Cloning of radish Rf (restoration of fertility) genes using TAC vector and proteome analysis.

使用 TAC 载体和蛋白质组分析克隆萝卜 Rf（生育力恢复）基因。

基本信息

批准号：
14360007
负责人：
TERACHI Toru
金额：
$ 9.22万
依托单位：
Kyoto Sangyo University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14360007/
关键词：
Cytoplasmic male-sterility Restoration of fertility gene Radish Ogura cytoplasm PPR motif Raphanus sativus Rf 雄性不稔オグラ型 Rf遺伝子 orf687 ミトコンドリア TACベクター国際研究者交流

项目摘要

This project aimed at cloning and characterization of Rf (restoration of fertility) gene(s) for Ogura male-sterility from both Japanese wild radish and European cultivars of radish, in order to clarify the structure, function, polymorphism and evolution of the Rf gene. After a pioneering work of Koizuka et al. (2003) where the first Rf gene was cloned from a Chinese variety of radish, it appears that the Rf gene encodes a protein of 687 amino acids (orf687) containing 16 PPR motifs. In this project, we cloned two orf687 homologues, each from a Japanese wild radish plant collected in a Tomioka population of Kumamoto prefecture, Japan, and from an European cultivar Comet. In addition, we characterized rf alleles in a Japanese cultivar Uchiki-Gensuke and a male-sterile wild radish taken in Fukuoka prefecture, Japan.Nucleotide sequencing of orf687 homologues revealed that all homologues encode a protein of 687 acids containing 16 PPR motifs. Comparison of deduced amino acid sequences among homologues revealed that three amino acid substitutions are present in the sixth PPR motif of orf687 from a Japanese wild radish, being compared with those of plants with rf allele. Two of three amino acid substitutions result in the charge difference of ORF687 protein, suggesting that these amino acid changes produce a functional difference between Rf and rf. The nucleotide sequence of orf687 in an European cultivar Comet also revealed possible 14 amino acid changes in Comet from those of plants with rf allele. Since orf687 in European cultivars is highly polymorphic, we are continuing to study on polymorphisms in Rf/rf genes of European cultivars of radish.For functional analysis of orf687, we have cloned orf687 of Comet and Uchiki-Gensuke into a binary vector, to transfer them into tobacco by Agrobacterium method. Transgenic tobacco with orf687 are now growing.

本项目旨在从日本野萝卜和欧洲萝卜品种中克隆和鉴定小仓雄性不育的Rf（生育力恢复）基因，以阐明Rf基因的结构、功能、多态性和进化。。经过小冢等人的开创性工作。 (2003)，第一个 Rf 基因是从中国萝卜品种中克隆出来的，Rf 基因似乎编码 687 个氨基酸的蛋白质 (orf687)，其中包含 16 个 PPR 基序。在这个项目中，我们克隆了两个 orf687 同源物，分别来自日本熊本县富冈种群中收集的日本野生萝卜植物和欧洲品种彗星。此外，我们还对日本品种 Uchiki-Gensuke 和日本福冈县的雄性不育野生萝卜中的 rf 等位基因进行了表征。orf687 同源物的核苷酸测序显示，所有同源物都编码含有 16 个 PPR 基序的 687 个酸的蛋白质。与具有 rf 等位基因的植物相比，同源物之间推导的氨基酸序列的比较表明，来自日本野萝卜的 orf687 的第六个 PPR 基序中存在三个氨基酸取代。三个氨基酸取代中的两个导致 ORF687 蛋白的电荷差异，表明这些氨基酸变化产生了 Rf 和 rf 之间的功能差异。欧洲品种Comet中orf687的核苷酸序列还揭示了Comet中可能有14个氨基酸与带有rf等位基因的植物相比发生了变化。由于欧洲萝卜品种orf687具有高度多态性，我们正在继续研究欧洲萝卜品种Rf/rf基因的多态性。为了对orf687进行功能分析，我们将Comet和Uchiki-Gensuke的orf687克隆到二元载体中，将其转入二元载体中。通过农杆菌法将它们转化为烟草。含有 orf687 的转基因烟草目前正在种植。