番茄CYP71基因的克隆及其功能的初步分析

CYP71 Gene Cloning and Identification of Its Function in Tomato

分析番茄的EST数据库,获得一条在番茄果实中表达的EST序列CYP71,通过RT-PCR分析表明,该基因在番茄的叶、花、果实中均有表达,其中幼果中表达最强,并受到乙烯的负调控,推测可能与果实的发育成熟相关。进而通过RACE(rapid amplification of cDNA ends)的方法获得了全长为1637bp的番茄CYP71基因(GenBank accession No.GQ370622)。该片段包括一个完整的开放阅读框(1488bp),编码495个氨基酸。通过系统进化树分析,属于CYP71家族。将番茄的CYP71基因定向克隆到植物表达载体pBI121中,获得CYP71基因的正、反义植物表达载体pBI121-CYP71s和pBI121-CYP71as,为深入研究该基因的功能奠定基础。

According to bioinformatics analysis of the EST database of tomato, an EST sequence CYP71 expressed in fruit of tomato was selected. RT-PCR analysis showed that this sequence was expressed in leaves, flowers and fruit, but most highly in young fruit, and was down regulated by the treatment of ethylene. Presumably, this sequence is related to fruit development and maturation. A full length cDNA （GenBank accession No. CQ370622） of CYP 71 with 1 637 bp was amplified from tomato fruit by using rapid amplification of cDNA ends（RACE） technique. Sequence analysis showed that the cDNA contained a 1 488 bp ORF and encoded a protein consisting of 495 amino acid residues. Phylogenetic analysis indicated that this cDNA fragment belonged to CYP71 family of P450. It was successfully cloned into plant expression vector pBI121 to produce sense and antisense plant expression vectors pBI121-CYP71s and pBI121-CYP71as, which provides a basis for study of the function of CYP71 gene.