利用酵母双杂交系统筛选本氏烟中与RGSV P5互作的蛋白

Screening of RGSV P5 Interactive Proteins in Nicotiana benthamiana by Two-hybrid System of Yeast

【目的】水稻草状矮化病是由水稻草状矮化病毒引起的,给粮食产量造成了严重威胁。RGSV P5是该病毒的沉默抑制子,在抵抗寄主RNA沉默中发挥重要作用。筛选、鉴定与水稻草状矮缩病毒(RGSV)沉默抑制子P5互作的寄主蛋白,为揭示水稻草状矮化病毒致病的分子机制提供理论基础,为该病毒病的防治提供有效策略。【方法】利用酵母双杂交技术筛选与水稻草状矮化病毒P5互作的寄主蛋白。提取感染水稻草状矮化病毒的水稻叶片的总RNA。依据P5基因的CDS序列设计特异性引物。利用RT-PCR技术获得P5基因,将P5基因构建到酵母诱饵表达载体pGBKT7上,利用双酶切鉴定诱饵质粒。将诱饵载体pGBKT7、pGBKT7-P5、重组质粒pGBKT7-P5/pGADT7分别转化到酵母感受态细胞Y2H Gold中,通过观察其在缺陷培养基SD/-Trp、SD/-Leu-Trp/上的生长情况及在SD/-Ade/-His/-Leu/-Trp/X-α-gal上的显色情况检测诱饵质粒的毒性和自激活活性。将烟草酵母cDNA文库质粒转化到含诱饵载体pGBKT7-P5的酵母感受态细胞中,先后用不同缺陷型培养基SD/-Leu/-Trp、SD/-Ade/-His/-Leu/-Trp和SD/-Ade/-His/-Leu/-Trp/X-α-gal筛选后获得阳性克隆。经酵母质粒提取、转化、测序和Blast等分子生物学方法最终获得互作蛋白的序列。【结果】RT-PCR扩增得到P5基因,其大小为576 bp。成功构建诱饵载体pGBKT7-P5。表达的诱饵蛋白P5对酵母菌没有毒性和自激活活性。诱饵蛋白P5从本氏烟酵母cDNA文库中进行大量筛选,获得15个阳性克隆,分析后最终得到7个与P5互作的本氏烟蛋白。经生物信息学分析它们分别为线粒体孔蛋白VDAC、ADP核糖基化因子GTP水解酶激活蛋白ArfGAP、囊泡突触结合蛋白Syt-2、延伸因子eEF1A、脯氨酸合成酶共转录的细菌同源蛋白PROSC、非特征蛋白C9orf78及一种未知蛋白。【结论】本研究成功筛选到7个与水稻草状矮化病毒P5互作的寄主因子。这些互作的寄主因子主要参与转运、生物或非生物胁迫、胁迫应答等生物学过程。深入研究这些互作因子将为解析寄主蛋白参与调控病毒的复制、运动、致病等分子机制提供理论基础,为水稻草状矮化病毒病的防治提供新的思路。

[Objective]Rice grassy stunt disease caused by Rice grassy stunt virus(RGSV)has frequently threatened rice production in many Asian countries.P5 is the primary viral suppressor of RNA silencing(VSR)of RGSV and plays an crucial role in interfering with host RNA silencing response.Host proteins in Nicotiana benthamiana interacting with P5 protein,the RNA silencing suppressor of Rice grassy stunt virus(RGSV),were screened and identified in order to reveal the pathogenic mechanism of RGSV in plant,which will lay the foundation for effective prevention and treatment of rice grassy stunt disease.[Method]The GAL4-based yeast two-hybrid system was used to screen and identify the interacting proteins with RGSV P5 in Nicotiana benthamiana.Total RNA was extracted from rice leaves infected by RGSV.Specific primers were designed according to the CDS sequence of P5 gene and P5 gene was obtained by RT-PCR.Then P5 gene was constructed into the yeast bait expression vector pGBKT7 which was verified by digestion and transformed into Y2H Gold competent cell.The cytotoxicity of P5 protein was detected by the colony growth of pGBKT-P5 on the DO Supplement medium SD/-Trp.The colony growth and color development of pGBKT-P5/pGADT7 on the DO Supplement medium SD/-Leu/-Trp,SD/-Ade/-His/-Leu/-Trp/X-α-gal,was observed to determine whether there was autonomous activation of P5.Positive clones were obtained after screening with different defective medium SD/-Ade/-His/-Leu/-Trp and SD/-Ade/-His/-Leu/-Trp/X-α-gal.The sequence of the interacting protein was obtained by yeast plasmid extraction,transformation,sequencing and Blast.[Result]The expressed bait protein P5 had no toxicity and self-activation activity to yeast.The bait protein P5 was screened from the yeast cDNA library of N.benthamiana,and 15 positive clones were obtained.7 proteins interacting with P5 were finally obtained.Bioinformatics analysis showed that they were mitochondrial pore protein VDAC,ADP ribosylation factor GTP hydrolase activator protein ArfGAP,vesicle synaptic binding protein Syt-2,elongation factor eEF1 A,proline synthase co-transcribed bacterial homologous protein PROSC,non-characteristic protein C9orf78 and an unknown protein.[Conclusion]The results of the study showed that 7 interacting proteins with P5 were identified,which were involved in vital biological processes,including transcription,response to abiotic/biotic stimulus,stress and protein metabolism.The results of this study will provide an important theoretical basis for the mechanisms of RGSV on movement,replication,pathogenesis and new strategies for controlling rice grassy stunt disease.