中国农业科学2013，Vol.46Issue(12)：2599-2606,8.DOI:10.3864/j.issn.0578-1752.2013.12.022
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
摘要
Abstract
[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 GS 115 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.关键词
玉米大斑病菌/生物信息学分析/真核表达/MAPKKey words
Setosphaeria turcica/ bioinformatics method/ eukaryotic expression/ MAPK引用本文复制引用
巩校东,范钰,李坡,杨阳,张长志,田兰,张晓玉,范永山,韩建民..玉米大斑病菌STK2的基因组定位、蛋白质结构预测及其表达[J].中国农业科学,2013,46(12):2599-2606,8.基金项目
国家自然科学基金(30471126)、河北省自然科学基金(C2010001854,C2012204033) （30471126）
