酵母双杂交筛选灰飞虱体内水稻黑条矮缩病毒p10的互作蛋白

Screening of RBSDV p10 Interacting Proteins in Small Brown Planthoppers by Yeast Two Hybrid System

为获取灰飞虱体内传播RBSDV相关介体因子,以RBSDV编码的外层外壳蛋白p10为诱饵,采用酵母双杂交方法从灰飞虱cDNA文库中筛选与RBSDV p10互作的蛋白。将RBSDVp10基因构建至pGBKT7载体获得诱饵表达载体pGBKT7-p10。自激活实验结果表明,p10蛋白对酵母细胞无毒性,也不具有自激活活性。通过pGADT7-cDNA文库质粒转化含有pGBKT7-p10的酵母AH109筛选获得灰飞虱cDNA文库中与p10互作的蛋白。文库筛选共获得326个阳性克隆。测序结果表明这些阳性克隆编码14种不同的互作蛋白,包括Actin 1,GAPDH,RACK等。这些互作蛋白参与胞吞胞吐作用和膜融合等过程,可能与病毒在介体内的循回和增殖相关。

To reveal protein factors in Laodelphax striatellus Fallen（the small brown planthopper, SBPH） that related to Rice black-streaked dwarf virus （RBSDV） transmission, the outer capsid protein pl0 encoded by RBSDV S10 was selected as bait to screen the SBPH cDNA library. RBSDV plO gene was inserted to pGBKT7 vector and the bait plasmid pGBKT7-pl0 was constructed. The result of auto-activation test showed that pl0 could not autonomously activate the expression of reporter genes in yeast and bad no toxicity to yeast cell. To obtain the interacting proteins with pl0, the pGADT7-cDNA library plasmids were transformed to the yeast AH109 which contains pGBKTT-pl0. Three hundred and twenty-six positive clones were acquired. The sequencing results show that these positive clones encode 14 proteins, including Actin 1, GAPDH and RACK. These interacting proteins were involved in several processes such as endocytosis, exocytosis and membrane fusion. The interactions between pl0 and these proteins may be related to the viral circulation and proliferation in insect vector.