AU722880B1 - Peptide fragments derived from CWPM23, a protein involved in freezing resistance of winter wheat - Google Patents
Peptide fragments derived from CWPM23, a protein involved in freezing resistance of winter wheat Download PDFInfo
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- AU722880B1 AU722880B1 AU48842/99A AU4884299A AU722880B1 AU 722880 B1 AU722880 B1 AU 722880B1 AU 48842/99 A AU48842/99 A AU 48842/99A AU 4884299 A AU4884299 A AU 4884299A AU 722880 B1 AU722880 B1 AU 722880B1
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- 108090000623 proteins and genes Proteins 0.000 title claims description 61
- 102000004169 proteins and genes Human genes 0.000 title claims description 50
- 102000007079 Peptide Fragments Human genes 0.000 title claims description 28
- 108010033276 Peptide Fragments Proteins 0.000 title claims description 28
- 230000008014 freezing Effects 0.000 title claims description 25
- 238000007710 freezing Methods 0.000 title claims description 25
- 241000209140 Triticum Species 0.000 title claims description 23
- 235000021307 Triticum Nutrition 0.000 title claims description 22
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 8
- 239000003550 marker Substances 0.000 claims description 7
- 108010052285 Membrane Proteins Proteins 0.000 claims description 4
- 102000018697 Membrane Proteins Human genes 0.000 claims description 4
- 239000012634 fragment Substances 0.000 claims description 4
- 241000894007 species Species 0.000 claims 2
- 235000018102 proteins Nutrition 0.000 description 38
- 239000012071 phase Substances 0.000 description 18
- 210000000170 cell membrane Anatomy 0.000 description 12
- 239000000243 solution Substances 0.000 description 11
- 238000000034 method Methods 0.000 description 10
- 241000196324 Embryophyta Species 0.000 description 8
- 239000000872 buffer Substances 0.000 description 8
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- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 8
- 125000003275 alpha amino acid group Chemical group 0.000 description 7
- IDOQDZANRZQBTP-UHFFFAOYSA-N 2-[2-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=CC=C1OCCO IDOQDZANRZQBTP-UHFFFAOYSA-N 0.000 description 6
- 229920004929 Triton X-114 Polymers 0.000 description 6
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- 244000098338 Triticum aestivum Species 0.000 description 4
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- 238000009395 breeding Methods 0.000 description 4
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- 241001465754 Metazoa Species 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 3
- 239000003599 detergent Substances 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
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- 239000013615 primer Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 2
- 239000011536 extraction buffer Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000005191 phase separation Methods 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- ILXAOQAXSHVHTM-UHFFFAOYSA-M sodium;2-amino-2-(hydroxymethyl)propane-1,3-diol;chloride Chemical compound [Na+].[Cl-].OCC(N)(CO)CO ILXAOQAXSHVHTM-UHFFFAOYSA-M 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- CNKBMTKICGGSCQ-ACRUOGEOSA-N (2S)-2-[[(2S)-2-[[(2S)-2,6-diamino-1-oxohexyl]amino]-1-oxo-3-phenylpropyl]amino]-3-(4-hydroxyphenyl)propanoic acid Chemical compound C([C@H](NC(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 CNKBMTKICGGSCQ-ACRUOGEOSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000020897 Formins Human genes 0.000 description 1
- 108091022623 Formins Proteins 0.000 description 1
- GWCJMBNBFYBQCV-XPUUQOCRSA-N Gly-Val-Ala Chemical compound NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O GWCJMBNBFYBQCV-XPUUQOCRSA-N 0.000 description 1
- LXKNSJLSGPNHSK-KKUMJFAQSA-N Leu-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N LXKNSJLSGPNHSK-KKUMJFAQSA-N 0.000 description 1
- LZHJZLHSRGWBBE-IHRRRGAJSA-N Leu-Lys-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O LZHJZLHSRGWBBE-IHRRRGAJSA-N 0.000 description 1
- VTJUNIYRYIAIHF-IUCAKERBSA-N Leu-Pro Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(O)=O VTJUNIYRYIAIHF-IUCAKERBSA-N 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108700001094 Plant Genes Proteins 0.000 description 1
- FHJQROWZEJFZPO-SRVKXCTJSA-N Pro-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 FHJQROWZEJFZPO-SRVKXCTJSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 239000012722 SDS sample buffer Substances 0.000 description 1
- 108091006629 SLC13A2 Proteins 0.000 description 1
- DOFAQXCYFQKSHT-SRVKXCTJSA-N Val-Pro-Pro Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DOFAQXCYFQKSHT-SRVKXCTJSA-N 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 238000003277 amino acid sequence analysis Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000022472 cold acclimation Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000007914 freezing tolerance Effects 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000012135 ice-cold extraction buffer Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229940050929 polyethylene glycol 3350 Drugs 0.000 description 1
- 235000013809 polyvinylpolypyrrolidone Nutrition 0.000 description 1
- 229920000523 polyvinylpolypyrrolidone Polymers 0.000 description 1
- TYPBXRFGTISSFB-UHFFFAOYSA-M potassium;3-morpholin-4-ylpropane-1-sulfonic acid;hydroxide Chemical compound [OH-].[K+].OS(=O)(=O)CCCN1CCOCC1 TYPBXRFGTISSFB-UHFFFAOYSA-M 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000012134 supernatant fraction Substances 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 108010015385 valyl-prolyl-proline Proteins 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Botany (AREA)
- Peptides Or Proteins (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Description
AUSTRALIA
Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s) KYUSHU UNIVERSITY Invention Title: PEPTIDE FRAGMENTS DERIVED FROM CWPM23, A PROTEIN INVOLVED IN FREEZING RESISTANCE OF WINTER WHEAT The following statement is a full description of this invention, including the best method of performing it known to me/us: -1A PEPTIDE FRAGMENTS DERIVED FROM CWPM23, A PROTEIN INVOLVED IN FREEZING RESISTANCE OF WINTER WHEAT BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to CWPM23 protein, which is a membrane protein of winter wheat and involved in resistance against freezing. Especially, this invention relates to purification, partial amino acid sequence determination and usage of the CWPM23 protein.
2. Description of Related Art Freezing injury of plants have been considered to be mainly caused by irreversible damage of plasma membranes when freeze-induced dehydration and shrinkage of cells occur, in accordance to formation of extracellular ice crystals. It is speculated that, from autumn to winter, through acquisition of resistance against freezing, a specific protein might be induced to stabilize plasma membrane, which results in avoidance of freezing injury.
For molecular breeding of organisms with resistance against such freezing injury, efforts have been performed on incorporation and expression of a plant gene encoding a protein induced under low-temperature conditions.
SUMMARY OF THE INVENTION Despite of it, most of the proteins, induced under low-temperature conditions, are hydrophilic. Moreover, such proteins are not localized in plasma membrane, where freezing injury actually occurs. It has been reported that, the effect to incorporate a gene encoding the hydrophilic protein is not sufficient to render resistance against freezing. The difficulty of purification prevented functional analysis and utilization of hydrophobic proteins.
Therefore, identification and purification of membrane-localized proteins, induced under low-temperature conditions, are indispensable.
98133 (10-349,459) CWPM23 protein is a plasma membrane-localized protein of winter wheat, induced specifically during the process of cold acclimation. The protein is accumulated in plasma membrane at its seedling, in parallel to development of freezing tolerance. Induction of the CWPM23 protein is significant in winter wheat, whereas the protein is not induced under low-temperature conditions in spring wheat with sensitivity to freezing. The knowledge would indicate close relationship between marked resistance against freezing observed in winter wheat and expression of the CWPM23 protein. Therefore, incorporation of CWPM23 gene into other plants would enable production of crops with prominent resistance against freezing and aridity. Therefore, the inventors prepared plasma membrane fraction of winter wheat, purified the CWPM23 protein from the fraction and performed protease treatment, in the purpose to obtain peptide fragments of the protein. Furthermore, amino acid sequence of the protein was analyzed for partial sequence determination.
A gene encoding the CWPM23 protein can be isolated, by polymerase chain reaction (PCR) using oligonucleotide primers designed according to the partial amino acid sequence thus determined. Isolated CWPM23 gene deserves great value at breeding of various crops. That is, crops with prominent resistance against freezing and aridity can be produced by genetic incorporation of the CWPM23 gene into other plants. Not only breeding of plants, transgenic microorganism or animals wherein said gene is incorporated could be produced. Using such technique, improvement of cell viability on the occasion of freezing can be achieved. When incorporated into animals, this gene will be available for improvement of freshness of foods, such as frozen meat or fish. In addition, when incorporated into a microorganism, this gene will be available for storage of fermented intermediate, such as bread dough, under frozen condition. Moreover, the CWPM23 protein is induced significantly in winter wheat species with high 98133 (10-349,459) -3resistance against freezing, such as Chihokukomugi or Norstar. The knowledge indicates that, the CWPM23 gene is available for breeding of wheats, as a marker of crops with high resistance against freezing.
These and other objects and advantages of the invention will become more apparent upon a reading of the detailed description and drawing.
BRIEF DESCRIPTION OF THE DRAWING Fig. 1 is a photograph showing the result of electrophoresis of isolated CWPM23 protein.
DETAILED DESCRIPTION OF EMBODIMENTS (Preparation of crude microsome fraction) Preparation of crude microsome fraction and plasma membrane fraction, derived from winter wheat, was performed according to the method described by Zhou et al. (Zhou et al. (1994) Plant Cell Physiol.35:175-182).
Aerial part (shoot) of low-temperature-adapted winter wheat (Triticum aestivum L.cv.Chihokukomugi) was sliced into 3mm width, and then immersed into ice-cold extraction buffer [50mM MOPS-KOH (pH7.6), 330mM sucrose, 5mM EGTA, 3mM EDTA, 30mM NaC1, 10mM NaF, casein hydrolyzate, 2.5mM K 2
P
2 0 5 PVPP, 2mM PMSF, BHT]. 10ml of the extraction buffer was used for 30g (wet weight under fresh condition) of wheat sample. Immediately after that, wheat sample in the extraction buffer was homogenized by Polytron PT 10-35, and the homogenate was filtrated through four piles of gauzes. The filtrate was centrifuged at 10,000g for 20 min to recover supernatant. The supernatant was centrifuged at 142,000g for 40 min and pellet thus recovered was suspended into suitable volume (about 30-50ml) of SKP buffer [10mM K-phosphate (pH7.6), 330mM sucrose, ImM EGTA, ImM DTT]. The suspension was centrifuged again at 10,000g for 20 min to recover supernatant. The supernatant was centrifuged once again at 200,000g for 20 min and pellet was 98133 (10-349,459) -4recovered. The pellet thus obtained was microsome fraction, and the fraction was suspended into small volume of SKP buffer.
(Preparation of plasma membrane fraction) The crude microsome fraction suspension, prepared by the method described above, was added into ice-cold polymer solution used for aqueous two phase partitioning system [final concentration Dextran polyethylene glycol 3350, 30mM NaCI, SKP buffer] (Zhou et al.(1994)Plant cell Physiol.35:175-182). At that time, 10g of said polymer solution was added for 30g of plant material. The mixture was stirred vigorously, and then centrifuged at 1,000g for 5 min. Upper phase of separated polymer solution (polyethylene glycol phase in which the plasma membrane fraction is concentrated) was recovered. As an independent sample, aqueous two phase partitioning was performed in the same manner to obtain lower phase (dextran phase). For the preparation of the fresh dextran phase, only SKP buffer was added into the polymer solution, instead of the crude microsome fraction. The polyethylene glycol phase containing the plasma membrane fraction was added into the fresh dextran phase thus prepared.
Aqueous two phase partitioning was performed once again according to the method described above. Polyethylene glycol phase thus recovered was diluted to more than twice volume by SKP buffer, and then centrifuged at 200,000g for 20 min to recover membrane fraction with rich content of plasma membrane. The membrane fraction was suspended into SKP buffer, and centrifuged again. The pellet thus recovered was plasma membrane fraction, which was re-suspended into SKP buffer.
(Solubilization of the CWPM23 protein) The plasma membrane fraction suspension thus prepared (corresponding to 1 mg of protein) was centrifuged at 260,000g to recover pellet, and the pellet was suspended into 300 pl of Tris-NaCl solution 98133 (10-349,459) Tris-HCl (pH7.4), 150mM NaCI]. Into the solution, 300 -il of Tris-NaCl solution containing 2% Triton X-114 was added. After sufficient mixing of the solution, the solution was cooled on ice for 30 min to solubilize the protein and it was centrifuged at 260,000g to harvest supernatant fraction (Triton X-114 solubilized fraction). Then, Triton X-114 phase separation was performed essentially according to the procedure of Brusca and Radolf et al.(Brusca and Radolf (1994) Methods Enzymol.228:1 82 -193). The Triton X-114 solubilized fraction was settled in an incubator maintained at 30 0 C for min, to separate Triton X-114 phase. The sample was centrifuged at 6,400g for 10 min to recover upper phase (aqueous phase). At the same time, the Tris- NaCI solution containing 1% of Triton X-114 was separated in the same manner to recover fresh lower phase (detergent phase). The aqueous phase in Swhich CWPM23 was concentrated was added into the fresh lower phase (detergent phase) thus recovered. The mixture was cooled sufficiently on ice and mixed thoroughly until detergent was dissolved. Phase separation was performed again in the same manner as mentioned above for separation of the aqueous phase. Into the aqueous phase thus recovered, four volume of acetone was added and mixed thoroughly, then settled at -80 0 C for two hours. The sample was centrifuged at 10,000g for 10 min to recover pellet. SDS-sample buffer was added into the pellet to solubilize protein.
(SDS-PAGE of the CWPM23 protein) About 2.5 gpg of the solubilized protein, obtained according to the method described above, was used as the sample for SDS polyacrylamide gel electrophoresis (SDS-PAGE), for separation of target protein (Laemmli (1970) Nature 227:680-685). The concentration of acrylamide used for SDS-PAGE was 13%. After the electrophoresis, the gel was immersed in Coomassie brilliant blue (CBB) solution for staining of protein bands. The band corresponding to the CWPM23 protein was cut off from the gel by a razor, and 98133 (10-349,459) -6immersed into sufficient volume of buffer for electroelution. The target protein was electro-eluted from the gel by an electroeluter (Bio-Rad:Model 422). The fraction thus obtained was used for Tricine-SDS-polyacrylamide gelelectrophoresis (Tricine-SDS-PAGE), with the acrylamide concentration of 16.2%T and 3%C, to separate the target protein (Schagger and Von Jagow (1987) Anal.Biochem. 166:368-379). A portion of the CWPM23 fraction, obtained by electro-elution of the target protein by the method described above using the gel after electrophoresis, was used for SDS-PAGE to confirm its purity (Fig. 1).
(Determination of partial amino acid sequence of the CWPM23 protein) Peptide fragments were obtained by lysil-endopeptidase (Wako Junyaku) treatment of the CWPM23, and the fragments were separated by Tricine-SDS-PAGE. Then they were transferred to polyvinylidenefluoride (PVDF) membrane by electro-blotting and detected by CBB staining. The detected bands were cut-off and amino-acid sequence analysis by gas-phase sequencer (Shimazu:PPSQ-10) was performed on three peptides (CP23F1,2,3).
The partial amino acid sequences of the three peptides derived from the CWPM23 were determined.
CP23F1 IGVAAE CP23F2 ILLLKL CP23F3 LKVKFYVPPFLPIIPVVG The CWPM23 protein, involved in resistance of winter wheat against freezing, was purified and partial amino acid sequences of the protein were determined. The oligonucleotide primer designed according to the partial amino acid sequence can be used as a primer for PCR. Therefore, PCR using the primer will enable isolation of a gene encoding CWPM23 protein.
Moreover, by incorporation of the gene, freezing resistance might be rendered to animals, plants or microorganisms.
98133 (10-349,459) -7- Sequence list Applicant name: President of Kyusyu university Title of invention: Peptide fragments derived from CWPM23, a protein involved in freezing resistance of winter wheat Number of sequence: 1 Sequence length: 6 Sequence type: Amino acid Topology: Linear Type of sequence: Peptide Original source: Triticum aestivum L.cv.Chihokukomugi CWPM23 protein Sequence: lie Gly Val Ala Ala Glu 1 Number of sequence: 2 Sequence length: 6 Sequence type: Amino acid Topology: Linear Type of sequence: Peptide Original source: Triticum aestivum L.cv.Chihokukomugi CWPM23 protein Sequence: lie Leu Leu Leu Lys Leu Number of sequence: 3 Sequence length: 18 Sequence type: Amino acid Topology: Linear 98133 (10-349,459) -8- Type of sequence: Peptide Original source: Triticum aestivum L.cv.Chihokukomugi CWPM23 protein Sequence: Leu Lys Val Lys Phe Tyr Val Pro Pro Phe 1 Leu Pro lie lie Pro Val Val Gly For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.
98133 (10-349,459)
Claims (14)
1. A peptide fragment characterized in that, the peptide fragment consists of the following sequence, Ile-Gly-Val-Ala-Ala-Glu.
2. A peptide fragment as claimed in claim 1, characterized in that, the peptide fragment is derived from CWPM23 which is a membrane protein of winter wheat involved in resistance of winter wheat against freezing.
3. A DNA fragment encoding the peptide fragment as claimed in claim 1.
4. A peptide marker for CWPM23 protein, which is involved in resistance against freezing, identified by a peptide fragment as claimed in claim 1. A use of the peptide fragment as claimed in claim 1, as a marker for CWPM23 protein.
6. A peptide fragment characterized in that, the peptide fragment consists of the following sequence, Ile-Leu-Leu- Leu-Lys-Leu.
7. A peptide fragment as claimed in claim 6, characterized in that, the peptide fragment is derived from CWPM23 which is a membrane protein of winter wheat involved in resistance of winter wheat against freezing.
8. A DNA fragment encoding the peptide fragment as claimed in claim 6.
9. A peptide marker for CWPM23 protein, which is involved in resistance against rreezmg, identitied by a peptide fragment as claimed in claim 6. A use of the peptide fragment as claimed in claim 6, as a marker for CWPM23 protein.
11. A peptide fragment characterized in that, the peptide fragment consists of the following sequence, Leu-Lys-Val-Lys-Phe-Tyr-Val-Pro-Pro- Phe-Leu-ProIl-Ile-Ile-ProVa-Val-Val-Gly. 10
12. A peptide fragment as claimed in claim 11, characterised in that, the peptide fragment is derived from CWPM23 which is a membrane protein of winter wheat involved in resistance of winter wheat against freezing.
13. A DNA fragment encoding the peptide fragment as claimed in claim 11.
14. A peptide marker for CWPM23 protein, which is involved in resistance against freezing, identified by a peptide fragment as claimed in claim 11.
15. A use of the peptide fragment as claimed in claim 11, as a marker for CWPM23 protein.
16. A peptide fragment according to any one of claims 1, 6 and 11 substantially as herein described with reference to the Examples. Dated this 5th day of June 2000 KYUSHU UNIVERSITY By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia H:\cintae\Keep\speci\speci 48842.99.doc 5/06/00
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10-349459 | 1998-12-09 | ||
| JP10349459A JP2995297B1 (en) | 1998-12-09 | 1998-12-09 | Peptide fragment of winter wheat freeze-resistance related protein CWPM23 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU722880B1 true AU722880B1 (en) | 2000-08-10 |
Family
ID=18403899
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU48842/99A Ceased AU722880B1 (en) | 1998-12-09 | 1999-09-21 | Peptide fragments derived from CWPM23, a protein involved in freezing resistance of winter wheat |
Country Status (4)
| Country | Link |
|---|---|
| JP (1) | JP2995297B1 (en) |
| AU (1) | AU722880B1 (en) |
| CA (1) | CA2286312A1 (en) |
| FR (1) | FR2787112A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1668141B1 (en) * | 2003-09-29 | 2012-11-07 | Monsanto Technology, LLC | Methods for enhancing drought tolerance in plants and methods thereof |
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1998
- 1998-12-09 JP JP10349459A patent/JP2995297B1/en not_active Expired - Fee Related
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1999
- 1999-09-21 AU AU48842/99A patent/AU722880B1/en not_active Ceased
- 1999-09-30 FR FR9912205A patent/FR2787112A1/en active Pending
- 1999-10-26 CA CA 2286312 patent/CA2286312A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| CA2286312A1 (en) | 2000-06-09 |
| JP2995297B1 (en) | 1999-12-27 |
| FR2787112A1 (en) | 2000-06-16 |
| JP2000169500A (en) | 2000-06-20 |
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