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

本研究旨在克隆和鉴定日本野生萝卜和欧洲萝卜Ogura雄性不育恢复基因（Rf），以阐明Rf基因的结构、功能、多态性和进化。在Koizuka等人（2003）的开创性工作之后，其中第一个Rf基因是从中国萝卜品种中克隆的，似乎Rf基因编码含有16个PPR基序的687个氨基酸的蛋白质（orf 687）。在这个项目中，我们克隆了两个orf 687同源物，每个从日本野生萝卜植物收集在富冈人口的熊本县，日本，从欧洲品种彗星。此外，我们的特点rf等位基因在日本栽培品种内木玄介和雄性不育野萝卜采取的福冈县，Japan.orf687同源物的核苷酸序列分析表明，所有同源物编码的蛋白质的687个酸含有16个PPR基序。同源物之间的推导的氨基酸序列的比较显示，在第六PPR基序的日本野萝卜orf 687存在三个氨基酸取代，与那些与rf等位基因的植物相比。ORF 687蛋白质的电荷差异是由两个氨基酸取代引起的，表明这些氨基酸的改变导致了Rf和rf之间的功能差异。欧洲品种Comet的orf 687的核苷酸序列也揭示了Comet中可能有14个氨基酸的变化，从那些具有rf等位基因的植物。鉴于orf 687在欧洲萝卜品种中具有高度多态性，我们继续研究欧洲萝卜品种Rf/rf基因的多态性，并将Comet和Uchiki-Gensuke的orf 687克隆到双元载体中，通过农杆菌介导法将其导入烟草中，进行功能分析。带有orf 687的转基因烟草正在生长。

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.