真菌蛋白激发子PevD1互作蛋白的酵母双杂交筛选及融合蛋白的原核表达

Screening of Interacting Proteins with Fungal Elicitor PevD1 by Yeast Two Hybrid System and High Expression of Recombinant in E. coli

PevD1是大丽轮枝菌(Verticillium dahliae)发酵液中分离纯化的一种新蛋白激发子,能激发烟草和棉花的免疫防御反应,提高抗病性。旨在深入研究PevD1的作用机理,利用酵母双杂交系统,以PevD1为诱饵蛋白,筛选拟南芥中PevD1的互作蛋白,经复筛、回转验证得到3个候选互作蛋白基因R1,R2和R4。利用同源克隆技术获得烟草中互作蛋白编码基因NR1、NR2和NR4;经酵母双杂交验证其中的NR2蛋白能与PevD1特异性结合。利用原核表达系统,成功构建了重组表达载体pMAL-C2XNR2并转化大肠杆菌细胞,经IPTG诱导获得了可溶性表达的NR2融合蛋白。

PevD I, a fungal protein elicitor, was secreted from FerticiEium dahEae and could induce hypersensitive responses （ HR ） and systemic acquired resistances （ SAR ） in tobacco plant. In this study, we screened its interaction partners using yeast two hybrid system with Arabidopsis thaliana cDNA library. Three potential interacting proteins were obtained. Homologous genes NR1, NR2 and NR4 in Nicotiana tobacum were cloned. Yeast two-hybrid binding assays confirmed NR2 protein strangely binding with PevD1. The entire coding region of NR2 gene was cloned into the bacterial expression vectors pMAL-C2X. After IPTG induction and SDS-PAGE analysis showed that the target protein was expressed at a high level in bacterial cells.