玉米大斑病菌STK2的基因组定位、蛋白质结构预测及其表达

Localization of STK2 of Setosphaeria turcica in the Genome,Characterization of Its Protein Structure and Expression in Eukaryotic Cells

【目的】确定玉米大斑病菌STK2在基因组中的位置;解析目的蛋白质Stk2的结构特征;构建STK2真核表达载体,获得真核表达体系中的胞外分泌蛋白。【方法】利用生物信息学方法,确定STK2在玉米大斑病菌基因组中的确切位置,解析Stk2蛋白质的结构特征;根据STK2的ORF序列及真核表达载体pPIC9K的多克隆位点设计引物,构建真核表达载体,采用电击转化法将重组质粒转入宿主菌GS115中进行诱导表达,利用SDS-PAGE检测并鉴定目的蛋白质。【结果】玉米大斑病菌STK2的ID为91433,该基因位于scaffold_3正链的1561986-1563262位置;Stk2蛋白具有MAPK类蛋白激酶的特征性保守结构域,其二级结构主要以α螺旋和无规则卷曲为主,β折叠较少且主要存在于N端,其三级结构具有1个较小的N端域和1个较大的C端域;成功构建了STK2的真核表达载体pPIC9K-STK2,筛选获得了抗性转化子;在毕赤酵母菌中,经甲醇诱导得到了1个分子量约为41 kD的蛋白质组分且被分泌至胞外,该蛋白质的大小与理论分子量相符,推测Stk2蛋白已在宿主菌中表达并被分泌至胞外。【结论】玉米大斑病菌STK2位于scaffold_3正链的1561986-1563262位置;Stk2具有MAPK激酶的所有特征性保守结构域,属于典型的MAPK蛋白激酶;该基因能在真核细胞中表达,获得了可溶性分泌蛋白质,为Stk2蛋白的多克隆抗体制备及基因的功能研究奠定基础。

【Objective】The objectives of this study is to determine the location in genome,and to investigate the protein structure of STK2 of Setosphaeria turcica.Meanwhile,a eukaryotic expression vector of STK2 was constructed and expressed in Pichia pastoris to obtain secreted protein in extracellular.【Method】 The location of STK2 and protein structures were analyzed using bioinformatics method.Primers were designed according to multiple cloning sites in vector pPIC9K and the ORF sequence of STK2.A recombinant eukaryotic expression vector was constructed and transformed into host yeast strain GS115 by electroporation.The aiming protein expressed in yeast cell through induction and was identified by SDS-PAGE.【Result】 The identity of STK2 was 91433,locating between 1561986 and 1563262 in the positive-strand of scaffold_3 in the genome of S.turcica.Stk2 shared characteristic conserved domains of MAPK-like protein kinase.α-helices and random coils were its main elements of the secondary structure of Stk2,β strands were lesser and mainly located in N ends in this structure.The 3D structure was composed of a smaller N-terminal lobe and a bigger C-terminal lobe.The eukaryotic expression vector of pPIC9K-STK2 was successfully constructed,and the transformants were screened according to their resistance.A protein,with its molecular weight of about 41 kD,was obtained after induced by methanol,while no corresponding proteins were found in control,suggesting STK2 had been secreted to extracellular of the yeast cells.【Conclusion】STK2 is located between 1561986 and 1563262 in the positive-strand of scaffold_3 in the genome of S.turcica.The characteristics of Stk2 were analyzed,and showed that Stk2 shared all of conserved domains of MAPK kinase.The results also revealed that STK2 was successfully expressed in yeast cell and soluble protein was got in the experiment.These results will lay a foundation for the preparation of Stk2 polyclonal antibody and for the study of the functions of STK2.