蝉拟青霉疏水蛋白PChyd基因表达模式分析及敲除载体构建

Analysis of Expression Pattern and Construction of Knockout Vector of PChyd Gene of Paecilomyces cicadae

为深入研究昆虫病原真菌蝉拟青霉疏水蛋白PChyd基因的功能,根据蝉拟青霉基因组信息克隆疏水蛋白PChyd基因,对该基因序列进行生物信息学分析,使用qRT-PCR技术对其在不同培养条件或阶段下的表达模式进行分析,并通过酶切酶连的方法构建了该基因的敲除载体。结果表明:PChyd基因的开放阅读框序列全长303 bp,编码100 aa,包含22 aa的信号肽序列和70 aa疏水蛋白功能区域。系统发育分析显示该基因与粗糙虫草菌亲缘关系最近。qRT-PCR结果显示PChyd基因在PDA培养的菌丝体、诱导的附着胞、诱导的芽生孢子中表达量显著高于另外2个样品,其中芽生孢子表达量最高,暗示该基因在蝉拟青霉侵染初期和在昆虫血腔中定殖阶段可能具有重要作用。凝胶电泳结果表明,成功构建了该基因的敲除载体,扩增出含有上臂、HPH、下臂的3356 bp左右的片段。本研究为进一步探究蝉拟青霉疏水蛋白PChyd基因的致病机理、生防工程菌的改造奠定了基础。

In order to investigate the function of PChyd of Paecilomyces cicadae,the gene was cloned according to the genomic data of P.cicadae,and the bioinformatics of the gene were analyzed by the NCBI database and the Expasy online tools.The expression pattern of PChyd was analyzed under different culture conditions or stages by qRT-PCR.The knockout vector of the gene was constructed by molecular biological methods.The open reading frame of PChyd was 303 bp encoding 100 aa.Domain analysis revealed that the gene contained a 22 aa signal peptide region and a 70 aa hydrophobin functional region.Phylogenetic analysis showed that the relationship of P.cicadae PChyd was close to Cordyceps confragosa hydrophobin.Expression pattern analysis showed that the expression level of PChyd in mycelium in PDA,appressorium and blastospore were significantly higher than that of the other two samples,and the expression level was the highest in blastospore among five samples,which indicating that this gene had important function in the initial stage of the infection of P.cicadae and in the colonization stage of insect blood.The results of gel electrophoresis and sequencing showed that the knockout vector of the gene was successfully constructed.The research would lay a foundation for further exploring of the pathogenic mechanism of PChyd and engineering this entomopathogenic fungus P.cicadae.