樱桃自交不亲和S9-单元型特异的F-box基因克隆及其表达分析

Cloning and Expression Analysis of the Self-Incompatibility S9-Haplotype Specific F-box Genes in Cherry(Prunus avium)

目的克隆李属甜樱桃自交不亲和性花粉S-决定子基因,为今后果树配子体自交不亲和性机理研究奠定理论基础。方法根据GenBank登录的16个S-locusF-box同源基因保守区设计兼并引物,利用RT-PCR、RACE等手段,从甜樱桃品种红灯花粉cDNA中克隆到两个编码376-氨基酸多肽的全长基因。结果GenBankBlast分析显示,克隆的两个基因中一个基因编码的蛋白产物与数据库甜樱桃自交不亲和性S3-单元型特异的PaSFB3(AB096857)编码的氨基酸序列完全一致。另一个基因编码一新的PaSFB同源序列,其推测的氨基酸序列N-端同SFB3一样具有明显的F-box基序,与PaSFB1～6的一致性为76%～82%。研究显示该基因在花粉组织中特异性表达,并表现出S9-单元型特异的连锁信号。结论新基因为甜樱桃自交不亲和性花粉S-决定子候选基因PaSFB家族中一新成员,命名为PaSFB9(GenBank登录号:DQ422809),红灯自交不亲和基因型确定为S3S9。

[ Objective] The purpose of this study is to isolate the pollen S-determiant genes involving self-incompatibility in cherry and settle some basic theories for the researches of self-incompatibility mechanism. [Method] A pair of consensus primers were designed according to the cDNA sequences of 16 S-locus F-box genes published in GenBank. With the RT-PCR and RACE methods, two full length genes coding a kind of polypeptides containing 376 amino acids were cloned in pollen cDNA of cherry cultivar Hongdeng. [Result] GerLBank blast showed that one of the genes coded the same amino acid sequence with the self-incompatibility S3-haplotype-specific SFB （S-locus F-box） gene, SFB3 （AB096857）. The other gene coded a novel F-box protein showed no identical sequence in NCBI database and shared 76%-82% amino acid identity with PaSFB 1-6. The experimental results showed that the novel gene expressed specifically in pollen and showed the S9-haplotype-specific linkage signal. [Conclusion] These characters suggested that the novel gene was a new member of pollen S-determinant gene family, PaSFB, in cherry, and was named PaSFB9 （GenBank accession number： DQ422809）. The S-genotype of Hongdeng was confirmed as S3S9.