玉米大斑病菌Stga-2及其启动子的克隆与基因表达分析

Isolation and Characterization of Stga-2 Gene from Setosphaeria turcica and Its Expression in vitro

【目的】克隆玉米大斑病菌(Setosphaeria turcica)Gα亚基(Heterotrimeric Gproteinalpha subunit)基因及其启动子,并对其进行异源表达,为深入研究Gα基因的表达规律、基因功能和编码蛋白的特性奠定基础。【方法】首先通过简并引物PCR法获得Gα基因的同源片段,再利用Genome walking技术克隆片段的5′端和3′端侧翼序列,最后采用RT-PCR法扩增基因全长,并对基因结构和上游调控元件进行生物信息学分析。同时,利用pET原核表达系统获得基因的编码产物。【结果】获得1个玉米大斑病菌Gα基因—Stga-2的全长序列及其上游部分启动子区,并利用pET原核表达系统对Stga-2成功进行了异源表达。生物信息学分析表明,Stga-2全长1318bp,由4个内含子和5个外显子组成,编码产物包含356个氨基酸残基。在347bp的上游侧翼序列中未发现典型的TATA-box及CAAT-box,但在起始密码子上游30bp处发现了转录起始子特征序列及位于其上游能够起始真菌基因转录的C+T丰富区域,同时还有转录因子Sp1的识别位点、CCA基序(CCAmotif)及CCG重复基序(CCG repeats)等顺式作用元件。【结论】玉米大斑病菌Stga-2及其启动子的成功克隆与表达进一步丰富了植物病原真菌的生物信息学资源,为深入了解植物病原真菌信号转导途径奠定了基础。

Objective The aim of this research was to clone heterotrimeric G protein alpha subunit gene in Setosphaeria turcica and express it in vitro.Method The homologous fragment of Gα gene was isolated by degenerate PCR,the 5＇and 3＇ flanking sequences were cloned using Genome walking,and the full length cDNA was obtained by RT-PCR.Gene structure and putative cis-acting elements were analyzed by bioinformatics methods and then the gene was expressed in vitro by pET expression system.Result The gene named as Stga-2,which encoded a protein of 356 amino acid residues,was 1 318 bp and interrupted by four introns.There wasn＇t typical TATA-box and CAAT-box in its upstream sequence,while the transcriptional initiator and C＋T rich region were observed.Other cis-acting elements,such as Sp1-binding site,CCA motif and CCG repeats,were also found here.At last,Stga-2 was expressed in vitro by pET system.Conclusion Cloning of Stga-2 gene and its promoter region in S.turcica enriched the biological information resource of filamentous fungi,and laid a foundation for the functional analysis about signal transduction pathway in phytopathogenic fungi.