玉米大斑病菌StRTG2基因结构、原核表达及表达模式分析

Analysis of the Gene Structure,Prokaryotic Expression and Expression Pattern of StRTG2 in Setosphaeria turcica

为明确玉米大斑病菌StRTG2基因功能以及其在病菌不同发育时期的表达模式。利用酵母ScRTG2基因编码的氨基酸序列进行BlastP,在玉米大斑病菌中得到了其同源基因序列,将其命名为StRTG2;分别以玉米大斑病菌野生型01-23的全基因组DNA及cDNA为模板,对该基因进行克隆;利用生物信息学技术对该基因编码的蛋白StRtg2进行理化性质分析、结构域预测、亚细胞定位预测;利用MEGA 7.0对Rtg2进行进化关系分析;扩增目的片段的ORF序列,构建该基因原核表达载体,通过转化大肠杆菌BL21后,使用IPTG进行诱导表达;最后利用RNA-Seq数据库分析StRTG2基因在玉米大斑病菌关键生长发育时期(菌丝、分生孢子、芽管、附着胞和侵入钉)的表达模式。主要研究结果如下:克隆了该基因发现其长度为1695 bp,不含有内含子;该基因编码的蛋白由564个氨基酸组成,理论等电点值(pI)是6.96,具有典型的Ppx-Gppa保守结构域,亚细胞定位预测结果显示,该蛋白在细胞各部位均有分布,分布于细胞质中的可能性最大;对该蛋白进行进化关系分析表明,玉米大斑病菌StRtg2蛋白与番茄匍柄霉菌中同源蛋白的同源性最高,其次是酿酒酵母;原核表达该基因,SDS-PAGE胶显示该蛋白的大小约为62 ku,与预期大小相符;表达模式分析发现,该基因在病菌发育的5个时期均有表达,其中芽管和侵入钉时期表达量显著降低。为进一步解析StRTG2基因的功能研究奠定基础。

In order to clarify the function of the StRTG2 gene of Setosphaeria turcica and its expression pattern in different developmental stages of the pathogen,the amino acid sequence encoded by the yeast ScRTG2 gene was used for BlastP,and the homologous gene sequence in Setosphaeria turcica was obtained,and named it as StRTG2;The whole genome DNA and cDNA of wild type 01-23 of Setosphaeria turcica were used respectively as a template to clone the gene;Use bioinformatics technology to analyze the physicochemical properties,domain prediction,and subcellular location prediction of StRtg2;Use MEGA 7.0 to analyze the evolutionary relationship of Rtg2;Amplify the ORF sequence of StRTG2 to construct a prokaryotic expression vector of the gene.After transforming into E.coli BL21,IPTG was used to induce express;Finally,the RNA-Seq database was used to analyze the main growth and development stages of the StRTG2 gene(hyphae,conidia,germ tubes,appressorium and invading nails).The main results were as follows:The gene was cloned and found to be 1695 bp in length without introns;the protein encoded by the gene was composed of 564 amino acids,with a theoretical isoelectric point(pI)of 6.96,and had a typical Ppx-Gppa conserved domain.The cell location prediction results showed that the protein was distributed in all parts of the cell,and the possibility of being distributed in the cytoplasm was the greatest;The analysis of the evolutionary relationship of the protein showed that the StRtg2 protein had the highest homology with the homologous protein of Stemphylium lycopersici,followed by Saccharomyces cerevisiae;Prokaryotic expression of the gene,SDS-PAGE gel showed that the size of the protein was 62 ku,which was in line with the expected size;Analysis of the expression pattern revealed that the gene was expressed in the 5 stages of pathogen development,among them,the expression of the germ tube and the invading nail period was significantly reduced.This research can lay the foundation for further analysis of the function of StRTG2 gene.