酵母双杂交系统筛选与水稻条纹病毒Pc2互作的寄主蛋白基因片段

Screening of Host Proteins Interacting with Pc2 of Rice Stripe Virus by Yeast Two-hybrid System

水稻条纹病毒编码的Pc2蛋白在病毒侵染及宿主症状发生中起作用。研究其与寄主间的分子互作有助于揭示病毒侵染与致病的分子机制。分别将编码Pc2N端（Pc2-N）与Pc2C端（Pc2-C）的基因片段克隆到酵母双杂交诱饵载体pGBKT7上．并构建水稻叶片cDNA文库．然后分别用Pc2-N和Pc2-C为诱饵筛选文库寻找与其互作的寄主因子。结果表明．诱饵载体中插入的Pc2-N和Pc2-C编码框和氨基酸序列均正确。Pc2-N对酵母菌株AHl09无毒性和自主激活能力，但Pc2-C对酵母具有细胞毒性。文库滴度为4x107CFU／mL．且大多数插入片段在500～2000bp之间。利用Pc2-N为诱饵筛选水稻cDNA文库．获得有8个候选阳性克隆。经序列测定和BLAST分析结果表明．这些克隆共编码5种蛋白．主要为胁迫相关蛋白与光系统相关蛋白。

Rice stripe virus encoded Pc2 protein is involved in the viral infection and symptom formation of rice. Study on the interaction between Pc2 and host is essential to elucidate the molecular mechanism of viral infection. In this study, the encoding region of Pe2-N or Pc2-C was fused to the pGBKT7, the rice eDNA library was constructed and screened by the Pc2-N or Pc2-C as a bait to identify proteins that interacted with Pc2. The results showed that the coding regions and the amino acid sequences of insert Pc2-N and Pc2-C were correct. Pc2-N had neither toxicity nor self activating effect on AH109, while Pc2-C was toxic to AH109. The eDNA library had a titer o f 4x107 CFU/mL, and most of insert fragments were between 500 bp to 2 000 bp in size. Eight candidate positive clones were obtained by screening with Pe2-N as a bait. Their eDNA Sequences were obtained and BLAST results showed that these cDNAs encoded 5 proteins, which mainly were the proteins involved in photosystem and stress.