玉米大斑病菌腺苷酸环化酶基因的克隆与功能分析

【目的】获得玉米大斑病菌（Setosphaeria turcica）cAMP信号转导途径中腺苷酸环化酶基因（StAC），利用基因敲除技术明确其在病菌致病过程中的功能。【方法】应用简并引物PCR和Genome Walking技术克隆玉米大斑病菌腺苷酸环化酶基因（StAC）DNA全长序列，并对该基因进行生物信息学分析；利用Southern blotting验证基因拷贝数；通过基因敲除技术创制StAC的缺失突变体，研究玉米大斑病菌cAMP信号转导途径中StAC的功能。【结果】StAC的DNA全长为6 816 bp，由5个外显子和4个内含子组成；ORF为6 018 bp，编码2 005个氨基酸，同源序列比对发现StAC与小麦黄斑叶枯病菌（Pyrenophora tritici-repentis）的AC基因有96%的同源性；Southern blotting证明StAC在玉米大斑病菌基因组中以单拷贝形式存在；基因功能分析表明，StAC缺失突变菌株Δstac气生菌丝灰白色，不产生分生孢子，毒素活性明显减弱，致病力下降，且在渗透胁迫条件下，菌株的抗逆能力增强，色素合成发生了改变。【结论】StAC主要调控玉米大斑病菌的产孢、致病性、高渗胁迫反应、毒素活性及色素的合成代谢。

Cloning and Functional Analysis of StAC Gene in Setosphaeria turcica

【Objective】 To understand the function of adenylate cyclase (AC) during regulating the fungal pathogenicity, the gene encoding AC in S. turcica was cloned and knocked out.【Method】Degenerated primer-PCR and genome walking were used to obtain the full length DNA of StAC in S. turcica. The structure and homology sequence alignment of StAC were analyzed by bioinformatics methods and its copy number was verified by Southern blotting. Furthermore, the function of StAC was analyzed by gene knockout technology.【Result】StAC, encoding a protein of 2 005 amino acid residues and including 5 exons and 4 introns, was 6 816 bp of DNA and 6 018 bp of ORF. The nucleotide sequence of StAC gene showed 96% identity with its homology in Pyrenophora tritici-repentis. Southern blotting showed that there was only single copy of StAC in genome of S. turcica. The phenotypic analysis showed that the aerial hyphae of StAC knockout mutant named Δstac were gray. The mutant failed to sporulate, and the toxin activity and pathogenicity on leaves of susceptible host was significantly reduced, but the resistance against hyperosmotic stress was enhanced.【Conclusion】It is suggested that StAC is involved in many procedures of S. turcica, including conidial formation, pathogenicity, hyperosmotic stress response, HT-toxin activity and the biosynthesis metabolism of pigmen.