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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.

本项目旨在克隆日本野生萝卜和欧洲萝卜品种小姑雄性不育恢复育性基因（Rf）并对其进行鉴定，以阐明Rf基因的结构、功能、多态性及其进化。在Koizuka等人（2003）的开创性工作之后，他们从一个中国品种的萝卜中克隆了第一个Rf基因，似乎Rf基因编码一个含有16个PPR基序的687个氨基酸（orf687）的蛋白质。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的一种日本野生萝卜，以及一种欧洲品种彗星。此外，我们还对日本栽培品种内奇根介和日本福冈县采集的雄性不育野生萝卜的rf等位基因进行了鉴定。对orf687同源物的核苷酸测序显示，所有同源物编码含有16个PPR基序的687个氨基酸的蛋白。对日本野萝卜orf687基因的第6个PPR基序进行了氨基酸序列比对，并与具有rf等位基因的植物进行了比较。ORF687蛋白的3个氨基酸替换中有2个氨基酸的替换导致了ORF687蛋白的电荷差异，提示这些氨基酸的改变导致了Rf和Rf之间的功能差异。欧洲彗星品种orf687的核苷酸序列也显示，彗星与具有rf等位基因的植物可能有14个氨基酸的变化。由于orf687在欧洲品种中具有高度多态性，我们将继续研究欧洲品种萝卜Rf/ Rf基因的多态性。为了分析orf687的功能，我们将Comet和Uchiki-Gensuke的orf687克隆到一个二元载体中，通过农杆菌法将其转移到烟草中。含有orf687的转基因烟草正在生长。