橡胶树炭疽菌CsPbs2基因的原核表达分析

Prokaryotic Expression Analysis of CsPbs2 Gene from Colletotrichum siamense

HOG MAPK途径参与植物病原真菌生长发育、致病和对杀菌剂敏感性等过程,CsPbs2基因是HOG MAPK信号通路的关键成员。为研究CsPbs2蛋白的互作蛋白及其调控机制,本研究克隆了橡胶树炭疽菌(Colletotrichum siamense) CsPbs2基因,构建融合蛋白表达载体,利用SDS-PAGE和Western Blot检测蛋白表达。结果显示橡胶树炭疽菌(C. siamense)的CsPbs2基因全长2 051 bp,编码667个氨基酸,包含个1个内含子,具有丝氨酸/苏氨酸蛋白激酶催化结构域。CsPbs2基因在进化关系上与球孢白僵菌(Beauveria bassiana)、禾谷镰刀菌(Fusarium graminearum)、尖孢镰刀菌(Fusarium oxysporum)、稻瘟病菌(Magnaporthe oryzae)的Pbs2基因有最近的亲缘关系。构建的GST-CsPbs2蛋白融合表达载体在供试条件下(IPTG:0.6 mmol/L, 0.8 mmol/L,1.0 mmol/L温度16℃和25℃)均可较好诱导表达,GST-CsPbs2融合蛋白大小约为96 kD。用GST亲和层析柱纯化获得蛋白,经Western Blot检测显示该蛋白可与GST单克隆抗体特异性结合。本研究成功构建了GSTCsPbs2蛋白融合表达载体,纯化得到GST-CsPbs2融合蛋白,为后续利用Pull-down技术钓取Cs Pbs2的互作蛋白提供基础。

In phytopathogenic fungi, the high osmolarity glycerol (HOG) response pathway plays an important role in growth and development, pathogenesis, fungicide sensitivity and so on. CsPbs2 gene plays an important role in HOG MAPK signaling Pathway. Currently, molecular characteristics of fungi which has important influence on the pathogenicity and fungicidal resistance of rubber trees very little is knowned by people. In order to understand the regulatory mechanism of Pbs2 protein in Colletotrichum siamense from rubber tree. In this study, C. siamense CsPbs2 gene was cloned by homologous cloning method. Prokaryotic expressed of fusion protein in Escherichia coli BL21 (DE3) was detected by SDS-PAGE and Western Blot. The results showed that the C. siamense CsPbs2 gene contained 1 introns and encoded 667 amino acids, it has a serine/threonine protein kinases catalytic domain of S TKc. Phylogenetic clustering suggested that there is a close relationship between CsPbs2 gene and Pbs2 gene of Beauveria bassiana, Fusarium graminearum, Fusarium oxysporum, Magnaporthe oryzae and GST-Pbs2 fusion expression vectors were constructed on the skeleton vectors of pGST. And prokaryotic expressed of fiision protein in Escherichia coll BL21 (DE3) was detected by SDS-PAGE and Western Blot. SDS-PAGE detection showed that GST-Pbs2 protein could be expressed successfully in Escherichia coli, induced by 0.6 mmol/L, 0.8 mmol/L or 1 mmol/L ITPT on 16°C or 25°C was the best optimized condition for the fusion proteins expression, and the sizes of the fusion proteins were 96 kD. Western Blot showed that purified fusion protein could be recognized by GST antibodies. The results showed that the recombinant GST-CsPbs2 protein was successfully constructed and the purification of GST-CsPbs2 protein was get. This research will provide a basis for the subsequent use of Pull-down technology to catch CsPbs2 interaction protein.