稻瘟病菌一假定几丁质酶在毕赤酵母中的表达  

Expression of a putative Magnaporthe oryzae chitinase in Pichia pastoris

作　　者：周洁[1] 陈鑫[1] 郑祥梓[1] 林成增[1] 王宗华[1] 鲁国东[2] 

机构地区：[1]福建农林大学生命科学学院 [2]福建农林大学植物保护学院,福建福州350002

出　　处：《福建农林大学学报（自然科学版）》2009年第6期603-607,共5页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：国家自然科学基金(30471132;30500325);福建省自然科学基金(2007J0048)资助项目

摘　　要：本研究从稻瘟病菌菌株Guy11中提取总RNA,利用RT-PCR方法扩增到一假定几丁质酶基因MGG_08054.6的cDNA序列,将其亚克隆至带有His-tag标签的酵母表达载体pPIC3.5K中,构成重组质粒pPIC3.5K-ws,电击转化至毕赤酵母菌菌株GS115,通过MD-MM平板筛选及PCR验证获得重组酵母转化子GS115-PICH-ws;阳性转化子在甲醇诱导下,成功分泌出重组蛋白.SDS-PAGE分析表明,重组蛋白分子质量为48 ku,与其理论计算值基本相符;Ni-NTA法分离纯化后的重组蛋白经DNS法测定具有几丁质酶活性.Chitinase may participate in regulating cell wall dynamics,mediating the growth and colonization of fungi.In this study,cDNA of a putative chitinase gene（MGG_08054.6） was amplified by RT-PCR from the total RNA of Magnaporthe oryzae strain Guy 11.It was then subcloned into the Pichia expression vector pPIC3.5K with a RGS-His-tag,generating a recombinant plasmid,pPIC 3.5K-ws.To express the fusion protein,linearized pPIC3.5K-ws was transformed into P.pastoris strain GS115 by electroporation.Positive recombinant clone GS115-PICH-ws was obtained after MD plate screening and PCR detection.The Pichia cells were induced by methanol to express the secreted recombinant protein.The relative molecular weight of recombinant protein determined by SDS-PAGE was about 48 ku,almost equal to the theoretical molecular weight.Recombinant protein was purified by Ni-NTA column,and showed weak chitinase activity assayed by the DNS method.These results provide a basis for further investigation of biological and biochemical properties of chitinase in M.oryzae.

关 键 词：稻瘟病菌 几丁质酶 毕赤酵母 表达 

分 类 号：Q556.2[生物学—生物化学]