Molecular mechanism of the recognition of the style to pollen with discriminate S-genotype in gametophytic self-incompatibility of apple

苹果配子体自交不亲和性S基因型花粉花柱识别的分子机制

• 批准号: 15580088
• 负责人: OKUNO Toshikatsu
• 金额: $ 2.43万
• 依托单位: Hirosaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15580088/
• 关键词: Self-incompatibility; apple; S-RNase; Affinity chromatography; Protein interaction; Surface plasmon resonance; Affinity column; リンゴ自家不和合性; S-RNase固定化クロマトグラフィー; 抗S-RNase抗体カラム; S-RNase相互作用タンパク; S-RNase酵素活性

To elucidate a molecular mechanism of gametophytic self-incompatibility of apple the interactive proteins to the S-RNase were screened from the pollen with distinctive S-genotype. The used varieties of apples is Stalking-Delicious (SD)(S-genotype : Sc, Se), Golden-Delicious (GD)(Sa, Sb).○Se-and Sc-RNase as S-RNase from SD were purified by our previously described method.○From germinated pollen of the varieties, SD and GD, crude extracts of soluble proteins and total RNA fractions were prepared.○The specific activities of the purified RNases were compared with other RNases (RNase A and T2) by using various RNA as substrate. The S-RNases showed very low values in specific activity than that of RNase A.○An affinity column of which Se-RNase bound to Hi-Trap (Pharmacia) was prepared. The crude pollen extracts were treated with the column and two absorbed proteins showed molecular mass of 48 and 66 KD on SDS-PAGE were obtained These proteins were confirmed to be a substance which interacts s … More pecifically with Se S-RNase by the immunodetection using Se-RNase antibody. Both substances, however, did not change the activity of the S-RNase to the tube elongation of pollen with the same S-genotype or alternative one.○The proteins, 48 and 66 KD of Mr, were also obtained by immune precipitation method.○The interaction of Se-RNase with soluble pollen proteins was examined by surface plasm on resonance method using Biacore 2000 (Biacore) with a Se-RNase binding sensor tip. For all fractions separated by DE chromatography of pollen crude extracts the protein interactions were measured. Only two fractions containing 48 or 66 KD of Mr were showed large Ka values and slow depression of Kd.In this studies the presence of interactive proteins to S-RNase from pollen extracts were confirmed by affinity column, immune precipitation, and surface plasmon resonance methods. These substances, however, did not inhibit the RNase activity of S-RNase in vitro an assay system, and therefore, additional substances derived from pollen may need to recognize the difference between self and cross pollen. Less

为阐明苹果配子体自交不亲和性的分子机理，从不同S基因型的花粉中筛选与S-RNase相互作用的蛋白质。苹果的品种是Stalking-Delicious（SD）（S基因型：Sc，Se），Golden-Delicious（GD）（Sa，Sb）.○从SD中纯化了Se-和Sc-RNase作为S-RNase.○从SD和GD的萌发花粉中，制备可溶性蛋白质粗提物和总RNA组分。○将纯化的RNA酶与其他RNA酶的比活性进行比较（RNase A和T2）。S-RNase比RNase A的比活性非常低。○制备Se-RNase与Hi-Trap（Pharmacia）结合的亲和柱。花粉粗提液经柱层析后，得到两种吸附蛋白，SDS-PAGE显示其分子量分别为48和66 KD。 ...更多信息 通过使用Se-RNase抗体的免疫检测，特别是与Se S-RNase结合。用免疫沉淀法获得了分子量为48和66 KD的蛋白质。用Biacore 2000（Biacore）表面等离子体共振法研究了Se-RNase与花粉可溶性蛋白质的相互作用。对于花粉粗提物的DE色谱分离的所有级分的蛋白质相互作用进行了测量。只有两个组分含有48或66 KD的先生表现出大的Ka值和缓慢的kd.In本研究中的存在相互作用的蛋白质从花粉提取物的S-RNase亲和柱，免疫沉淀，和表面等离子体共振方法证实。然而，这些物质在体外测定系统中不抑制S-RNase的RNase活性，因此，可能需要来自花粉的其他物质来识别自交和杂交花粉之间的差异。少

项目成果

Biosynthesis of Lambertellols Based on the High Specific Incorporation of the 13-C labeled Acetate and Their Biological Properties

基于13-C标记乙酸盐高特异性掺入的兰伯特醇生物合成及其生物学性质

• 发表时间: 2004
• 期刊: J.Amer.Chem.Soc. 126(6)
• 作者: Ken-ichi Nihei;Kimiko Hashimoto;Kazuo Miyairi;Toshikatsu Okuno;T.Murakami et al.
• 通讯作者: T.Murakami et al.

Synthesis of thiosaccharides employing the Pummerer rearrangement of tetrahydrothiopyran oxides

• DOI: 10.1016/j.tet.2004.06.006
• 发表时间: 2004-08
• 期刊: Tetrahedron
• 影响因子: 2.1
• 作者: Junji Fujita;H. Matsuda;Kazunori Yamamoto;Yasuharu Morii;M. Hashimoto;T. Okuno;K. Hashimoto

Lamberteroll C a labile and novel Biosynthetic congener of lambertellols A and B.

Lamberteroll C 是兰伯特醇 A 和 B 的不稳定且新颖的生物合成同系物。

• 发表时间: 2005
• 期刊: Bioorg Med.Chem.Lett. 15
• 作者: Takanori Murakami;Akane Sasaki;Ed Fukushi;Jun Kawabata;Masaru Hashimoto;Toshikatsu Okuno
• 通讯作者: Toshikatsu Okuno

S-RNase from Self-incompatible and - compatible Cultivars : purification, Cloning Enzymic Properties, and Pollen tube Growth

来自自交不亲和和亲和品种的 S-RNase：纯化、克隆酶特性和花粉管生长

• 发表时间: 2002
• 期刊: Biosci.Biotechnol.Biochem. 66(6)
• 作者: Takanori Murakami;Yoshifumi Morikawa;Masaru Hashimoto;Toshikatsu Okuno;Yukio Harada;J.Fujita et al.;N.katoh et al.
• 通讯作者: N.katoh et al.

S-RNases from Self-incompatible and -compatible Apple Cultivars: Purification, Cloning, Enzymic Properties, and Pollen Tube Growth Inhibitory Activity

• DOI: 10.1271/bbb.66.1185
• 发表时间: 2002-01
• 期刊: Bioscience, Biotechnology, and Biochemistry
• 作者: Naoki Katoh;K. Goto;Junpei Asano;K. Fukushima;Kenji Yamada;A. Kasai;T. Li;Makoto Takanoha;K. Miyairi;T. Okuno