橡胶树炭疽菌CsHog1基因的克隆和原核表达分析

Cloning and Prokaryotic Expression of CsHog1 Gene from Colletotrichum siamense

HOG MAPK途径在植物病原真菌生长发育、致病过程和对杀菌剂敏感性等方面发挥重要功能,但在不同的病原真菌中具有一定差异。为研究Hog1蛋白在橡胶树炭疽菌(Colletotrichum siamense)中的功能和调控机制,本研究利用同源克隆法克隆获得橡胶树炭疽菌(C. siamense)的CsHog1基因,构建GST-CsHog1融合表达载体,利用SDS-PAGE和WesternBlot检测蛋白表达情况。结果表明:橡胶树炭疽菌(C.siamense)的CsHog1基因大小1383 nt,编码459个氨基酸,包含5个内含子,具有丝氨酸/苏氨酸蛋白激酶催化结构域;聚类分析显示橡胶树炭疽菌的CsHog1基因编码的氨基酸序列与黄瓜炭疽菌的ClOsc1(Hog1同源基因)同源性最高。所构建的蛋白融合表达载体在供试条件下(IPTG0.3、0.5、0.8、1.0 mmol/L;温度16、25、28℃)均可较好诱导表达,GST-CsHog1融合蛋白大小约为70 ku。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。

The high osmolarity glycerol(HOG) response pathway plays important roles in growth and development,pathogenesis, fungicide sensitivity etc. in phytopathogenic fungi, but its function may vary in different pathogenic fungi.In this study, sHog1 was cloned by homologous cloning from Colletotrichum siamense isolated from rubber trees in order to understand the role and regulatory mechanisms of Hog1 protein. GST-Hog1 fusion protein expression vectors were also constructed and expressed in Escherichia coli BL21(DE3). The expressed fusion proteins were analyzed on SDS-PAGE and Western Blotting. The results showed that CsHog1 contained 5 introns and encoded 459 amino acids and had the Serine/Threonine protein kinases catalytic domain STKc. Phylogenetic clustering showed that CsHog1 shared the highest similarity to ClOsc1(Hog1 homologue) of C. lagenarium. SDS-PAGE analysis showed that GST-Hog1 protein was successfully expressed in E. coli under the induction of IPTG at 0.3, 0.5, 0.8 and 1.0 mmol/L and at incubation temperature of 16, 25 or 28 ℃. The size of the fusion proteins was 70 ku which was identical to the expected molecular weight. Western Blotting showed that the purified fusion protein could be recognized by GST antibodies.