橡胶树白粉菌分支酸变位酶基因克隆及蛋白表达纯化  

Cloning, Prokaryotic Expression and Purification of Chorismate Mutase from Oidium heveae

作　　者：刘耀 何其光[1] 廖小淼 徐良向 刘文波[1] 林春花[1] 李潇[1] 郑服丛[1] 缪卫国[1] Liu Yao;He Qiguang;Liao Xiaomiao;Xu Liangxiang;Liu Wenbo;Lin Chunhua;Li Xiao;Zheng Fucong;Miao Weiguo(Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests,Ministry of Education,Institute of Plant Protection,Hainan University,Haikou,570228)

机构地区：[1]海南大学植物保护学院,热带农林生物灾害绿色防控教育部重点实验室,海口570228

出　　处：《基因组学与应用生物学》2020年第3期1120-1127,共8页Genomics and Applied Biology

基　　金：国家自然科学基金(No.31660033,No.31560495);国家重点研发计划(No.2018YFD0201105);海南自然科学基金创新研究团队项目(2016CXTD002);海南省重点研发计划项目(ZDYF2016208);现代农业产业技术体系建设专项资金项目(No.CARS-33-GW-BC1)共同资助。

摘　　要：由橡胶树白粉菌引起的橡胶树白粉病是橡胶树的重要的叶部病害之一,严重影响橡胶的产量。然而目前对橡胶树白粉菌的致病机理研究匮乏。分支酸变位酶是莽草酸途径的关键酶,能够将分支酸转化为预苯酸,为植物提供氨基酸及大量代谢产物,在植物抗病中起到非常重要的作用。而植物病原物在致病过程当中能够分泌分支酸变位酶影响植物莽草酸途径,从而抑制植物的防卫反应。因此研究橡胶树白粉菌分支酸变位酶在其致病过程中的功能具有一定的意义。试验利用同源比对分析在橡胶树白粉菌基因组中获得一个分支酸变位酶同源蛋白,并用PCR克隆获得橡胶树白粉菌分支酸变位酶基因,命名为OHCmu。后续构建GST-OHCmu融合原核表达载体,筛选最优诱导条件,并利用GST亲和层析柱对蛋白进行纯化。结果表明橡胶树白粉菌OHCmu基因大小843 bp,具有1个内含子,编码263个氨基酸;具有d5csma_结构域,属于Chorismate mutaseⅡ蛋白家族;GST-OHCmu融合蛋白外源诱导表达在供试条件下(IPTG:0.8 mmol/L, 16℃)可以有较好的表达,获得融合蛋白大小约为56 kD。经过GST亲和层析柱纯化、切割GST标签后,顺利获得浓度较高、较纯的橡胶树白粉菌OHCmu蛋白。研究结果为后续OHCmu蛋白的特性及致病机理研究提供参考。Rubber powdery mildew caused by Oidium heveae is one of the important leaf diseases of rubber trees,which seriously affects rubber production. But the pathogenic mechanism of Oidium heveae is still lacking.Chorismate mutase is a key enzyme in the shikimate pathway. It can transform the chorismate into prephenate,providing amino acids and a large number of metabolites for plants, and playing a very important role in plant disease resistance. Many plant pathogens have the ability to secrete chorismate mutases into host plant and affect the plant shikimate pathway. Thereby inhibiting the defense response of plants and promoting the infection of pathogens. Therefore, it is great significance to study the function of chorismate mutase from Oidium heveae. The OHCmu gene was cloned by homologous cloning method and GST-OHCmu fusion expression vectors was constructed. The optimal induction condition was selected and purified by GST affinity chromatography column.The results showed that the gene size of OHCmu was 843 bp contained 1 intron and encoding 263 amino acids. It has d5 csma_domain which belong to Chorismate mutase Ⅱ family. GST-HOCmu protein could be expressed successfully in Escherichia coli, and the size of the fusion protein was 56 kD. Finally, high yield and high purity OHCmu protein was obtained. The results of this study laid a foundation for subsequent studies on the characteristics and pathogenesis of OHCmu protein.

关 键 词：橡胶树 白粉菌 原核表达 蛋白纯化 

分 类 号：S763.7[农业科学—森林保护学]