玉米大斑病菌亲环素基因的克隆及表达规律分析

Cloning and Expression Analysis of Setosphaeria turcica Cyclophilin Gene

利用简并引物PCR结合RACE技术获得S. turcica中亲环素基因的全长,并通过Real-time PCR技术检测该基因在病菌侵染结构发育过程中的表达模式。结果表明,玉米大斑病菌亲环素基因开放阅读框全长1 125 bp,3′UTR 154 bp,5′UTR 93 bp,编码374个氨基酸,将此基因命名为CyPs1,并将其cDNA序列提交GenBank,获得登录号EU679371.1,Protein ID为ACD62431.1。系统发育树分析显示,CyPs1与玉米小斑病菌(Bipolaris maydis)、蓝莓枯枝病菌(Neofusicoccum parvum)等物种的亲环素同源性可达到90%以上。该基因在病菌分生孢子萌发、附着胞形成及侵染阶段均有表达,至附着胞形成和侵染菌丝形成阶段,转录水平分别升至分生孢子时期的2倍和3倍。

Cyclophilin is a highly conserved protein family and involved in the development and function of pathogenic fungi.This study aimed to obtain the cyclophilin gene and clarify cyclophilin regulating mechanism during the morphogenesis and pathogenicity of Setosphaeria turcica.The full length of the cyclophilin gene was obtained through degenerate polymerase chain reaction(PCR)combined with rapid-amplification of c DNA ends(RACE)technique and its expression patterns were detected at different developmental stages of S.turcica based on quantitative real-time PCR(q RT-PCR)analysis.The main results were as follows,the full-length open reading frame(ORF)of the cyclophilin gene was 1 125 bp[with a 3′-untranslated region(UTR)of 154 bp and a 5′-UTR of93 bp]and encoded a protein of 374 amino acids.The gene was named as Cy Ps1 and submitted to Gen Bank with the accession no.EU679371.1 and Protein ID ACD62431.1.phylogenetic tree analysis showed that it contained more than 90%of the homologues in Bipolaria maydis and Neofusicoccum parvum respectively;in addition.Cy Ps1 continuously expressed during the developmental process of S.turcica,with the transcription levels at appressorium formation and invasive mycelial morphogenesis stages were respectively increased to 2 and 3 times of that at conidia.