バラ科植物における配偶体型自家不和合性の分子機構

• Project/Area Number: 11238204
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Osaka University
• Principal Investigator: 乗岡 茂巳 (乗岡 茂已) 大阪大学, 大学院・生命機能研究科, 教授 (70198638)
• Project Period (FY): 1999 – 2003
• Project Status: Completed (Fiscal Year 2003)
• Budget Amount: ¥54,500,000 (Direct Cost: ¥54,500,000); Fiscal Year 2003: ¥8,500,000 (Direct Cost: ¥8,500,000); Fiscal Year 2002: ¥8,500,000 (Direct Cost: ¥8,500,000); Fiscal Year 2001: ¥8,500,000 (Direct Cost: ¥8,500,000); Fiscal Year 2000: ¥11,000,000 (Direct Cost: ¥11,000,000); Fiscal Year 1999: ¥18,000,000 (Direct Cost: ¥18,000,000)
• Keywords: 自家不和合性 / 自他認識 / リボヌクレアーゼ / 花粉管伸長 / 受精 / 高等植物 / バラ科 / ニホンナシ / S-RNase / 立体構造 / 蛋白質間相互作用 / リボタクレアーゼ / バラ科植物 / 自家認識

Research Abstract

平成14年度にin vitro花粉管伸長阻害実験系と二次元電気泳動法を用いて花粉管伸長阻害時に特異的に発現しているタンパク質を解析した。その結果、数十のタンパク質が花粉管伸長阻害時に特異的に発現していたが、F-boxタンパク質やプロテアソームなどは検出されなかった。そこで、安定同位体を用いたICAT(isotope-Coded affinity tag)試薬として最近開発されたNBS(2-nitrobenzenesulfenyl)試薬を用い、平成14年度の二次元電気泳動法では検出できなかったタンパク質の同定を行い、花粉管伸長阻害時に発現しているタンパク質のカタログ化を進めた。花粉はS3ホモ花粉を用い、in vitro花粉管伸長阻害実験系で培地にS3-RNaseを添加して花粉管伸長阻害を起こした。次に、花粉管からタンパク質を抽出し、NBS試薬を反応させ(NBSはタンパク質中のトリプトファン側鎖を特異的に修飾する試薬である)、質量分析計で測定した。ところで、S-RNaseを培地に添加して伸長阻害を起こした花粉管タンパク質を安定同位体C13原子6個を含むNBS試薬で修飾し、S-RNaseを培地に添加せず伸長阻害を起こしていない花粉管タンパク質を通常のNBS試薬で修飾し、両タンパク質を等量ずつ混ぜて一連の反応を行えば、それぞれのペプチドが質量分析チャート上で6マス差をもつダブレットピークとして現れる。6マス重いピークが高ければ花粉管伸長阻害時に発現量が増加するタンパク質、低ければ発現量が減少するタンパク質を示している。実験の結果、伸長阻害時に特に発現量が増加した6種類のペプチドが検出された。さらに、S4ホモ花粉と,S4-RNaseについても同様の実験を行い、特に発現量が増加した6種類のペプチドが検出された。現在、MS/MS de novo sequencing法により各ピークのアミノ酸配列決定を試みている。

Research Products

• [Publications] Takeshi Takasaki: "Sequence of the S_9-RNase cDNA and PCR-RFLP system for discriminating S_1 to S_9-haplotypes in Japanese pear."Euphytica. 135. 157-167 (2004)
• [Publications] Yuegang Zhou: "Nucleotide sequence of pdn3 gene encoding a plant defensin-like protein in pollen grains of Japanese pear(Pyrus pyrifolia)."J.Phant Physiol.Mol.Biol.. 29(4). 360 (2003)
• [Publications] Yuegang Zhou: "Nucleotide sequence of a cDNA(pdn2)encoding a plant defensin-like protein in pollen grains of Japanese pear(Pyrus pyrifolia)."J.Phant Physiol.Mol.Biol.. 29(1). 80 (2003)
• [Publications] Yuegang Zhou: "Nucleotide sequence of a cDNA encoding plant defensin-like protein (PDN1) in pollen grains of Japanese Pear"J. Plant Physiol. Mol. Biol.. 28(5). 407 (2002)
• [Publications] D.M.R.K.K.Dissanayake: "Cis-regulatory elements for pistil specific expression in S-RNase promoter region of Japanese Pear"Acta Horticulture. 587. 459-465 (2002)
• [Publications] Carlos Castillo: "S-RNase based PCR-RFLP system for S-genotype identification in Japanese Pear"Acta Horticulture. 587. 449-458 (2002)
• [Publications] Daiji Kiyozumi: "Pollen UDP-glucose pyrophosphorylase showing polymorphism well-correlated to S-genotype of Pyruspyrifolia, Japanese Pear"Sex. Plant Reprod.. 14. 315-323 (2002)
• [Publications] Katsuhiko Kondo: "Cultivated tomato has defects in both S-RNase and HT protein genes required for stylar function of self-incompatibility"Plant Journal. 29. 627-636 (2002)
• [Publications] Carlos Castillo: "Cloning of the S_8-RNase (S_8-allele) of Japanese pear (Pyrus pyrifolia Hakai)"Plant Biotechnology. 19. 1-6 (2002)
• [Publications] Koh Ida: "The 1.55Å resolution structure of Nicotiana alata SF_<11>-RNase associated with gametophytic self-incompatibility"J. Mol. Biol.. 314. 103-112 (2001)
• [Publications] Takanori Matsuura: "Crystal structure at 1.5Å resolution of Pyrus pyrifolia pistil ribonuclease responsible for gametophytic self-incompatibility"J. Biol. Chem.. 276. 45261-45269 (2001)
• [Publications] Naoko Norioka: "Sequence comparison of the 5'-flanking regions of Pyrus pyritolia S-RNase associated with gametophytic self-incompatibility"Sex. Plant Reprod.. 13. 289-291 (2001)
• [Publications] Garlos Castillo: "Reconsideration of S-genotype. assignments and discovery of a new allele based on S-RNase PCR-RFLPs in Japanese pear cultivars"Breeding Science. 51. 5-11 (2001)
• [Publications] Takanori Matsuura: "Purification and crystallization of Japanese Pear S-RNase associated with gametophytic self-incompatibility"Acta Crystallographica. D57. 172-173 (2001)
• [Publications] Koh Ida: "Crystallization and preliminary X-ray crystallographic analysis of S-allelic glycoprotein SF_<11>-RNase from Nicotiana alata"Acta Crystallographica. D57. 143-144 (2001)
• [Publications] Niranjali Gamage: "Expression of a fusion protein of Pyrus pyrifolia S-RNase with glutathione-S-transferase in E.coli"Biotechnology Letters. 22. 1413-1417 (2000)
• [Publications] 乗岡茂巳: "花!性と生殖の分子生物学"日向康吉. 258 (2001)
• [Publications] Carlos Castillo et al.: "Reconsidreration of S-genotype assignment, discovery of a new allele based on S-RNase PCR-RFLPs in Japanese pear cultivars"Breeding Science. 51. 5-11 (2001)
• [Publications] Naoko Norioka et al.: "Sequence comparison of the 5' flanking regions of Pyrus prifolia S-RNases associated with gametophytic self-incompatibility"Sexual Plant Reproduction. 13. 289-291 (2001)
• [Publications] Takanori Matsuura et al.: "Crystal structure at 1.5Å resolution of Pyrus pyrifolia ribonuctease responsible for gametophytic self-incompatibility"Journal of Biological Chemistry. 276. 45261-45269 (2001)
• [Publications] Koh Ida et al.: "The 1.55Å resolution structure of Nicotiana alata S_<F11>-RNase associated with gametophytic self-incompatibility"Journal of Molecular Biology. 314. 103-112 (2001)
• [Publications] Carlos Castillo et al.: "Cloning of the S_8-RNase (S_8-ailele) of Japanese pear (Phrus pyrifolia Nakai)"Plant Biotechnology. 19. 1-6 (2002)
• [Publications] Niranjali Gamage: "Expression of a fusion protein of Pyrus pyrifolia S-RNase with glutathione-S-transferase in E.coli."Biotechnology Letters. 22. 1413-1417 (2000)
• [Publications] Koh Ida: "Crystallization and preliminary X-ray crystallographic analysis of S-allelic glycoprotein SFn-RNase from Nicotiana alata."Acta Crystallographica. D57. 143-144 (2001)
• [Publications] Takanori Matsuura: "Purification and crystallization of Japanese pear S-RNase associated with gametophytic self-incompatibility."Acta Crystallographica. D57. 172-173 (2001)
• [Publications] 乗岡茂已: "花-性と生殖の分子生物学"日向康吉(学会出版センター). 258 (2001)
• [Publications] 乗岡茂已: "基礎生化学実験講座"大島泰郎(東京化学同人). 282 (2001)
• [Publications] Akiko Sasaki: "Partial purification and characterization of a novel endo-β-mannosidase acting on N-linked sugar chaing from Lilium longflorum Thumb"Journal of Biochemistry. 125. 363-367 (1999)
• [Publications] Takeshi Ishimizu: "PCR-based method of determination of S-genotype of Japanese pear"Theoretical and Applied Genetics. 98. 961-967 (1999)
• [Publications] Takeshi Ishimizu: "Presence of Asn-linked N-acetylglucosamine and chitobiose in Pyruspyrifolia S-RNases associated with gametophytic self-incompatibility"European Journal of Biochemistry. 263. 624-634 (1999)
• [Publications] 乗岡茂巳: "生産と技術"生産技術誌. 4 (1999)
• [Publications] 乗岡茂巳: "タンパク質実験ノート(下)分離同定から一次構造の決定まで"羊土社. 12 (1999)