稻瘟病菌MGG-01005的表达纯化与生物信息学分析  

Expression,purification and bioinformatics analysis of Magnaporthe oryzae MGG-01005

作　　者：赵秀平 王双[1] 闫星伊 段强 张帅 陈永胜[1] 李国瑞[1] ZHAO Xiuping;WANG Shuang;YAN Xingyi;DUAN Qiang;ZHANG Shuai;CHEN Yongsheng;LI Guorui(College of Life Science and Food Science, Inner Mongolia University for Nationalities, Inner Mongolia Industrial Engineering Research Center of Universities for Castor, Inner Mongolia Key Laboratory of Castor Breeding, Inner Mongolia Collaborative Innovation Center for Castor Industry, Inner Mongolia Engineering Research Center of Industrial technology Innovation of Castor, Tongliao 028043, China)

机构地区：[1]内蒙古民族大学生命科学与食品学院,内蒙古自治区高校蓖麻产业工程技术研究中心,内蒙古自治区蓖麻育种重点实验室,内蒙古自治区蓖麻产业协同创新中心,蓖麻产业技术创新内蒙古自治区工程研究中心,内蒙古通辽028043

出　　处：《浙江农业学报》2021年第3期470-478,共9页Acta Agriculturae Zhejiangensis

摘　　要：MGG-01005是与稻瘟病菌的菌丝生长有重要关系的基因,对其进行一般理化性质、结构域、功能位点预测等一系列生物信息学分析,构建了原核表达载体pETM13-MGG-01005,利用IPTG诱导重组蛋白表达,并通过镍离子亲和层析、阴离子交换层析以及分子筛层析进行蛋白纯化。结果显示,该蛋白相对分子质量约为16471.49 u,编码153个氨基酸,含Tctex-1结构域,无跨膜结构和信号肽,无功能位点,存在磷酸活性位点,为不稳定亲水蛋白;该蛋白可被0.1 mmol·L^(-1) IPTG诱导表达,亲和层析的最适洗脱液组分为20 mmol·L^(-1) Tris-HCl,500 mmol·L^(-1) NaCl,80 mmol·L^(-1)咪唑;阴离子交换层析表明该蛋白对低盐条件具耐受性;分子筛层析具有形态均一且对称性良好的构象,最大洗脱峰出峰位置对应的蛋白相对分子质量约为35 ku,表明该蛋白以二聚体形式存在。本研究最终得到大量高纯蛋白,以期为进一步探索该蛋白的功能以及稻瘟病菌的后续相关研究奠定理论与实践基础。MGG-01005 is an important gene associated with mycelia growth of Magnaporthe oryzae.In this study,a series of bioinformatics analysis including general physical and chemical properties,structural domains,and functional site prediction were carried out to construct the prokaryotic expression vector PETM13-MGG-01005.IPTG was used to induce the expression of recombinant protein,and the protein was purified by nickel ion affinity chromatography,anion exchange chromatography and molecular sieve chromatography.The results showed that the molecular weight of the protein was about 16471.49 u,which encoded 153 amino acids.It contained the domain of Tctex-1,had no transmembrane structure and signal peptide,had no functional site,and had the active site of phosphoric acid,and was an unstable hydrophilic protein.The protein could be induced to express by 0.1 mmol·L^(-1) IPTG.The optimal eluent of affinity chromatography was divided into 20 mmol·L^(-1) Tris-HCl,500 mmol·L^(-1) NaCl,and 80 mmol·L^(-1) imidazole.Anion exchange chromatography showed that the protein was resistant to low salt conditions.The molecular sieve chromatography has the conformation of uniform morphology and good symmetry,and the molecular weight of the protein corresponding to the maximum elution peak is about 35 ku,indicating that the protein exists in the form of dimer.In this study,a large amount of high purity protein was obtained in order to lay a theoretical and practical foundation for further exploration of the function of this protein and subsequent related studies on Magnaporthe oryzae.

关 键 词：稻瘟病菌 原核表达 纯化 生物信息学分析 

分 类 号：S435.111.41[农业科学—农业昆虫与害虫防治]