenic callus and is substantially
not present in non-embryogenic tissue.
[0026] A further aspect of the present invention provides an
isolated polypeptide or biologically-active fragment thereof or a
variant or derivative of these, said polypeptide comprising the
amino acid sequence set forth in SEQ ID NO:2 or an amino acid
sequence having at least about 71% similarity to SEQ ID NO:2,
wherein said polypeptide is present in plant zygotic embryos or
embryogenic callus and is substantially not present in
non-embryogenic tissue.
[0027] Yet another aspect of the present invention is directed to
an isolated polypeptide comprising a sequence of amino acids
encoded by the nucleotide sequence substantially as set forth in
SEQ ID NO:1 or SEQ ID NO:3 or a nucleotide sequence having at least
about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form or a nucleotide sequence capable of hybridizing
to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form under low
stringency conditions.
[0028] Still another aspect of the present invention provides a
method for producing a recombinant polypeptide in a host cell or
tissue, said method comprising introducing into the said cell or
tissue an expression vector comprising a nucleic acid molecule
wherein said nucleic acid molecule comprises a sequence of
nucleotides substantially as set forth in SEQ ID NO:1 or SEQ ID
NO:3 or its complementary form, or a nucleotide sequence having at
least about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form or a nucleotide sequence capable of hybridizing
to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form under low
stringency conditions wherein said nucleic acid molecule is
operably linked to one or more regulatory sequences such that the
nucleic acid molecule is capable of being expressed in said cell or
tissue.
[0029] Even still another aspect of the invention provides a method
for modulating apoptotic processes in a cell or tissue, said method
comprising introducing into said cell or tissue an expression
vector comprising a nucleic acid molecule, said nucleic acid
molecule comprising a sequence of nucleotides substantially as set
forth in SEQ ID NO:1 or SEQ ID NO:3 or its complementary form, or a
nucleotide sequence having at least about 71% similarity to SEQ ID
NO:1 or SEQ ID NO:3 or its complementary form or a nucleotide
sequence capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3 or
its complementary form under low stringency conditions wherein said
nucleic acid molecule is operably linked to one or more regulatory
sequences such that the nucleic acid molecule is capable of being
expressed in said cell or tissue.
[0030] Even yet another aspect of the invention provides a method
for modulating apoptotic processes in a cell, said method
comprising administering to said cell an apoptotic
process-controlling effective amount of a recombinant polypeptide,
said polypeptide comprising the amino acid sequence set forth in
SEQ ID NO:2 or an amino acid sequence having at least about 71%
similarity to SEQ ID NO:2, said administration being for a time and
under conditions sufficient to modulate apoptosis.
[0031] Another aspect of the invention provides a method for
detecting embryogenic plant material, said method comprising
immobilizing a sample putatively containing RNA from the material
to be screened on a solid support and contacting said immobilized
RNA with a labelled nucleotide sequence capable of hybridizing to
all or part of an mRNA transcript corresponding to the nucleotide
sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or their
derivatives or homologues as defined herein and then detecting the
presence of said label.
[0032] A further aspect of the present invention contemplates an
antibody to a polypeptide, said polypeptide comprising a sequence
of amino acids substantially as set forth in SEQ ID NO:2 or an
amino acid sequence having at least 71% similarity to SEQ ID NO:2,
wherein said polypeptide is present in plant zygotic embryos or
embryogenic callus and is substantially not present in
non-embryogenic tissue.
[0033] Yet another aspect of the present invention contemplates a
method for detecting a polypeptide which is indicative of the
presence of embryogenic tissue in oil-palm or related plants, said
method comprising contacting the tissue or an extract thereof with
an antibody specific for said polypeptide or its derivatives or
homologues for a time and under conditions sufficient for an
antibody-polypeptide complex to form, and then detecting said
complex.
[0034] Still another aspect of the present invention contemplates a
pharmaceutical composition comprising the polypeptide having an
amino acid sequence as set forth in SEQ ID NO:2 or a functional
homologue thereof or a molecule having at least 71% similarity to
SEQ ID NO:2 and one or more pharmaceutically-acceptable carriers
and/or diluents.
[0035] Even still another aspect of the present invention is
directed to a regenerated differentiated plant comprising a nucleic
acid molecule encoding a polypeptide comprising an amino acid
sequence substantially as set forth in SEQ ID NO:2 or an amino acid
sequence having at least about 71% similarity to SEQ ID NO:2,
wherein said polypeptide is present in plant zygotic embryos or
embryogenic callus and is substantially not present in
non-embryogenic tissue.
[0036] Even yet another aspect of the present invention is directed
to a regenerated differentiated plant comprising a nucleic acid
molecule, wherein said nucleic acid molecule comprises a sequence
of nucleotides substantially as set forth in SEQ ID NO:1 or SEQ ID
NO:3 or its complementary form, or a nucleotide sequence having at
least about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form or a nucleotide sequence capable of hybridizing
to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form under low
stringency conditions.
[0037] Another aspect of the present invention provides an isolated
nucleic acid molecule having promoter activity wherein, in its
naturally occurring form, the promoter is operably linked to a
nucleotide sequence substantially as set forth in SEQ ID NO:1 or
SEQ ID NO:3 or a nucleotide sequence complementary thereto or a
nucleotide sequence capable of hybridizing to SEQ ID NO:1 or SEQ ID
NO:3 or its complementary form under low stringency conditions.
TABLE-US-00001 SUMMARY OF SEQUENCE IDENTIFIERS SEQUENCE IDENTIFER
DESCRIPTION SEQ ID NO: 1 Nucleotide coding sequence of embryogenic
specific polypeptide from oil-palm (OPEm1) SEQ ID NO: 2 Amino acid
sequence of embryogenic specific polypeptide from oil-palm (OPEm1);
corresponds to SEQ ID NO: 1 SEQ ID NO: 3 Nucleotide coding sequence
of embryogenic specific polypeptide from oil-palm (OPEm1) with 5'
and 3' non-transcribed sequences SEQ ID NO: 4 Amino acid sequence
of embryogenic specific polypeptide from oil-palm (OPEm1);
corresponds to SEQ ID NO: 3 SEQ ID NO: 5 Oligonucleotide primer
AGL15AtF SEQ ID NO: 6 Oligonucleotide primer AGL15AtR SEQ ID NO: 7
Amino acid sequence of 1-Cys peroxiredoxin from Hordeum vulgare
(barley) [HvPer1] SEQ ID NO: 8 Amino acid sequence of 1-Cys
peroxiredoxin from Arabidopsis thaliana (thalecress) [AtPer1] SEQ
ID NO: 9 Amino acid sequence of 1-Cys peroxiredoxin from Brassica
campestris (Chinese cabbage) [C2CPRX]
BRIEF DESCRIPTION OF THE FIGURES
[0038] FIG. 1a is a photographic representation showing reverse
transcription of zygotic embryo total RNA with primers AGL15AtF and
AGL15AtR resulting in the production of two bands, designated
RTPCR1 (.about.562 bp) and RTPCR2 (.about.501 bp) (left). The bands
were excised and purified (right).
[0039] FIG. 1b is a photographic representation showing Northern
analysis of RTPCR1 and RTPCR2. RTPCR1 was exclusively expressed in
embryogenic materials (ZE and EC). However, RTPCR2 was
constitutively expressed. ZE: zygotic embryo; EC: embryogenic
calli; NEC: non-embryogenic calli; YL: young leaves.
[0040] FIG. 2a is a photographic representation showing Northern
analysis of OPEm1 (top) and expression of 18S ribosomal cDNA
(bottom) as control. Each lane contains 10 .mu.g of total RNA from
different types of tissues. Lane 1: embryogenic calli from clone
FC1454; Lane 2: embryogenic calli of clone FC1454 that have lost
their embryogenic potential (ENP); Lane 3: embryogenic calli from
FC1501; Lane 4: ENP of FC1501; Lane 5: Non-embryogenic calli of
FC1501. Lane 6: embryogenic calli of FC1509; Lane 7: ENP of FC1509;
Lane 8: non-embryogenic calli of FC1509; Lane 9: embryogenic calli
from early suspension cultures; Lane 10: suspension cultures; Lane
11: white embryoids; Lane 12: green embryoids; Lane 13: bipolar
structures; Lane 14: immature 12 WAA zygotic embryos; Lane 15:
mature 15 WAA zygotic embryos; Lane 16: vegetative meristem; Lane
17: inflorescence from frond 17 and Lane 18: young leaves.
[0041] FIG. 2b is a graphical representation showing hydropathy
plot of OPEm1 with a calculated pI of 7.48.
[0042] FIG. 2c is a photographic representation showing Southern
analysis of OPEm1. Each lane contains 10 .mu.g genomic DNA digested
with: Lane 1: EcoRI; Lane 2: BamHI; Lane 3: HindIII; Lane 4: KpnI;
Lane 5: NotI; Lane 6: SfiI; Lane 7: SpeI and Lane 8: StuI. The
digests were run alongside a 1 kb DNA molecular weight marker
(Promega).
[0043] FIG. 3 is the nucleotide and deduced amino acid sequences of
OPEm1.
[0044] FIG. 4a is photographic representation showing a 3-D
structure of the monomer unit of the human peroxiredoxin,
C91S-hORF6. Each monomer consists of 224 amino acids with two
domains. Domain I is the larger N-terminal domain and Domain II is
the smaller C-terminal domain.
[0045] FIG. 4b is a photographic representation showing that human
peroxiredoxin exists as a tightly associated dimer.
[0046] FIG. 4c is a photographic representation showing the deduced
3-D structure of OPEm1, which has very high similarity to the human
peroxiredoxin.
[0047] FIG. 4d is a schematic representation depicting a topology
diagram of C91S-hORF6 monomer. The shaded area corresponds to the
thioredoxin fold.
[0048] FIG. 5 is the sequence alignment of OPEm1 with examples of
1-Cys and 2-Cys peroxiredoxins. The `*` denotes a single fully
conserved residue. The `:` denotes conservation of strong groups.
The `.` conservation of weak groups. Those without any symbol
denote no consensus (CLUSTALW, Biology Workbench Version 3.2,
University of Illinois, 1999). The amino acid sequences were
obtained from Genbank: HvPer1 (Hordeum vulgare, barley, P52572),
AtPer1 (Arabidopsis thaliana, thalecress, CAA63909) and C2CPRX
(Brassica campestris L. ssp. pekinensis, chinese cabbage).
[0049] FIG. 6 is the sequence alignment of OPEm1 with examples of
other members of 1-Cys peroxiredoxin. The `*` denotes a single
fully conserved residue. The `:` denotes conservation of strong
groups. The `.` denotes conservation of weak groups. Those without
any symbols denote no consensus. (CLUSTALW, Biology Workbench
Version 3.2, University of Illinois, 1999). The peroxiredoxin amino
acid sequences were obtained from Genbank: HvPer1 (Hordeum vulgare,
barley, P52572) and AtPer1 (Arabidopsis thaliana, thalecress,
CAA63909). The `#` and `@` denote the positively charged residue
His 38 and Arg 128 respectively, which are all found close to the
Cys 46. The PVCT region represents a specific characteristic of the
1-Cys peroxiredoxin. The basic residues at the terminal end of the
1-Cys peroxiredoxin align to the nuclear localization signal (NLS)
region that is not present in OPEm1. A coloured version of this
Figure where the PVCT region is in blue and the basic region is in
red is available from the Applicant upon request.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0050] The present invention is predicated in part on the
identification of a developmentally-regulated nucleic acid
molecule. The identification of the nucleic acid molecule permits
the discrimination of plant tissue at different developmental
stages. The nucleic acid molecule, therefore, permits
identification of a plant physiological process or tissue or other
plant material associated with a plant physiological process. More
particularly, the polynucleotide sequence is expressed only in
zygotic embryo and embryogenic callus. The nucleic acid molecule
and/or an amino acid sequence encoded thereby of the present
invention may be used as a means of discriminating embryogenic from
non-embryogenic material in plants, in particular monocot plants
and even more particularly in oil-palm and related plants. The term
"material" includes cells, tissue, clusters of cells, callus,
organelles, seeds, pollen and other plant parts. The nucleic acid
molecule of the present invention and an amino acid sequence
encoded thereby are both referred to herein as a "molecular
marker". Reference herein to a "molecular marker" is not to impart
any limitation as to its structure, location in a cell or its
use.
[0051] Reference to the term "discriminating" in relation to
embryogenic and non-embryogenic tissue includes reference to the
determination of a strong likelihood that certain tissue is
embryogenic as distinct from non-embryogenic on the basis of the
presence of the subject nucleic acid molecule or its expression
product. Reference to a "determination" includes reference to a
"prediction" or other reasoned deduction. Embryogenic material
includes inter alia zygotic embryo and embryogenic callus
material.
[0052] Accordingly, one aspect of the present invention provides an
isolated nucleic acid molecule encoding a polypeptide comprising an
amino acid sequence substantially as set forth in SEQ ID NO:2 or an
amino acid sequence having at least about 71% similarity to SEQ ID
NO:2, wherein said polypeptide is present in plant zygotic embryos
or embryogenic callus and is substantially not present in
non-embryogenic tissue.
[0053] In a related embodiment, the present invention provides an
isolated nucleic acid molecule comprising a sequence of nucleotides
substantially as set forth in SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form, or a nucleotide sequence having at least about
71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its complementary
form or a nucleotide sequence capable of hybridizing to SEQ ID NO:1
or SEQ ID NO:3 or its complementary form under low stringency
conditions.
[0054] Preferably the nucleic acid molecule is regulated
developmentally such that its presence may be used as a means of
discriminating embryogenic from non-embryogenic material.
[0055] Accordingly, in a preferred embodiment, the present
invention provides an isolated nucleic acid molecule capable of
discriminating embryogenic from non-embryogenic material, wherein
said nucleic acid molecule comprises a sequence of nucleotides
substantially as set forth in SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form, or a nucleotide sequence having at least about
71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its complementary
form or a nucleotide sequence capable of hybridizing to SEQ ID NO:1
or SEQ ID NO:3 or its complementary form under low stringency
conditions.
[0056] The term "nucleic acid molecule" includes a polynucleotide,
nucleotide or genetic sequence such as, but not limited to, mRNA,
RNA, cRNA, cDNA or DNA. Reference to a DNA molecule includes
genomic DNA.
[0057] By "isolated" is meant material that is substantially or
essentially free from components that normally accompany it in its
native state. For example, an "isolated nucleic acid molecule" as
used herein refers to a polynucleotide sequence, which has been
purified from the sequences which flank it in a naturally-occurring
state, e.g., a DNA fragment which has been removed from the
sequences which are normally adjacent to the fragment.
[0058] The isolated nucleotide sequence of the present invention
also extends to derivatives including, mutants and homologues of
the said sequence.
[0059] By "derivative" is meant any single or multiple nucleotide
deletions, additions or substitutions as well as mutants,
fragments, portions or parts of said isolated nucleic acid
molecule. All such deletions, additions, substitutions, mutants,
fragments, portions, or parts are encompassed by the term
"derivative". Particularly useful derivatives include alterations
to the 5' end portion of the polynucleotide sequence or the 3' end
portion or a nucleotide sequence spanning the 5' and 3' portions.
Synthetic derivatives may also be useful, for example, in
diagnostic assays. A derivative also conveniently includes a
polynucleotide sequence having less than 100% identity with the
nucleotide sequence set forth in SEQ ID NO:1, but which is capable
of hybridizing thereto or its complementary form under low
stringency conditions.
[0060] Terms such as "hybridization", "hybridizing" and the like
are used herein to denote the pairing of complementary nucleotide
sequences to produce a DNA-DNA hybrid or a DNA-RNA hybrid.
Complementary base sequences are those sequences that are related
by the base-pairing rules. In DNA, A pairs with T and C pairs with
G. In RNA, U pairs with A and C pairs with G. In this regard, the
terms "match" and "mismatch" as used herein refer to the
hybridization potential of paired nucleotides in complementary
nucleic acid strands. Matched nucleotides hybridize efficiently,
such as the classical A-T and G-C base pair mentioned above.
Mismatches are other combinations of nucleotides that do not
hybridize efficiently.
[0061] "Stringency" as used herein, refers to the temperature and
ionic strength conditions, and presence or absence of certain
organic solvents, during hybridization and washing procedures. The
higher the stringency, the higher will be the degree of
complementarity between immobilized target nucleotide sequences and
the labelled probe polynucleotide sequences that remain hybridized
to the target after washing.
[0062] "Stringency conditions" refers to temperature and ionic
conditions under which only nucleotide sequences having a high
frequency of complementary bases will hybridize. The stringency
required is nucleotide sequence dependent and depends upon the
various components present during hybridization and subsequent
washes, and the time allowed for these processes. Generally, in
order to maximize the hybridization rate, non-stringent
hybridization conditions are selected: about 20 to 25.degree. C.
lower than the thermal melting point (T.sub.m). The T.sub.m is the
temperature at which 50% of specific target sequence hybridizes to
a perfectly complementary probe in solution at a defined ionic
strength and pH. Generally, in order to require at least about 85%
nucleotide complementarity of hybridized sequences, highly
stringent washing conditions are selected to be about 5 to
15.degree. C. lower than the T.sub.m. In order to require at least
about 71% nucleotide complementarity of hybridized sequences,
moderately stringent washing conditions are selected to be about 15
to 30.degree. C. lower than the T.sub.m. Highly permissive (low
stringency) washing conditions may be as low as 50.degree. C. below
the T.sub.m, allowing a high level of mis-matching between
hybridized sequences. Those skilled in the art will recognize that
other physical and chemical parameters in the hybridization and
wash stages can also be altered to affect the outcome of a
detectable hybridization signal from a specific level of homology
between target and probe sequences.
[0063] Reference herein to a low stringency includes and
encompasses from at least about 0 to at least about 15% v/v
formamide and from at least about 1 M to at least about 2 M salt
for hybridization, and at least about 1 M to at least about 2 M
salt for washing conditions. Generally, low stringency is at from
about 25-30.degree. C. to about 42.degree. C. The temperature may
be altered and higher temperatures used to replace formamide and/or
to give alternative stringency conditions. Alternative stringency
conditions may be applied where necessary, such as medium
stringency, which includes and encompasses from at least about 16%
v/v to at least about 30% v/v formamide and from at least about 0.5
M to at least about 0.9 M salt for hybridization, and at least
about 0.5 M to at least about 0.9 M salt for washing conditions, or
high stringency, which includes and encompasses from at least about
31% v/v to at least about 50% v/v formamide and from at least about
0.01 M to at least about 0.15 M salt for hybridization, and at
least about 0.01 M to at least about 0.15 M salt for washing
conditions. In general, washing is carried out T.sub.m=69.3+0.41
(G+C) % (Marmur and Doty, 1962). However, the T.sub.m of a duplex
DNA decreases by 1.degree. C. with every increase of 1% in the
number of mismatch base pairs (Bonner and Laskey, 1974). Formamide
is optional in these hybridization conditions. Accordingly,
particularly preferred levels of stringency are defined as follows:
low stringency is 6.times.SSC buffer, 0.1% w/v SDS at 25-42.degree.
C.; a moderate stringency is 2.times.SSC buffer, 0.1% w/v SDS at a
temperature in the range 20.degree. C. to 65.degree. C.; high
stringency is 0.1.times.SSC buffer, 0.1% w/v SDS at a temperature
of at least 65.degree. C.
[0064] Suitably, the isolated nucleic acid molecule has at least
greater than 70% (for example, 71%), preferably at least about 75%,
more preferably at least about 80%, more preferably yet at least
about 85%, still more preferably at least about 90% and even still
more preferably at least about 95% or above (e.g. 96% or 97% or 98%
or 99%) sequence similarity to the nucleotide sequence set forth in
SEQ ID NO:1 or SEQ ID NO:3.
[0065] The term "similarity" as used herein includes exact identity
between compared sequences at the nucleotide or amino acid level.
Where there is non-identity at the nucleotide level, "similarity"
includes differences between sequences which result in different
amino acids that are nevertheless related to each other at the
structural, functional, biochemical and/or conformational levels.
Where there is non-identity at the amino acid level, "similarity"
includes amino acids that are nevertheless related to each other at
the structural, functional, biochemical and/or conformational
levels. In a particularly preferred embodiment, nucleotide and
sequence comparisons are made at the level of identity rather than
similarity.
[0066] Terms used to describe sequence relationships between two or
more polynucleotides or polypeptides include "reference sequence",
"comparison window", "sequence similarity", "sequence identity",
"percentage of sequence similarity", "percentage of sequence
identity", "substantially similar" and "substantial identity". A
"reference sequence" is at least 12 but frequently 15 to 18 and
often at least 25 or above, such as 30 monomer units, inclusive of
nucleotides and amino acid residues, in length. Because two
polynucleotides may each comprise (1) a sequence (i.e. only a
portion of the complete polynucleotide sequence) that is similar
between the two polynucleotides, and (2) a sequence that is
divergent between the two polynucleotides, sequence comparisons
between two (or more) polynucleotides are typically performed by
comparing sequences of the two polynucleotides over a "comparison
window" to identify and compare local regions of sequence
similarity. A "comparison window" refers to a conceptual segment of
typically 12 contiguous residues that is compared to a reference
sequence. The comparison window may comprise additions or deletions
(i.e. gaps) of about 20% or less as compared to the reference
sequence (which does not comprise additions or deletions) for
optimal alignment of the two sequences. Optimal alignment of
sequences for aligning a comparison window may be conducted by
computerized implementations of algorithms (GAP, BESTFIT, FASTA,
and TFASTA in the Wisconsin Genetics Software Package Release 7.0,
Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or
by inspection and the best alignment (i.e. resulting in the highest
percentage homology over the comparison window) generated by any of
the various methods selected. Reference also may be made to the
BLAST family of programs as for example disclosed by Altschul et
al. (1997). A detailed discussion of sequence analysis can be found
in Unit 19.3 of Ausubel et al. (1998).
[0067] The terms "sequence similarity" and "sequence identity" as
used herein refers to the extent that sequences are identical or
functionally or structurally similar on a nucleotide-by-nucleotide
basis or an amino acid-by-amino acid basis over a window of
comparison. Thus, a "percentage of sequence identity", for example,
is calculated by comparing two optimally aligned sequences over the
window of comparison, determining the number of positions at which
the identical nucleic acid base (e.g. A, T, C, G, I) or the
identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val,
Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and
Met) occurs in both sequences to yield the number of matched
positions, dividing the number of matched positions by the total
number of positions in the window of comparison (i.e., the window
size), and multiplying the result by 100 to yield the percentage of
sequence identity. For the purposes of the present invention,
"sequence identity" will be understood to mean the "match
percentage" calculated by the DNASIS computer program (Version 2.5
for windows; available from Hitachi Software engineering Co., Ltd.,
South San Francisco, Calif., USA) using standard defaults as used
in the reference manual accompanying the software. Similar comments
apply in relation to sequence similarity.
[0068] A particularly preferred embodiment of the instant invention
provides an isolated nucleic acid molecule comprising a
polynucleotide sequence substantially as set forth in SEQ ID NO:1
or SEQ ID NO:3. Although the present invention is particularly
exemplified with respect to oil-palm, this is done with the
understanding that the instant invention encompasses any monocot
plant. Reference herein to a monocot includes any member of the
plant family Gramineae, Palmae, Juncaceae and Achenes, but is not
limited to cereals, grasses, maize, sugar cane, oats, wheat, barley
as well as oil-palm.
[0069] In a convenient embodiment, reference to a nucleic acid
molecule includes reference to a "gene". The term "gene" is used in
its broadest sense and includes reference to a polynucleotide
sequence such as a cDNA corresponding to the exons of a gene.
Accordingly, reference herein to a Agene.apprxeq. is to be taken to
include:-- [0070] (i) a classical genomic gene consisting of
transcriptional and/or translational regulatory sequences and/or a
coding region and/or non-translated sequences (i.e. introns, 5'-
and 3'-untranslated sequences); or [0071] (ii) mRNA or cDNA
corresponding to the coding regions (i.e. exons) and 5'- and
3'-untranslated sequences of the gene; and/or [0072] (iii) a
structural region corresponding to the coding regions (i.e. exons)
optionally further comprising untranslated sequences and/or a
heterologous promoter sequence which consists of transcriptional
and/or translational regulatory regions capable of conferring
expression characteristics on said structural region.
[0073] The term "gene" is also used to describe synthetic or fusion
molecules encoding all or part of a functional product, in
particular, a sense or antisense mRNA product or a peptide,
oligopeptide or polypeptide or a biologically-active protein.
Reference to a "gene" also includes reference to a "synthetic
gene".
[0074] The term "synthetic gene" refers to a non-naturally
occurring gene as hereinbefore defined which preferably comprises
at least one or more transcriptional and/or translational
regulatory sequences operably linked to a structural gene
sequence.
[0075] The term "structural gene" shall be taken to refer to a
nucleotide sequence, which is capable of being transmitted to
produce mRNA and optionally, encodes a peptide, oligopeptide,
polypeptide or biologically active protein molecule. Those skilled
in the art will be aware that not all mRNA is capable of being
translated into a peptide, oligopeptide, polypeptide or protein;
for example, if the mRNA lacks a functional translation start
signal or alternatively, if the mRNA is antisense mRNA. The present
invention clearly encompasses synthetic genes comprising nucleotide
sequences, which are not capable of encoding peptides,
oligopeptides, polypeptides or biologically-active proteins. In
particular, the present inventors have found that such synthetic
genes may be useful, for example, in diagnostic assays of gene
expression in cells, tissues or organs of a eukaroytic
organism.
[0076] The term "structural gene region" refers to that part of a
synthetic gene, which is expressed in a cell, tissue or organ under
the control of a promoter sequence to which it is operably
connected. A structural gene region may be operably under the
control of a single promoter sequence or multiple promoter
sequences. Accordingly, the structural gene region of a synthetic
gene may comprise a nucleotide sequence, which is capable of
encoding an amino acid sequence or is complementary thereto. In
this regard, a structural gene region, which is used in the
performance of the instant invention, may also comprise a
nucleotide sequence which encodes an amino acid sequence yet lacks
a functional translation initiation codon and/or a functional
translation stop codon and, as a consequence, does not comprise a
complete open reading frame. In the present context, the term
"structural gene region" also extends to a non-coding nucleotide
sequences, such as 5'-upstream or 3'-downstream sequences of a gene
which would not normally be translated in a eukaryotic cell which
expresses said gene.
[0077] In another aspect, the invention is directed to a vector
comprising the nucleic acid molecule as broadly described above.
The vector comprising the nucleic acid molecule may be in isolated
form or may exist as an extrachromosomal element or all or part of
the vector may be integrated into the genome of a host cell. The
vector may also be packaged for sale in a kit with instructions for
use inter alia as a diagnostic agent or in an assay system.
[0078] In a preferred embodiment, the instant invention provides a
vector comprising a nucleic acid molecule having a sequence of
nucleotides substantially as set forth in SEQ ID NO:1 or SEQ ID
NO:3 or its complementary form, or a nucleotide sequence having at
least about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form or a nucleotide sequence capable of hybridizing
to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form under low
stringency conditions.
[0079] A particularly preferred embodiment of the present invention
provides a vector comprising a nucleic acid molecule capable of
discriminating embryogenic from non-embryogenic material, wherein
said nucleic acid molecule comprises a sequence of nucleotides
substantially as set forth in SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form, or a nucleotide sequence having at least about
71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or a nucleotide
sequence capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3 or
its complementary form under low stringency conditions.
[0080] Furthermore, the vector may comprise a nucleic acid molecule
which when transcribed generates a mRNA which is antisense relative
to the transcript generated by SEQ ID NO:1 or SEQ ID NO:3 or its
related sequence. A related sequence includes a nucleotide sequence
having at least about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3
or its complementary form or a nucleotide sequence capable of
hybridizing to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form
under low stringency conditions. A related sequence, therefore,
includes a derivative and homologue.
[0081] In a further related embodiment, the invention is directed
to a vector comprising a polynucleotide sequence as broadly
described above wherein the polynucleotide sequence is operably
linked to one or more regulatory sequences, including but not
limited to a promoter sequence and/or a transcription terminator
sequence.
[0082] By "operably linked" is meant that transcriptional and
translational regulatory nucleic acids are positioned relative to a
functional coding region in such a manner that the functional
coding region is transcribed and optionally the polypeptide is
translated. The term "functional" includes a nucleotide sequence
which encodes a peptide, polypeptide or protein, or which exhibits
some other function such as but not limited to binding to DNA or
RNA.
[0083] By "vector" is meant a nucleic acid molecule, preferably a
DNA molecule derived, for example, from a plasmid, bacteriophage,
or plant virus, into which a nucleic acid sequence may be inserted
or cloned. A vector may also be a form of genetic construct. A
vector preferably contains one or more unique restriction sites and
may be capable of autonomous replication in a defined host cell
including a target cell or tissue or a progenitor cell or tissue
thereof, or be integrable with the genome of the defined host such
that the cloned sequence is reproducible. Accordingly, the vector
may be an autonomously replicating vector, i.e. a vector that
exists as an extrachromosomal entity, the replication of which is
independent of chromosomal replication, e.g. a linear or closed
circular plasmid, an extrachromosomal element, a minichromosome, or
an artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
which, when introduced into a cell, is integrated into the genome
of the recipient cell and replicated together with the
chromosome(s) into which it has been integrated. A vector system
may comprise a single vector or plasmid, two or more vectors or
plasmids, which together contain the total DNA to be introduced
into the genome of the host cell, or a transposon. The choice of
the vector will typically depend on the compatibility of the vector
with the cell into which the vector is to be introduced. The vector
may also include a selectable marker such as an antibiotic
resistance gene that can be used for selection of suitable
transformants. Examples of such resistance genes are well known to
those of skill in the art.
[0084] Reference herein to a "promoter" is to be taken in its
broadest context and includes the transcriptional regulatory
sequences of a classical genomic gene, including the TATA box which
is required for accurate transcription initiation, with or without
a CCAAT box sequence and additional regulatory elements (i.e.
upstream activating sequences, enhancers and silencers) which alter
gene expression in response to developmental and/or external
stimuli, or in a tissue-specific manner. A promoter is usually, but
not necessarily, positioned upstream or 5', or a structural gene
region, the expression of which it regulates. Furthermore, the
regulatory elements comprising a promoter are usually positioned
within 2 kb of the start site of transcription of the gene.
[0085] In the present context, the term "promoter" is also used to
describe a synthetic or fusion molecule, or derivative which
confers, activates or enhances expression of a nucleic acid
molecule in a cell. The term "expression" encompasses transcription
to a mRNA molecule alone or both transcription and translation to a
corresponding amino acid sequence. By "mRNA" is meant either a
sense or antisense mRNA molecule.
[0086] Preferred promoters may contain additional copies of one or
more specific regulatory elements, to further enhance expression of
the sense molecule and/or to alter the spatial expression and/or
temporal expression of said sense molecule. For example, regulatory
elements which confer copper inducibility may be placed adjacent to
a heterologous promoter sequence driving expression of a sense
molecule, thereby conferring copper inducibility on the expression
of said molecules.
[0087] Placing a nucleic acid molecule under the regulatory control
of a promoter sequence means positioning the said molecule such
that expression is controlled by the promoter sequence. Promoters
are generally positioned 5' (upstream) to the genes that they
control. In the construction of heterologous promoter/structural
gene combinations, it is generally preferred to position the
promoter at a distance from the gene transcription start site that
is approximately the same as the distance between that promoter and
the gene it controls in its natural setting, i.e. the gene from
which the promoter is derived. As is known in the art, some
variation in this distance can be accommodated without loss of
promoter function. Similarly, the preferred positioning of a
regulatory sequence element with respect to a heterologous gene to
be placed under its control is defined by the positioning of the
element in its natural setting, i.e. the genes from which it is
derived. Again, as is known in the art, some variation in this
distance can also occur.
[0088] Examples of promoters suitable for use in the synthetic
genes of the present invention include viral, fungal, bacterial,
animal and plant derived promoters capable of functioning in plant,
animal, insect, fungal, yeast or bacterial cells. The promoter may
regulate the expression of the structural gene component
constitutively, or differentially with respect to cell, the tissue
or organ in which expression occurs or, with respect to the
developmental stage at which expression occurs, or in response to
external stimuli such as physiological stresses, or pathogens, or
metal ions, amongst others.
[0089] Preferably, the promoter is capable of regulating expression
of a nucleic acid molecule in a eukaryotic cell, tissue or organ,
at least during the period of time over which the target gene is
expressed therein and more preferably also immediately preceding
the commencement of detectable expression of the target gene in
said cell, tissue or organ.
[0090] Accordingly, strong constitutive promoters are particularly
useful for the purposes of the present invention or promoters,
which may be induced by virus infection or the commencement of
target gene expression.
[0091] Plant-operable and animal-operable promoters are
particularly preferred for use in the construct of the present
invention. Examples of preferred promoters include the
bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter,
lac operator-promoter, tac promoter, SV40 late promoter, SV40 early
promoter, RSV-LTR promoter, CMV IF promoter, CaMV 35S promoter,
SCSV promoter, SCBV promoter and the like.
[0092] In consideration of the preferred requirement for high-level
expression which coincides with expression of the target gene or
precedes expression of the target gene, it is highly desirable that
the promoter sequence is a constitutive strong promoter such as the
CMV-IE promoter or the SV40 early promoter sequence, the SV40 late
promoter sequence, the CaMV 35S promoter, or the SCBV promoter,
amongst others. Those skilled in the art will readily be aware of
additional promoter sequences other than those specifically
described.
[0093] In the present context, the terms "in operable connection
with" or "operably under the control" or similar shall be taken to
indicate that expression of the structural gene region or multiple
structural gene region is under the control of the promoter
sequence with which it is spatially connected; in a cell, tissue,
organ or whole organism.
[0094] The construct preferably contains additional regulatory
elements for efficient transcription, for example, a transcription
termination sequence.
[0095] The term "terminator" refers to a DNA sequence at the end of
a transcriptional unit which signals termination of transcription.
Terminators are 3'-non-translated DNA sequences containing a
polyadenylation signal, which facilitates the addition of
polyadenylate sequences to the 3'-end of a primary transcript.
Terminators active in plant cells are known and described in the
literature. They may be isolated from bacteria, fungi, viruses,
animals and/or plants or synthesized de novo.
[0096] As with promoter sequences, the terminator may be any
terminator sequence which is operable in the cells, tissues or
organs in which it is intended to be used.
[0097] The present invention further extends to the promoter of the
gene sequence defined by SEQ ID NO:1 or SEQ ID NO:3. Accordingly,
another aspect of the present invention contemplates an isolated
nucleic acid molecule having promoter activity wherein, in its
naturally occurring form, the promoter is operably linked to a
nucleotide sequence substantially as set forth in SEQ ID NO:1 or
SEQ ID NO:3 or a nucleotide sequence complementary thereto or a
nucleotide sequence capable of hybridizing to SEQ ID NO:1 or SEQ ID
NO:3 or its complementary form under low stringency conditions.
[0098] Examples of terminators particularly suitable for use in the
synthetic genes of the present invention include the SV40
polyadenylation signal, the HSV TK polyadenylation signal, the CYC1
terminator, ADH terminator, SPA terminator, nopaline synthase (NOS)
gene terminator of Agrobacterium tumefaciens, the terminator of the
cauliflower mosaic virus (CaMV) 35S gene, the zein gene terminator
from Zea mays, the Rubisco small subunit gene (SSU) gene terminator
sequences, subclover stunt virus (SCSV) gene sequence terminators,
any rho-independent E. coli terminator, or the lacZ alpha
terminator, amongst others.
[0099] In a particularly preferred embodiment, the terminator is
the SV40 polyadenylation signal or the HSV TK polyadenylation
signal which are operable in animal cells, tissues and organs,
octopine synthase (OCS) or nopaline synthase (NOS) terminator
active in plant cells, tissue or organs, or the lacZ alpha
terminator which is active in prokaryotic cells.
[0100] Those skilled in the art will be aware of additional
terminator sequences, which may be suitable for use in performing
the invention. Such sequences may readily be used without any undue
experimentation.
[0101] Another aspect provides a host cell containing the nucleic
acid molecule of the present invention. In one embodiment the said
nucleic acid molecule is conveniently comprised within a vector as
hereinbefore described. In another embodiment, all or part of the
nucleic acid molecule of the present invention may be integrated
into the DNA of the host cell. Suitably, the host cell is a
bacterium or other prokaryote, or a plant cell or other eukaryote.
In a particularly preferred embodiment, the plant is oil-palm or a
related plant. A related plant is one which includes a plant having
similarity at the genetic, biochemical, immunological,
physiological or behavoural levels to oil-palm plants. Genetic
similarity, for example, includes similar codon usage, genetic
organization and nucleotide similarity (e.g. at least about 71%
similarity over defined regions).
[0102] Accordingly, a further aspect of the instant invention
provides a host cell comprising a nucleic acid molecule encoding a
polypeptide comprising an amino acid sequence substantially as set
forth in SEQ ID NO:2 or an amino acid sequence having at least
about 71% similarity to SEQ ID NO:2, wherein said polypeptide is
present in plant zygotic embryos or embryogenic callus and is
substantially not present in non-embryogenic tissue.
[0103] The present invention also contemplates the production of
recombinant proteins, polypeptides or peptides in a host cell. A
reference herein to "proteins", "polypeptides" or "peptides" is a
reference to a polymer of amino acid residues and to variants of
the same. The terms "proteins", "polypeptides" and "peptides" are
used interchangeably. The production of recombinant polypeptides is
useful, for example, to generate molecules for production of
antibodies for use as a diagnostic agent or as a potential
therapeutic.
[0104] Accordingly, another aspect of the present invention
provides an isolated polypeptide or biologically-active fragment
thereof or a variant or derivative of these, said polypeptide
comprising the amino acid sequence set forth in SEQ ID NO:2 or an
amino acid sequence having at least about 71% similarity to SEQ ID
NO:2, wherein said polypeptide is present in plant zygotic embryos
or embryogenic callus and is substantially not present in
non-embryogenic tissue.
[0105] In a related embodiment, the present invention is directed
to an isolated polypeptide comprising a sequence of amino acids
encoded by the nucleotide sequence substantially as set forth in
SEQ ID NO:1 or SEQ ID NO:3 or a nucleotide sequence having at least
about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form or a nucleotide sequence capable of hybridizing
to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form under low
stringency conditions.
[0106] In one embodiment, without wishing to limit the present
invention to any one theory or mode of operation, the polypeptide
may be useful as an antioxidant for the modulation of cellular
apoptotic processes. Cell wall metabolism involves deposition of
insoluble proteins that can be observed as thickening of the cell
wall surrounding the proembryos. The insolubization process has
been linked to the presence of hydrogen peroxide which accumulates
during metabolic processes. Hence, in a particular embodiment, the
nucleic acid molecule of the present invention encodes an
antiboxidant only found in embryogenic tissues, and which may be
responsible for protecting proembryos from being destroyed by the
accumulation of hydrogen peroxide within cells. In a particularly
preferred embodiment, the polypeptide of the present invention
encodes a peroxiredoxin useful for the modulation of cellular
apoptotic processes. In another embodiment, the polypeptide is
useful as an immunological agent to generate antibodies useful as
diagnostic markers.
[0107] By "biologically active fragment" is meant a fragment of a
full-length parent polypeptide which fragment retains the activity
of the parent polypeptide. A biologically active fragment may
therefore comprise peroxiredoxin activity, which protects tissues
from reactive oxygen species (ROS). Alternatively, or in addition,
the fragment may retain one or more epitopes for generating
antibodies therefor. As used herein, the term "biologically active
fragment" includes deletion mutants and small peptides, for
example, of at least 10, preferably at least 20 and more preferably
at least 30 contiguous amino acids, which comprise the above
activities. Peptides of this type may be obtained through the
application of standard recombinant nucleic acid techniques or
synthesized using conventional liquid or solid phase synthesis
techniques. For example, reference may be made to solution
synthesis or solid phase synthesis as described, for example, in
Chapter 9 entitled "Peptide Synthesis" by Atherton and Shephard
which is included in a publication entitled "Synthetic Vaccines"
edited by Nicholson and published by Blackwell Scientific
Publications. Alternatively, peptides can be produced by digestion
of a polypeptide of the invention with proteinases such as
endoLys-C, endoArg-C, endoGlu-C and staphylococcus V8-protease. The
digested fragments can be purified by, for example, high
performance liquid chromatographic (HPLC) techniques.
[0108] Hence, another aspect of the present invention provides a
method for producing a recombinant polypeptide in a host cell or
tissue, said method comprising introducing into the said cell or
tissue an expression vector comprising a nucleic acid molecule
wherein said nucleic acid molecule comprises a sequence of
nucleotides substantially as set forth in SEQ ID NO:1 or SEQ ID
NO:3 or its complementary form, or a nucleotide sequence having at
least about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form or a nucleotide sequence capable of hybridizing
to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form under low
stringency conditions wherein said nucleic acid molecule is
operably linked to one or more regulatory sequences such that the
nucleic acid molecule is capable of being expressed in said cell or
tissue.
[0109] According to another aspect of the invention, there is
provided a method for modulating apoptotic processes in a cell or
tissue, said method comprising introducing into said cell or tissue
an expression vector comprising a nucleic acid molecule, said
nucleic acid molecule comprising a sequence of nucleotides
substantially as set forth in SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form, or a nucleotide sequence having at least about
71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its complementary
form or a nucleotide sequence capable of hybridizing to SEQ ID NO:1
or SEQ ID NO:3 or its complementary form under low stringency
conditions wherein said nucleic acid molecule is operably linked to
one or more regulatory sequences such that the nucleic acid
molecule is capable of being expressed in said cell or tissue.
[0110] In an alternative embodiment, the present invention
contemplates a method for modulating apoptotic processes in a cell,
said method comprising administering to said cell an apoptotic
process-controlling effective amount of a recombinant polypeptide,
said polypeptide comprising the amino acid sequence set forth in
SEQ ID NO:2 or an amino acid sequence having at least about 71%
similarity to SEQ ID NO:2, said administration being for a time and
under conditions sufficient to modulate apoptosis.
[0111] The terms "modulating" and "modulate" include up-regulating
and down-regulating expression of the subject nucleic acid molecule
or levels of the instant polypeptide. Inducing apoptosis may be
useful in the treatment of plants and animals (including humans) of
cancers, galls and other outgrowths. Preventing apoptosis may be
important for treating neurodegenerative disorders or other
necrotic conditions. Accordingly, the present invention further
contemplates a composition, such as a pharmaceutical composition,
comprising the polypeptide of the instant invention or comprising
genetic molecules capable of encoding said polypeptide. Such
composition generally also comprises one or more pharmaceutically
acceptable carriers and/or diluents.
[0112] Means of introducing vectors into cells or tissues (i.e.
transfecting or transforming target cells) are well-known to those
skilled in the art.
[0113] The constructs described supra are capable of being modified
further, for example, by the inclusion of marker nucleotide
sequences encoding a detectable marker enzyme or a functional
analogue or derivative thereof, to facilitate detection of the
synthetic gene in a cell, tissue or organ in which it is expressed.
According to this embodiment, the marker nucleotide sequences will
be present in a translatable format and expressed, for example, as
a fusion polypeptide with the translation product(s) of any one or
more of the structural genes or alternatively as a non-fusion
polypeptide.
[0114] Those skilled in the art will be aware of how to produce the
synthetic genes described herein and of the requirements for
obtaining the expression thereof, when so desired, in a specific
cell or cell-type under the conditions desired. In particular, it
will be known to those skilled in the art that the genetic
manipulations required to perform the present invention may require
the propagation of a genetic construct described herein or a
derivative thereof in a prokaryotic cell such as an E. coli cell or
a plant cell or an animal cell.
[0115] The constructs of the present invention may be introduced to
a suitable cell, tissue or organ without modification as linear
DNA, optionally contained within a suitable carrier, such as a
cell, virus particle or liposome, amongst others. To produce a
genetic construct, the synthetic gene of the invention is inserted
into a suitable vector or opisome molecule, such as a bacteriophage
vector, viral vector or a plasmid, cosmid or artificial chromosome
vector which is capable of being maintained and/or replicated
and/or expressed in the host cell, tissue or organ into which it is
subsequently introduced.
[0116] Accordingly, another aspect of the present invention
provides a genetic construct comprising a nucleic acid molecule
encoding a polypeptide comprising an amino acid sequence
substantially as set forth in SEQ ID NO:2 or an amino acid sequence
having at least about 71% similarity to SEQ ID NO:2, wherein said
polypeptide is present in plant zygotic embryos or embryogenic
callus and is substantially not present in non-embryogenic
tissue.
[0117] In a related aspect of the invention, there is provided a
genetic construct which at least comprises a nucleic acid molecule
comprising a sequence of nucleotides substantially as set forth in
SEQ ID NO:1 or SEQ ID NO:3 or its complementary form, or a
nucleotide sequence having at least about 71% similarity to SEQ ID
NO:1 or SEQ ID NO:3 or its complementary form or a nucleotide
sequence capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3 or
its complementary form under low stringency conditions and one or
more origins of replication and/or selectable marker gene
sequences.
[0118] Genetic constructs are particularly suitable for the
transformation of a eukaryotic cell to introduce novel genetic
traits thereto, in addition to the provision of resistance
characteristics to viral pathogens. Such additional novel traits
may be introduced in a separate genetic construct or,
alternatively, on the same genetic construct which comprises the
synthetic genes herein described. Those skilled in the art will
recognize the significant advantages, in particular in terms of
reduced genetic manipulations and tissue culture requirements and
increased cost-effectiveness of including genetic sequences which
encode such additional traits and the synthetic genes described
herein in a single genetic construct.
[0119] Usually, an origin of replication or a selectable marker
gene suitable for use in bacteria is physically-separated from
those genetic sequences contained in the genetic construct which
are intended to be expressed or transferred to a eukaryotic cell,
or integrated into the genome of a eukaryotic cell.
[0120] As used herein, the term "selectable marker gene" includes
any gene which confers a phenotype on a cell on which it is
expressed to facilitate the identification and/or selection of
cells which are transfected or transformed with a genetic construct
of the invention or a derivative thereof.
[0121] Suitable selectable marker genes contemplated herein include
the ampicillin-resistance gene (Amp.sup.r), tetracycline-resistance
gene (Tc.sup.r), bacterial kanamycin-resistance gene (Kan.sup.r),
is the zeocin resistance gene (Zeocin is a drug of the bleomycin
family which is trade mark of InVitrogen Corporation), the AURI-C
gene which confers resistance to the antibiotic aureobasidin A,
phosphinothricin-resistance gene, neomycin phosphotransferase gen
(nptII), hygromycin-resistance gene, .E-backward.-glucuronidase
(GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green
fluorescent protein-encoding gene or the luciferase gene, amongst
others.
[0122] Preferably, the selectable marker gene is the nptII gene or
Kan.sup.r gene or green fluorescent protein (GFP)-encoding
gene.
[0123] Those skilled in the art will be aware of other selectable
marker genes useful in the performance of the present invention and
the subject invention is not limited by the nature of the
selectable marker gene.
[0124] The present invention extends to all genetic constructs
essentially as described herein, which include further genetic
sequences intended for the maintenance and/or replication of said
genetic construct in prokaryotes or eukaryotes and/or the
integration of said genetic construct or a part thereof into the
genome of a eukaryotic cell or organism.
[0125] Standard methods described supra may be used to introduce
the constructs into the cell, tissue or organ, for example,
liposome-mediated transfection or transformation, transformation of
cells with attenuated virus particles or bacterial cells, cell
mating, transformation or transfection procedures known to those
skilled in the art.
[0126] Additional means for introducing recombinant DNA into plant
tissue or cells include, but are not limited to, transformation
using CaCl.sub.2 and variations thereof, direct DNA uptake into
protoplasts, PEG-mediated uptake to protoplasts, microparticle
bombardment, electroporation, microinjection of DNA, microparticle
bombardment of tissue explant or cells, vacuum-infiltration of
tissue with nucleic acid, or in the case of plants, T-DNA-mediate
transfer from Agrobacterium to the plant tissue.
[0127] For microparticle bombardment of cells, a microparticle is
propelled into a cell to produce a transformed cell. Any suitable
ballistic cell transformation methodology and apparatus can be used
in performing the present invention. Exemplary apparatus and
procedures are disclosed by Stomp et al. (U.S. Pat. No. 5,122,466)
and Sanford and Wolf (U.S. Pat. No. 4,945,050). When using
ballistic transformation procedures, the genetic construct may
incorporate a plasmid capable of replicating in the cell to be
transformed.
[0128] Examples of microparticles suitable for use in such systems
include 1 to 5 .mu.m gold spheres. The DNA construct may be
deposited on the microparticle by any suitable technique, such as
by precipitation.
[0129] In a further embodiment of the present invention, the
genetic constructs described herein are adapted for integration
into the genome of a cell in which it is expressed. Those skilled
in the art will be aware that, in order to achieve integration of a
genetic sequence or genetic construct into the genome of a host
cell, certain additional genetic sequences may be required. In the
case of plants, left and right border sequences from the T-DNA of
the Agrobacterium tumefaciens Ti plasmid will generally be
required.
[0130] According to another aspect of the invention, there is
provided a transformed plant cell containing an expression vector
as broadly herein described. The term "plant cell" as used herein
refers to protoplasts or other cells derived from plants,
gamete-producing cells, and cells which regenerate into whole
plants. Plant cells include cells in plants as well as protoplasts
or other cells in culture. By "plant tissue" is meant
differentiated and undifferentiated tissue derived from roots,
shoots, pollen, seeds, tumour tissue, such as crown galls, and
various forms of aggregations of plant cells in culture, such as
embryos and calluses.
[0131] In a still further aspect, the invention provides a
regenerated differentiated plant consisting of plant cells
containing an expression vector as broadly herein described. Plants
may conveniently be regenerated from transformed plant cells or
tissues or organs on hormone-containing media and the regenerated
plants may take a variety of forms, such as chimeras of transformed
cells and non-transformed cells; clonal transformants (e.g. all
cells transformed to contain the expression cassette); grafts of
transformed and untransformed tissue (e.g. a transformed root stock
grafted to an untransformed scion in citrus species). Transformed
plants may be propagated by a variety of means, such as by clonal
propagation or classical breeding techniques. For example, a first
generation (or T1) transformed plants may be selfed to give
homozygous second generation (or T2) transformed plants, and the T2
plants further propagated through classical breeding
techniques.
[0132] Accordingly, yet another aspect of the invention is directed
to a regenerated differentiated plant comprising a nucleic acid
molecule encoding a polypeptide comprising an amino acid sequence
substantially as set forth in SEQ ID NO:2 or an amino acid sequence
having at least about 71% similarity to SEQ ID NO:2, wherein said
polypeptide is present in plant zygotic embryos or embryogenic
callus and is substantially not present in non-embryogenic
tissue.
[0133] In a related embodiment, the present invention is directed
to a regenerated differentiated plant comprising a nucleic acid
molecule, wherein the nucleic acid molecule comprises a sequence of
nucleotides substantially as set forth in SEQ ID NO:1 or SEQ ID
NO:3 or its complementary form, or a nucleotide sequence having at
least about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form or a nucleotide sequence capable of hybridizing
to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form under low
stringency conditions.
[0134] As used herein, "plant" and "differentiated plant" refer to
a whole plant or plant part containing differentiated plant cell
types, tissues and/or organ systems. Plantlets and seeds are also
included within the meaning of the foregoing terms. Plants included
in the invention are any plants amenable to transformation
techniques, including angiosperms, gymnosperms, monocotyledons and
dicotyledons. In a most preferred embodiment, the plant is oil-palm
or a related plant.
[0135] In yet another aspect, the invention provides oil-palm
harvested from a differentiated plant as broadly described
above.
[0136] The nucleic acid molecule of the present invention is
useful, inter alia, to distinguish embryogenic from non-embryogenic
material. Accordingly, embryogenic material may be detected in
vitro by screening for expression of the subject nucleic acid
molecule. As defined above, expression may result in transcript or
translation product or both. A range of assays may be employed to
detect nucleic acid transcript or translation products. These
assays are well known to those skilled in the art and particularly
useful assays are described below.
[0137] Accordingly, one aspect of the present invention
contemplates a method for detecting embryogenic plant material,
said method comprising screening for expression of a nucleic acid
molecule, said nucleic acid molecule comprising a sequence of
nucleotides substantially as set forth in SEQ ID NO:1 or SEQ ID
NO:3 or its complementary form, or a nucleotide sequence having at
least about 71% similarity to SEQ ID NO:1 or SEQ ID NO:3 or its
complementary form or a nucleotide sequence capable of hybridizing
to SEQ ID NO:1 or SEQ ID NO:3 or its complementary form under low
stringency conditions wherein expression of said nucleic acid
molecule is indicative of the presence of embryogenic material.
[0138] Reference to "material" includes reference to cells,
tissues, callus and/or organelles or related tissue. The expression
"detecting" embryogenic plant material includes distinguishing
between embryogenic and non-embryogenic material.
[0139] The assay may be conducted in any number of ways. For
example, mRNA transcript may be detected as the expression product.
In one method, Northern blot analysis may be used.
[0140] According to this embodiment, there is provided a method for
detecting embryogenic plant material, said method comprising
immobilizing a sample putatively containing RNA from the material
to be screened on a solid support and contacting said immobilized
RNA with a labelled nucleotide sequence capable of hybridizing to
all or part of an mRNA transcript corresponding to the nucleotide
sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or their
derivatives or homologues as defined herein and then detecting the
presence of said label.
[0141] The label may be any reporter molecule capable of providing
an identifiable signal such as .sup.32P, .sup.35S, or other
radionucleotide, fluorogenic molecule, enzyme or other suitable
reporter molecule.
[0142] By "immobilized" means both a "dot blot" type assay or an
electrophoretic assay where the total RNA is subjected to
electrophoresis.
[0143] The probe is preferably a cDNA molecule including a fragment
(e.g. from about 8 nucleotides in length) or whole or substantially
whole length molecules corresponding to SEQ ID NO:1 or SEQ ID NO:3
or its complementary form. Alternatively, the probe is a RNA
molecule complementary to the target mRNA sequence.
[0144] Any number of variations may be performed to the assay
without departing from the scope or spirit of the invention.
[0145] In another embodiment, expression is determined by detecting
the translation product, i.e. a sequence of amino acids such as in
the form of a peptide, polypeptide or protein (encompassed herein
by the term "polypeptide").
[0146] In one useful embodiment, antibodies are generated to the
subject polypeptide. Such antibodies may be used in an immunoassay
to detect the instant polypeptide. The presence of the polypeptide
is indicative of embryogenic material.
[0147] Accordingly, another aspect of the present invention
contemplates an antibody to a polypeptide, said polypeptide
comprising a sequence of amino acids substantially as set froth in
SEQ ID NO:2 or an amino acid sequence having at least about 71%
similarity to SEQ ID NO:2, wherein said polypeptide is present in
plant zygotic embryos or embryogenic callus and is substantially
not present in non-embryogenic tissue.
[0148] Either monoclonal or polyclonal antibodies may be employed.
The use of monoclonal antibodies in an immunoassay is particularly
preferred because of the ability to produce them in large
quantities and the homogeneity of the product. The preparation of
hybridoma cell lines for monoclonal antibody production derived by
fusing an immortal cell line and lymphocytes sensitized against the
immunogenic preparation can be done by techniques which are well
known to those who are skilled in the art. (See, for example,
Douillard and Hoffman, (1981); Kohler and Milstein, (1975);
(1976).
[0149] Another aspect of the present invention contemplates a
method for detecting a polypeptide which is indicative of the
presence of embryogenic tissue in oil-palm or related plants, said
method comprising contacting the tissue or an extract thereof with
an antibody specific for said polypeptide or its derivatives or
homologues for a time and under conditions sufficient for an
antibody-polypeptide complex to form, and then detecting said
complex.
[0150] The presence of the polypeptide may be detected in any
number of ways such as by Western blotting and ELISA procedures. A
wide range of immunoassay techniques are available as can be seen
by reference to U.S. Pat. Nos. 4,016,043, 4,424,279 and
4,018,653.
[0151] In one assay, an unlabelled antibody specific to the
oil-palm polypeptide is immobilized on a solid substrate and the
sample to be tested brought into contact with the bound molecule.
After a suitable period of incubation, for a period of time
sufficient to allow formation of an antibody-polypeptide complex, a
second antibody specific to the polypeptide, labelled with a
reporter molecule capable of producing a detectable signal, is then
added and incubated, allowing time sufficient for the formation of
another complex of antibody-polypeptide-labelled antibody. Any
unreacted material is washed away, and the presence of the
polypeptide is determined by observation of a signal produced by
the reporter molecule. The results may either be qualitative, by
simple observation of the visible signal, or may be quantitated by
comparing with a control ample containing known amounts of
polypeptide. Variations on this assay include a simultaneous assay,
in which both sample and labelled antibody are added simultaneously
to the bound antibody. These techniques are well known to those
skilled in the art, including any minor variations as will be
readily apparent. In accordance with the present invention, the
sample is one which might contain the oil-palm polypeptide
including cell or callus extract or lysate. The sample is,
therefore, generally a biological sample.
[0152] In this assay, a first antibody having specificity for the
polypeptide or antigenic parts thereof, is either covalently or
passively bound to a solid surface. The solid surface is typically
glass or a polymer, the most commonly used polymers being
cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride
or polypropylene. The solid supports may be in the form of tubes,
beads, discs of microplates, or any other surface suitable for
conducting an immunoassay. The binding processes are well-known in
the art and generally consist of cross-linking covalently binding
or physically adsorbing, the polymer-antibody complex is washed in
preparation for the test sample. An aliquot of the sample to be
tested is then added to the solid phase complex and incubated for a
period of time sufficient (e.g. 2-40 minutes or overnight if more
convenient) and under suitable conditions (e.g. from room
temperature to about 38.degree. C. such as 25.degree. C.) to allow
binding of the antibody. Following the incubation period, the
antibody solid phase is washed and dried and incubated with a
second antibody specific for a portion of the polypeptide. The
second antibody is linked to a reporter molecule which is used to
indicate the binding of the second antibody to the polypeptide.
[0153] An alternative method involves immobilizing the target
molecules in the biological sample and then exposing the
immobilized target to specific antibody which may or may not be
labelled with a reporter molecule. Depending on the amount of
target and the strength of the reporter molecule signal, a bound
target may be detectable by direct labelling with the antibody.
[0154] Alternatively, a second labelled antibody, specific to the
first antibody is exposed to the target-first antibody complex to
form a target-first antibody-second antibody tertiary complex. The
complex is detected by the signal emitted by the reporter
molecule.
[0155] By "reporter molecule", as used in the present
specification, is meant a molecule which, by its chemical nature,
provides an analytically identifiable signal which allows the
detection of antigen-bound antibody. Detection may be either
qualitative or quantitative. The most commonly used reporter
molecules in this type of assay are either enzymes, fluorophores or
radionuclide containing molecules (i.e. radioisotopes) and
chemiluminescent molecules.
[0156] In the case of an enzyme immunoassay (ETA), an enzyme is
conjugated to the second antibody, generally by means of
glutaraldehyde or periodate. As will be readily recognized,
however, a wide variety of different conjugation techniques exist,
which are readily available to the skilled artisan. Commonly used
enzymes include horseradish peroxidase, glucose oxidase,
-galactosidase and alkaline phosphatase, amongst others. The
substrates to be used with the specific enzymes are generally
chosen for the production, upon hydrolysis by the corresponding
enzyme, of a detectable color change. Examples of suitable enzymes
include alkaline phosphatase and peroxidase. It is also possible to
employ fluorogenic substrates, which yield a fluorescent product
rather than the chromogenic substrates noted above. In all cases,
the enzyme-labelled antibody is added to the first
antibody-polypeptide complex, allowed to bind, and then the excess
reagent is washed away. A solution containing the appropriate
substrate is then added to the complex of
antibody-polypeptide-antibody. The substrate will react with the
enzyme linked to the second antibody, giving a qualitative visual
signal, which may be further quantitated, usually
spectrophotometrically, to give an indication of the amount of
hapten which was present in the sample. "Reporter molecule" also
extends to use of cell agglutination or inhibition of agglutination
such as red blood cells on latex beads, and the like.
[0157] Alternately, fluorescent compounds, such as fluorescein and
rhodamine, may be chemically coupled to antibodies without altering
their binding capacity. When activated by illumination with light
of a particular wavelength, the fluorochrome-labelled antibody
adsorbs the light energy, inducing a state to excitability in the
molecule, followed by emission of the light at a characteristic
color visually detectable with a light microscope. As in the EIA,
the fluorescent labelled antibody is allowed to bind to the first
antibody-polypeptide complex. After washing off the unbound
reagent, the remaining tertiary complex is then exposed to the
light of the appropriate wavelength, the fluorescence observed
indicates the presence of the hapten of interest Immunofluorescene
and EIA techniques are both very well established in the art and
are particularly preferred for the present method. However, other
reporter molecules, such as radioisotope, chemiluminescent or
bioluminescent molecules, may also be employed.
[0158] The present invention, therefore, provides in one
embodiment, a screening procedure to identify, detect or otherwise
discriminate between embryogenic and non-embryogenic material. In
this regard, substantial savings in time and cost may be made by
removing non-embryogenic material from tissue being used in in
vitro multiplication of oil-palm or related plants. The ability to
distinguish embryogenic cultures from the non-embryogenic cultures
at an early stage facilitates culling of cultures and thus
expensive laboratory space can be saved as well as months of labor.
Furthermore, the ability to control embryogenesis would meant that
cultures need not depend on random chance to attain their
embryogenic potential. The assay of the present invention may also
be automated or semi-automated where one or more steps are
controlled by a computer programme. Alternatively or in addition,
the present invention further provides a test kit for identifying
embryogenic material, said test kit in compartmental form comprises
in one compartment, an agent for detecting a nucleic acid or
polypeptide associated with embryogenic material in oil-palm plants
or related plants; a second or further compartments are adapted to
contain reagents including solid supports for detecting the subject
nucleic acid molecule or polypeptide.
[0159] The polypeptide of the present invention is also useful in
therapeutic treatments, such as in anti-aging.
[0160] Accordingly, another aspect of the present invention
contemplates a composition such as a pharmaceutical composition
comprising the polypeptide having an amino acid sequence as set
forth in SEQ ID NO:2 or a functional homologue thereof or a
molecule having at least 71% similarity to SEQ ID NO:2 and one or
more pharmaceutically acceptable carriers and/or diluents.
[0161] The preferred composition of the present invention is in the
form of a pharmaceutical composition.
[0162] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions (where water soluble) and sterile powders
for the extemporaneous preparation of sterile injectable solutions.
It must be stable under the conditions of manufacture and storage
and must be preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier can be a
solvent or dilution medium comprising, for example, water, ethanol,
polyol (for example, glycerol, propylene glycol and liquid
polyethylene glycol, and the like), suitable mixtures thereof and
vegetable oils. The proper fluidity can be maintained, for example,
by the use of superfactants. The preventions of the action of
microorganisms can be brought about by various anti-bacterial and
anti-fungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thirmerosal and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminium monostearate and
gelatin.
[0163] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with the active ingredient and optionally other active
ingredients as required, followed by filtered sterilization or
other appropriate means of sterilization. In the case of sterile
powders for the preparation of sterile injectable solutions,
suitable methods of preparation include vacuum drying and the
freeze-drying technique which yield a powder of active ingredient
plus any additionally desired ingredient.
[0164] When the active ingredient is suitably protected, it may be
orally administered, for example, with an inert diluent or with an
assimilable edible carrier, or it may be enclosed in hard or soft
shell gelatin capsule, or it may be compressed into tablets, or it
may be incorporated directly with the food of the diet or
administered via breast milk. For oral therapeutic administration,
the active ingredient may be incorporated with excipients and used
in the form of ingestible tablets, buccal tablets, troches,
capsules, elixirs, suspensions, syrups, wafers and the like. Such
compositions and preparations should contain at least 1% by weight
of active compound. The percentage of the compositions and
preparations may, of course, be varied and may conveniently be
between about 5 to about 80% of the weight of the unit. The amount
of active compound in such therapeutically useful compositions is
such that a suitable dosage will be obtained. Preferred
compositions or preparations according to the present invention are
prepared so that an oral dosage unit form contains between about
0.1 .mu.g and 200 mg of active compound. Alternative dosage amounts
include from about 1 .mu.g to about 1000 mg and from about 10 .mu.g
to about 500 mg. These dosages may be per individual or per kg body
weight. Administration may be per hour, day, week, month or
year.
[0165] The tablets, troches, pills, capsules, creams and the like
may also contain the components as listed hereafter. A binder such
as gum, acacia, corn starch or gelatin; excipients such as
dicalcium phosphate; a disintegrating agent such as corn starch,
potato starch, alginic acid and the like; a lubricant such as
magnesium stearate; and a sweetening agent such as sucrose, lactose
or saccharin may be added or a flavouring agent such as peppermint,
oil of wintergreen or cherry flavouring. When the dosage unit form
is a capsule, it may contain, in addition to materials of the above
type, a liquid carrier. Various other materials may be present as
coatings or to otherwise modify the physical form of the dosage
unit. For instance, tablets, pills or capsules may be coated with
shellac, sugar or both. A syrup or elixir may contain the active
compound, sucrose as a sweetening agent, methyl and propylparabens
as preservatives, a dye and flavouring such as cherry or orange
flavour. Of course, any material used in preparing any dosage unit
form should be pharmaceutically pure and substantially non-toxic in
the amounts employed. In addition, the active compound(s) may be
incorporated into sustained-release preparations and
formulations.
[0166] Pharmaceutically acceptable carriers and/or diluents include
any and all solvents, dispersion media, coatings, anti-bacterial
and anti-fungal agents, isotonic and absorption delaying agents and
the like. The use of such media and agents for pharmaceutical
active substances is well known in the art and except insofar as
any conventional media or agent is incompatible with the active
ingredient, their use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0167] It is particularly advantageous to incorporate the active
ingredient as a cream capable of preventing or delaying aging.
[0168] The present invention is further described by the following
non-limiting Examples.
Example 1
Reverse Transcription of PCR (RT-PCR)
[0169] A one-step RT-PCR was carried out with the use of the Titan
One Tube RT-PCR System from Boehringer Mannheim.
[0170] The reaction components for master mix 1 and master mix 2
were prepared in separate DEPC-treated tubes. The components in
master mix 1 were: 4 .mu.l of dNTP mix (10 mM)+1 .mu.l downstream
primer (10 .mu.M)+1 .mu.l upstream primer (10 .mu.M)+1 .mu.l
template RNA (.about.1 .mu.g zygotic embryo total RNA)+2.5 .mu.l
DTT solution (100 mM)+7 .mu.l RNase Inhibitor (40 U/.mu.l)+sterile
H.sub.2O to a final volume of 25 .mu.l. The primers used here were
the AGL15AtF (5'-AGGAGGATTGTGCAGAG-3' [SEQ ID NO:5]) and AGL15AtR
(5'-CAAACTCTCAGCTAGGCA-3' [SEQ ID NO:6]) based on Heck et al.
(1995).
[0171] In master mix 2, 10 .mu.l of the 5.times.RT-PCR buffer with
Mg.sup.2+ and 1 .mu.l of the enzyme mix (AMY reverse
transcriptase+Expand High Fidelity enzyme mix) were added together
in a total volume of 25 .mu.l made up by H.sub.2O. The whole 25
.mu.l of master mix 1 and master mix 2 were added into a 0.2 ml
thin-walled PCR tube on ice. The contents were mixed properly and
briefly centrifuged to collect the sample at the bottom of the
tube. The preparation was overlaid with 30 .mu.l mineral oil and
placed in a thermocycler, which had been equilibrated at 50.degree.
C. for 30 min, after which the programme went directly into
thermocycling.
[0172] The parameters had been set at: 94.degree. C. for 2 min; 10
cycles at (94.degree. C. for 30 sec; 50.degree. C. for 30 sec;
68.degree. C. for 45 sec); 25 cycles at (94.degree. C. for 30 sec;
50.degree. C. for 30 sec; 68.degree. C. for 45 sec+cycle elongation
of 5 sec for each cycle); a final extension at 68.degree. C. for 7
min. The RT-PCR product was analyzed on a 2% agarose gel. Bands
were excised from the gel and purified using the Concert Rapid Gel
Extraction kit (Clontech) and cloned into pCRScript (Stratagene).
This cloning kit is very similar to the Zero Blunt TOPO PCR cloning
kit except that clones are selected on LB+50 .mu.g/ml ampicillin
medium.
[0173] Two bands were obtained with AGL15AtF and AGL15AtR (FIG. 1).
Both bands were excised, the ends were polished and they were then
cloned into pCR-Script (Stratagene) and transformed into DH5.alpha.
competent cells. The successfully transformed clones were
designated RTPCR1 (for clone from fragment 1) and RTPCR2 (for clone
from fragment 2). RTPCR1 and RTPCR2 were cultured and plasmid
minipreps were done.
Example 2
Sequence Analysis
[0174] Clones to be sequenced were sent to ACGT (USA) and the
sequencing was performed on a single-run basis using universal
primers T3/T7 for most clones except for clones from the enriched
library, where the primers PN1/PN2 were used. Sequence analyses
were carried out using DNASIS (Hitachi software package, 1997) and
BLAST (Basic Local Alignment Search Tool) (Altshul et al., 1990;
1997), available via the internet at http://www.ncbi.nlm.nih.gov.
To analyze the sequences with their closely related counterparts,
the alignment of the sequences was done using the CLUSTALW
programme available from the Biology Workbench version 3.2 at
http://biology.nesa.uiuc.edu.
[0175] Both analyses gave similar results. RTPCR1 was shown to have
about 71% homology (DNASIS) to an embryo specific mRNA of Bromus
secalinas, which is found to be up-regulated in hydrated dormant
seeds (Goldmark et al., 1992), as well as a few other dormancy
related genes. RTPCR2, on the other hand was found to have a
homology (>71%) to an S-phase specific gene (Uchimiya et al.,
1994) which is involved in the cell cycle. Through the sequencing
results, it was realized that the reason two very distinct bands
were produced was because each band was generated by only one type
of primer. That is, RTPCR1 by AGL15AtR and RTPCR2 by AGL15AtF.
Example 3
Northern Hybridization
[0176] The different types of poly A.sup.+ RNA extracted were
electrophoresed on 2% v/v formaldehyde gels and transferred onto
nylon membranes using standard blotting techniques (Maniatis et
al., 1982). Two percent formaldehyde gels (120 ml) were prepared by
melting down 3 g of agarose in 110 ml of DEPC-treated H.sub.2O and
15 ml 10.times.MOPS buffer (200 mM MOPS (Ph 7.0), 10 mM EDTA, 50 mM
sodium acetate). When the mixture cooled down to about 55.degree.
C., 37% formaldehyde was added to a final concentration of 6%. The
contents were properly mixed and then poured into the gel casting
tray and allowed to set.
[0177] In the preparation of the RNA samples for analysis, about 10
to 15 .mu.g of total RNA was used for each different type of
tissue. The RNA samples used were in a small volume of H.sub.2O. At
times, the samples needed to be concentrated: the maximum volume of
RNA that can be accommodated was 4.8 .mu.l for each preparation. In
each tube, the required amount of RNA (if less than 4.8 .mu.l was
needed, H.sub.2O was added to a final volume of 4.8 .mu.l) was
added into 2 .mu.l of 10.times.MOPS buffer, 3.2 .mu.l of 37%
formaldehyde and 10 .mu.l of formamide, making the total volume 20
.mu.l. The samples were placed in a heating block at 65.degree. C.
for 15 min and immediately chilled on ice. Just before loading, 2
.mu.l of loading buffer (50% glycerol+1 mM EDTA+0.25% bromophenol
blue+0.25% xylene cyanole) was added into each tube. Gels were
electrophoresed in 1.times.MOPS buffer at a low voltage (20 to 30V)
until the bromophenol blue dye reached the bottom of the gel.
[0178] The gel was stained with 0.5 .mu.g/ml ethidium bromide in
200 mM ammonium acetate for 45 min to 1 hr. This was followed by
destaining with several changes of DEPC-treated H.sub.2O, until the
bands of the RNA marker (Gibco BRL) and the rRNA of the samples
were clearly visible. The individual bands of the marker were
marked by making a hole in them. Similarly, the rRNA bands (28S and
18S) of the samples were also randomly marked. The gels were rinsed
with DEPC-treated H.sub.2O several times to remove the
formaldehyde, followed by a final rinse with 2.times.SSC before
blotting. The gel was placed on its reverse side on the blotting
apparatus, making sure that no bubbles were trapped in between the
gel and the wick, which was made of 2 pieces of 3MM Whatman
chromatography paper that had been cut to size. The wick forms a
bridge on a glass plate placed across a container with 10.times.SSC
as the transfer buffer. A positively charged nylon membrane was cut
to size, along with 4 pieces of 3MM Whatman chromatography paper of
the same size, and pre-wetted in 2.times.SSC. First, the wet
membrane was placed carefully onto the gel, without trapping any
air bubbles, and this was followed by the pre-wetted paper. Another
4 pieces of dry 3MM Whatman chromatography paper and a stack of
paper towels were placed over this. A glass plate was placed right
at the top of this set-up and weights were added to keep them in
direct contact with each other. The transfer was allowed to occur
for at least 16 hr.
[0179] After the transfer was completed, the blotting set-up was
dismantled and the marks previously made on the gel were penciled
onto the membrane. The membrane was then rinsed in 2.times.SSC for
15 min and auto-crosslinked at 120,000 .mu.l of UV energy or
alternatively baked at 80.degree. C. for 1.5 to 2 hr.
[0180] The probes were radioactively labeled using the High Prime
kit (Boehringer Mannheim). As a control, 18S rRNA probe was also
hybridized to the RNA blots. The 18S rRNA probe was prepared by
double-digesting the pBG35 plasmid (Malaysian Palm Oil Board) with
KpnI (Promega) and EcoR1 (Promega) at 37.degree. C. overnight. The
digest contained 1.0 .mu.l plasmid pBG35, 2.0 .mu.l 10.times.
restriction buffer, 1.5 .mu.l EcoR1 (12 U/.mu.l), 1.5 .mu.l Kpn1
(12 U/.mu.l) and sterile H.sub.2O to a final volume of 20 .mu.l.
The digestion was electrophoresed on a 0.8% agarose gel and the
desired 1.6 kb band was excised from the gel and purified. Usually
in the case of Northern analysis, high stringency washes were
applied (up to 0.5.times.SSC+0.1% SDS at 65.degree. C. for 15
min).
[0181] An initial, simple Northern analysis was carried out on the
two RTPCR clones and interestingly, RTPCR1 was found to be
expressed only in zygotic embryo (ZE) and embryogenic callus (EC),
indicating that it may be an embryogenic related gene. By
comparison, RTPCR2 was constitutively expressed (FIG. 1b).
Example 4
(a) Further Characterization: Sequence of Full-Length Clone
[0182] Because of the interesting results obtained with RTPCR1,
this clone was further studied. RTPCR1 was only of partial length
when obtained through RT-PCR, therefore the zygotic embryo cDNA
library was screened to obtain its full-length clone. The resultant
clone was named as OPEm1. The nucleotide and deduced amino acid
sequences of OPEm1 is shown in FIG. 3. It contains an open reading
frame (ORF) from position 30 to 605 encoding a protein with 192
amino acids. A hydrophobic region occurs close to the
carboxyl-terminus of the predicted protein, which has a predicted
pI of 7.48 (FIG. 2b).
(b) Detailed Northern Analysis
[0183] A more detailed Northern blot was prepared to reconfirm the
previous results obtained (FIG. 2a). The transcript size of OPEm1
was determined to be approximately 1995 nucleotides (nt). The
expression pattern of OPEm1 further proves that this clone has the
potential to be exploited as an embryogenic marker because its
expression can be detected in all the embryogenic calli regardless
of their clonal differences, in suspension cultures (lanes 9 and
10), in embryoids (lanes 11 and 12) right up to somatic embryos in
the form of bipolar structures (lane 13). Expression signals in
lanes 9, 10 and 13 are a little faint but were still able to be
visibly detected on the autoradiograph. Expression was also
detected in zygotic embryo (lane 15). The expression of OPEm1 was
not found in cultures that had lost their embryogenic potential
(lanes 2, 4 and 7), non-embryogenic calli (lanes 5 and 8) as well
as other vegetative tissues such as the meristem, inflorescences
and young unexpended leaves of the oil-palm (lanes 16, 17 and 18
respectively).
Example 5
Southern Hybridization
[0184] Genomic DNA of the oil-palm extracted from young unexpanded
leave. The genomic DNA (5 to 10 .mu.g) was digested with several
different enzymes. Each digestion contained 30 .mu.l of genomic DNA
(10 .mu.g), 5 .mu.l of 10.times. restriction buffer, 5 .mu.l of BSA
(1 mg/ml), 5 .mu.l enzyme (EcoR1, BamH1, Hind III, Kpn1, Not1,
Sfi1, Spe1 and Stu1) and sterile H.sub.2O to a final volume of 50
.mu.l. The digestion was carried out at 37.degree. C. and complete
digestion was ensured by an overnight incubation. The digested DNA
was electrophoresed on a 1.0% agarose gel along side a 1 kb DNA
molecular weight marker (Promega). After the run, the gel was
photographed and holes were made to mark the positions of the bands
belonging to the marker. The gel was then immersed in depurination
solution (0.25 N HCl) for 10 min with gentle shaking. The solution
was decanted and the gel was rinsed several times with sterile
H.sub.2O, after which denaturation solution (0.5 M NaOH, 1.5 M
NaCl) was added to the gel and agitated for 30 min. This was again
followed by several rinses of sterile H.sub.2O and finally the gel
was neutralized with the neutralization solution (3 M NaCl, 0.5 M
Tris-HCl (Ph 7.4)) for 30 min.
[0185] A similar blotting apparatus as for the Northern was set up
for Southern analysis. Southern analysis showed that OPEm1 gene may
be a member of a multigene family (FIG. 2c).
Example 6
3D-Structure
[0186] Since the results of the Northern analyses seems to
contradict the function inferred for the gene based on its sequence
similarity to dormancy-related genes, it was decided that the
protein structure of OPEm1 should be determined, in order to try to
elucidate the possible function of the gene. FIG. 4a represents the
3-dimensional (3D) structure predicted for OPEm1 and it was evident
that it had a very similar structure to the monomer unit of a human
peroxiredoxin (Choi et al., 1998). The human peroxiredoxin (Prx),
C19S-hORF6, exists in the form of a homodimer (FIG. 4c).
[0187] The structure of OPEm1 can be explained based on the 3D
structure and topology diagram of the C19S-hORF6 which is shown in
FIGS. 4b and 4d, respectively. The monomer can be divided into two
domains, D1 and D2. D1, the larger of the two, is the N-terminal,
which contains the thioredoxin fold (active site of the enzyme).
This site has a .beta..alpha..beta. motif comprised of
four-stranded .beta.-sheets (.beta.3, .beta.4, .beta.6 and .beta.7)
and three flanking .alpha.-helices (.alpha.2, .alpha.4 and
.alpha.5). There are also two .beta.-strands (.beta.1 and .beta.2)
and a short .alpha.-helix (.alpha.1) at the N-terminus just before
the thioredoxin fold. After the .beta..alpha..beta. motif, an
.alpha.-helix (.alpha.3) and a .beta.-strand (.beta.5) are
inserted. In the case of the C19S-hORF6, D2 comprises three
.beta.-strands and one .alpha.-helix and it is connected to D1 by
the extended helix .alpha.5 and a following loop. In OPEm1, this
region is very short, having only two .beta.-sheets (8.beta. and
9.beta.) with a loop between them. This difference is also
reflected in FIG. 6, in which, by residue 189, their amino acid
sequences no longer show similarity with the group.
Example 7
Sequence Alignments
[0188] The deduced amino acid sequence of OPEm1 was compared with
other plant Prx sequences that have been isolated. FIG. 5 shows the
alignment of amino acid sequences 1-Cys and 2-Cys groups of Prx in
plants. It seems that OPEm1 is more closely related to 1-Cys rather
than to the 2-Cys group of Prx. This is reflected in FIG. 6, which
shows an alignment between OPEm1 and other 1-Cys Prx in plants.
They share similar sequences surrounding the first cysteine, which
also sets the 1-Cys apart from the 2-Cys group. In 1-Cys, the
sequences are PVCT, whereas in 2-Cys, they are represented by FVCP.
From FIG. 6, it was also observed that OPEm1's C-terminus differs
from the other 1-Cys Prx group. Based on the hydropathy plot (FIG.
2b), OPEm1 may be membrane bound at this region, unlike the other
1-Cys members. However, the same region in 1-Cys Prx members
indicates the presence of a nuclear localization signal that
facilitates the nuclear targeting function of the protein that is
missing from OPEm1.
[0189] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
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Sequence CWU 1
1
91573DNAoil palmCDS(1)..(573) 1atg ccg ggg cta acg atc ggc gac acg
atc ccg aac ctg gag gtg gag 48Met Pro Gly Leu Thr Ile Gly Asp Thr
Ile Pro Asn Leu Glu Val Glu1 5 10 15acc acg cac ggg aag atc cgg atc
cac gac tac gtc ggc gat ggt tgg 96Thr Thr His Gly Lys Ile Arg Ile
His Asp Tyr Val Gly Asp Gly Trp 20 25 30gcc atc atc ttc tcc cat ccc
gcg gat ttc aca ccc gtg tgc acg acg 144Ala Ile Ile Phe Ser His Pro
Ala Asp Phe Thr Pro Val Cys Thr Thr 35 40 45gag ctg ggg aag atg gcg
gcg tac gcg gag gag ttc gag aaa aga ggg 192Glu Leu Gly Lys Met Ala
Ala Tyr Ala Glu Glu Phe Glu Lys Arg Gly 50 55 60gtg aag ctg cta ggc
atc tcc tgc gac gat gtc aag tgc cac atg gaa 240Val Lys Leu Leu Gly
Ile Ser Cys Asp Asp Val Lys Cys His Met Glu65 70 75 80tgg atc aaa
gac gtc gag gcc tac acg ccc gga tgt cgc gta aca tat 288Trp Ile Lys
Asp Val Glu Ala Tyr Thr Pro Gly Cys Arg Val Thr Tyr 85 90 95cca att
gta gcc gac ccc aag agg gag gtg atc aaa ctg ctg aac atg 336Pro Ile
Val Ala Asp Pro Lys Arg Glu Val Ile Lys Leu Leu Asn Met 100 105
110gta gac cct gag gag aag gac tca aat ggg aac cag ctc ccg tca cgg
384Val Asp Pro Glu Glu Lys Asp Ser Asn Gly Asn Gln Leu Pro Ser Arg
115 120 125gcc ctt cat ata gtg ggc cct gat aag aag gtt aag ctg agc
ttt ctg 432Ala Leu His Ile Val Gly Pro Asp Lys Lys Val Lys Leu Ser
Phe Leu 130 135 140tac ccg gcg tcg acg ggg cgg aac atg gag gag gtg
gtc agg gtg ttg 480Tyr Pro Ala Ser Thr Gly Arg Asn Met Glu Glu Val
Val Arg Val Leu145 150 155 160gag tcg ctt cag aag acg atc aag tat
aag gtg gcg acc cca gcg aac 528Glu Ser Leu Gln Lys Thr Ile Lys Tyr
Lys Val Ala Thr Pro Ala Asn 165 170 175tgg aaa ccg ggg gag ccg gtg
gtg atc tcg ccc gag cgt gtc caa 573Trp Lys Pro Gly Glu Pro Val Val
Ile Ser Pro Glu Arg Val Gln 180 185 1902191PRToil palm 2Met Pro Gly
Leu Thr Ile Gly Asp Thr Ile Pro Asn Leu Glu Val Glu1 5 10 15Thr Thr
His Gly Lys Ile Arg Ile His Asp Tyr Val Gly Asp Gly Trp 20 25 30Ala
Ile Ile Phe Ser His Pro Ala Asp Phe Thr Pro Val Cys Thr Thr 35 40
45Glu Leu Gly Lys Met Ala Ala Tyr Ala Glu Glu Phe Glu Lys Arg Gly
50 55 60Val Lys Leu Leu Gly Ile Ser Cys Asp Asp Val Lys Cys His Met
Glu65 70 75 80Trp Ile Lys Asp Val Glu Ala Tyr Thr Pro Gly Cys Arg
Val Thr Tyr 85 90 95Pro Ile Val Ala Asp Pro Lys Arg Glu Val Ile Lys
Leu Leu Asn Met 100 105 110Val Asp Pro Glu Glu Lys Asp Ser Asn Gly
Asn Gln Leu Pro Ser Arg 115 120 125Ala Leu His Ile Val Gly Pro Asp
Lys Lys Val Lys Leu Ser Phe Leu 130 135 140Tyr Pro Ala Ser Thr Gly
Arg Asn Met Glu Glu Val Val Arg Val Leu145 150 155 160Glu Ser Leu
Gln Lys Thr Ile Lys Tyr Lys Val Ala Thr Pro Ala Asn 165 170 175Trp
Lys Pro Gly Glu Pro Val Val Ile Ser Pro Glu Arg Val Gln 180 185
1903873DNAoil palmCDS(28)..(600) 3cacgaggtga atcggagccg ttgaaag atg
ccg ggg cta acg atc ggc gac acg 54 Met Pro Gly Leu Thr Ile Gly Asp
Thr 1 5atc ccg aac ctg gag gtg gag acc acg cac ggg aag atc cgg atc
cac 102Ile Pro Asn Leu Glu Val Glu Thr Thr His Gly Lys Ile Arg Ile
His10 15 20 25gac tac gtc ggc gat ggt tgg gcc atc atc ttc tcc cat
ccc gcg gat 150Asp Tyr Val Gly Asp Gly Trp Ala Ile Ile Phe Ser His
Pro Ala Asp 30 35 40ttc aca ccc gtg tgc acg acg gag ctg ggg aag atg
gcg gcg tac gcg 198Phe Thr Pro Val Cys Thr Thr Glu Leu Gly Lys Met
Ala Ala Tyr Ala 45 50 55gag gag ttc gag aaa aga ggg gtg aag ctg cta
ggc atc tcc tgc gac 246Glu Glu Phe Glu Lys Arg Gly Val Lys Leu Leu
Gly Ile Ser Cys Asp 60 65 70gat gtc aag tgc cac atg gaa tgg atc aaa
gac gtc gag gcc tac acg 294Asp Val Lys Cys His Met Glu Trp Ile Lys
Asp Val Glu Ala Tyr Thr 75 80 85ccc gga tgt cgc gta aca tat cca att
gta gcc gac ccc aag agg gag 342Pro Gly Cys Arg Val Thr Tyr Pro Ile
Val Ala Asp Pro Lys Arg Glu90 95 100 105gtg atc aaa ctg ctg aac atg
gta gac cct gag gag aag gac tca aat 390Val Ile Lys Leu Leu Asn Met
Val Asp Pro Glu Glu Lys Asp Ser Asn 110 115 120ggg aac cag ctc ccg
tca cgg gcc ctt cat ata gtg ggc cct gat aag 438Gly Asn Gln Leu Pro
Ser Arg Ala Leu His Ile Val Gly Pro Asp Lys 125 130 135aag gtt aag
ctg agc ttt ctg tac ccg gcg tcg acg ggg cgg aac atg 486Lys Val Lys
Leu Ser Phe Leu Tyr Pro Ala Ser Thr Gly Arg Asn Met 140 145 150gag
gag gtg gtc agg gtg ttg gag tcg ctt cag aag acg atc aag tat 534Glu
Glu Val Val Arg Val Leu Glu Ser Leu Gln Lys Thr Ile Lys Tyr 155 160
165aag gtg gcg acc cca gcg aac tgg aaa ccg ggg gag ccg gtg gtg atc
582Lys Val Ala Thr Pro Ala Asn Trp Lys Pro Gly Glu Pro Val Val
Ile170 175 180 185tcg ccc gag cgt gtc caa tgaggaggcc aagcagatgt
tcccgcaggg 630Ser Pro Glu Arg Val Gln 190agttgagaat gtgaatctcc
catcgaagaa ggattacctc cgcttcacaa aagtctaatg 690ttgttgggcc
gtccgtgata tgttcataag tggtttctgg ggcccgactg tatactgtgt
750tgtcgtgtta tatgtttgtg ttggtatcat gtagtttgtg ccttagggga
gtttggatat 810taatttgtag tttatgttaa ttattaaagt ttttaccatg
agattaaaaa aaaaaaaaaa 870aaa 8734191PRToil palm 4Met Pro Gly Leu
Thr Ile Gly Asp Thr Ile Pro Asn Leu Glu Val Glu1 5 10 15Thr Thr His
Gly Lys Ile Arg Ile His Asp Tyr Val Gly Asp Gly Trp 20 25 30Ala Ile
Ile Phe Ser His Pro Ala Asp Phe Thr Pro Val Cys Thr Thr 35 40 45Glu
Leu Gly Lys Met Ala Ala Tyr Ala Glu Glu Phe Glu Lys Arg Gly 50 55
60Val Lys Leu Leu Gly Ile Ser Cys Asp Asp Val Lys Cys His Met Glu65
70 75 80Trp Ile Lys Asp Val Glu Ala Tyr Thr Pro Gly Cys Arg Val Thr
Tyr 85 90 95Pro Ile Val Ala Asp Pro Lys Arg Glu Val Ile Lys Leu Leu
Asn Met 100 105 110Val Asp Pro Glu Glu Lys Asp Ser Asn Gly Asn Gln
Leu Pro Ser Arg 115 120 125Ala Leu His Ile Val Gly Pro Asp Lys Lys
Val Lys Leu Ser Phe Leu 130 135 140Tyr Pro Ala Ser Thr Gly Arg Asn
Met Glu Glu Val Val Arg Val Leu145 150 155 160Glu Ser Leu Gln Lys
Thr Ile Lys Tyr Lys Val Ala Thr Pro Ala Asn 165 170 175Trp Lys Pro
Gly Glu Pro Val Val Ile Ser Pro Glu Arg Val Gln 180 185
190517DNAoil palm 5aggaggattg tgcagag 17618DNAoil palm 6caaactctca
gctaggca 187218PRTHordeum vulgare 7Met Pro Gly Leu Thr Ile Gly Asp
Thr Val Pro Asn Leu Glu Leu Asp1 5 10 15Ser Thr His Gly Lys Ile Arg
Ile His Asp Tyr Val Gly Asn Gly Tyr 20 25 30Val Ile Leu Phe Ser His
Pro Gly Asp Phe Thr Pro Val Cys Thr Thr 35 40 45Glu Leu Ala Ala Met
Ala Asn Tyr Ala Lys Glu Phe Glu Lys Arg Gly 50 55 60Val Lys Leu Leu
Gly Ile Ser Cys Asp Asp Val Gln Ser His Lys Glu65 70 75 80Trp Thr
Lys Asp Ile Glu Ala Tyr Lys Pro Gly Ser Lys Val Thr Tyr 85 90 95Pro
Ile Met Ala Asp Pro Asp Arg Ser Ala Ile Lys Gln Leu Asn Met 100 105
110Val Asp Pro Asp Glu Lys Asp Ala Gln Gly Gln Leu Pro Ser Arg Thr
115 120 125Leu His Ile Val Gly Pro Asp Lys Val Val Lys Leu Ser Phe
Leu Tyr 130 135 140Pro Ser Cys Thr Gly Arg Asn Met Asp Glu Val Val
Arg Ala Val Asp145 150 155 160Ser Leu Leu Thr Ala Ala Lys His Lys
Val Ala Thr Pro Ala Asn Trp 165 170 175Lys Pro Gly Glu Cys Val Val
Ile Ala Pro Gly Val Ser Asp Glu Glu 180 185 190Ala Lys Lys Met Phe
Pro Gln Gly Phe Glu Thr Ala Asp Leu Pro Ser 195 200 205Lys Lys Gly
Tyr Leu Arg Phe Thr Lys Val 210 2158216PRTArabidopsis thaliana 8Met
Pro Gly Ile Thr Leu Gly Asp Thr Val Pro Asn Leu Glu Val Glu1 5 10
15Thr Thr His Asp Lys Phe Lys Leu His Asp Tyr Phe Ala Asn Ser Trp
20 25 30Thr Val Leu Phe Ser His Pro Gly Asp Phe Thr Pro Val Cys Thr
Thr 35 40 45Glu Leu Gly Ala Met Ala Lys Tyr Ala His Glu Phe Asp Lys
Arg Gly 50 55 60Val Lys Leu Leu Gly Leu Ser Cys Asp Asp Val Gln Ser
His Lys Asp65 70 75 80Trp Ile Lys Asp Ile Glu Ala Phe Asn His Gly
Ser Lys Val Asn Tyr 85 90 95Pro Ile Ile Ala Asp Pro Asn Lys Glu Ile
Ile Pro Gln Leu Asn Met 100 105 110Ile Asp Pro Ile Glu Asn Gly Pro
Ser Arg Ala Leu His Ile Val Gly 115 120 125Pro Asp Ser Lys Ile Lys
Leu Ser Phe Leu Tyr Pro Ser Thr Thr Gly 130 135 140Arg Asn Met Asp
Glu Val Leu Arg Ala Leu Asp Ser Leu Leu Met Ala145 150 155 160Ser
Lys His Asn Asn Lys Ile Ala Thr Pro Val Asn Trp Lys Pro Asp 165 170
175Gln Pro Val Val Ile Ser Pro Ala Val Ser Asp Glu Glu Ala Lys Lys
180 185 190Met Phe Pro Gln Gly Phe Lys Thr Ala Asp Leu Pro Ser Lys
Lys Gly 195 200 205Tyr Leu Arg His Thr Glu Val Ser 210
2159272PRTBrassica campestri 9Met Ala Ser Val Ala Ser Ser Thr Thr
Leu Ile Ser Ser Ser Ala Ser1 5 10 15Val Leu Pro Ala Thr Lys Ser Ser
Leu Leu Pro Ser Pro Ser Leu Ser 20 25 30Phe Leu Pro Thr Leu Ser Ser
Pro Ser Pro Ser Ala Ser Leu Arg Ser 35 40 45Leu Val Pro Leu Pro Ser
Pro Gln Ser Ala Ser Ser Ser Arg Arg Ser 50 55 60Phe Ala Val Lys Gly
Gln Thr Asp Asp Leu Pro Leu Val Gly Asn Lys65 70 75 80Ala Pro Asp
Phe Glu Ala Glu Gly Val Phe Asp Gln Glu Phe Ile Lys 85 90 95Phe Ile
Lys Val Lys Leu Ser Asp Tyr Ile Gly Lys Lys Tyr Val Ile 100 105
110Leu Phe Phe Leu Pro Leu Asp Phe Thr Phe Val Cys Pro Thr Glu Ile
115 120 125Thr Ala Phe Ser Asp Arg Tyr Ala Glu Phe Glu Lys Leu Asn
Thr Glu 130 135 140Val Leu Gly Val Ser Val Asp Ser Val Ser Val Phe
Ser His Leu Ala145 150 155 160Gly Val Gln Thr Asp Arg Lys Phe Gly
Gly Leu Gly Asp Leu Asn Tyr 165 170 175Pro Leu Ile Ser Asp Val Thr
Lys Ser Ile Ser Lys Ser Phe Gly Val 180 185 190Leu Ile His Asp Gln
Gly Ile Ala Leu Arg Gly Leu Phe Ile Ile Asp 195 200 205Lys Glu Gly
Val Ile Gln His Ser Thr Ile Asn Leu Gly Ile Gly Arg 210 215 220Ser
Val Asp Glu Thr Met Arg Thr Leu Gln Ala Leu Gln Tyr Ile Gln225 230
235 240Glu Gly Pro Gly Glu Val Cys Pro Ala Gly Trp Lys Pro Gly Glu
Lys 245 250 255Ser Met Lys Pro Asp Pro Lys Leu Ser Lys Glu Leu Phe
Ser Ala Ile 260 265 270
* * * * *
References