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

点击翻译按钮获取中文摘要

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.