本生烟eIF4Es基因克隆及其酵母双杂交诱饵载体构建

Cloning eIF4Es from Nicotiana benthamiana and Construction of Its Recombinant pLexA Plasmid

由木薯褐色条斑病毒(Cassava brown steak virus,CBSV)和乌干达木薯褐色条斑病毒(Uganda Cassava brown steak virus,UCBSV)引起的木薯褐色条斑病毒病,是危害木薯的主要病害之一。目前尚无有效防治方法,并且特别是缺乏CBSV/UCBSV抗性育种材料。真核翻译起始因子4E(eukaryotie translation initiation factor4E,eIF4E)不仅参与蛋白质的翻译起始,并且Potyviruses的复制和翻译也依赖于eIF4E与病毒基因组连接蛋白(virus genome linked protein,VPg)的相互作用,目前发现的植物隐性抗病基因大部分都是eIF4E家族的等位基因。本研究利用生物信息学方法和RT-PCR技术获得本生烟(Nicotiana benthamiana)eIF4E家族的8个基因,聚类分析显示它们分别编码eIF4E、eIF4E的异构体和类似帽结合的蛋白。为筛选与CBSV/UCBSV相互作用的本生烟eIF4E家族蛋白,本研究进一步构建了这8个基因的Lex A-酵母双杂交系统的诱饵载体,并导入酵母细胞EGY48(p8op-LacZ)进行细胞毒性和自激活检测。结果显示导入重组质粒的EGY48(p8op-LacZ)酵母细胞在SD/-His/-Ura缺陷型培养基上生长良好,在SD/-Trp/-Ura缺陷型平板上不生长,并且在SD/Gal/Raf/-His/-Ura/X-Gal培养基上不显蓝色,说明重组质粒表达产物不仅对该酵母细胞无毒性,而且对下游报告基因无自激活作用,可用于该系统的互作蛋白筛选研究。本研究为利用酵母双杂交系统发现与CBSV/UCBSV相互作用的本生烟eIF4E基因,探索通过基因编辑与CBSV/UCBSV互作的寄主因子进行抗CBSV/UCBSV分子育种提供科学依据。

Cassava production is severely affected by cassava brown streak disease(CBSD)which is caused by Cassava brown steak virus(CBSV)and Uganda Cassava brown steak virus(UCBSV),which belong to the Ipomovirus of the Potyviridae family.However no chemical controls are available for viral diseases and lack of CBSV/CBSV resistant breeding materials.Eukaryotie translation initiation factor 4E(eIF4E)is not only involved in the initiationof protein translation,but also the replication and translation of Potyviruses is dependent on its interaction with the Virus genome linked protein(VPg).Currently,most of the recessive resistance genes found in plants are alleles of the eIF4E family.Eight eukaryotic initiation factor 4E(eIF4E)genes of Nicotiana benthamiana were found from Nicotiana benthamianagenomes by bioinformatic analysis.The Open Reading Frame(ORF)of each gene was cloned by RT-PCR with specific primers,and was further constructed into p LexA vector.To test the toxic and autonomous activation for these recombinant constructs,each of them was transformed into the yeast cell EGY48(p8op-LacZ)and the resulting yeast cells were cultivated by using SD/-His/-Ura,SD/-Trp/-Ura and SD/Gal/Raf/-His/-Ura/X-Gal medium as well.The results showed that the yeast cells with recombinant construct growed well by using SD/-His/-Ura medium while could not grow on SD/-Trp/-Ura plate,which indicate these recombinant constructs don’t have toxicity and self-activation effect of Trp gene.Furthure more none of them have self-activation effect of LacZ gene which indicated by no blue colour shown up when cultivated on SD/Gal/Raf/-His/-Ura/X-Gal medium.These results indicated these recombinant constructs could be used to protein-protein interaction screening by using yeast two-hybrid system.This study paves the way to find out the eIF4E-like factors which interact with CBSV/UCBSV and also contributes to explore new virus resistant breeding methods through editing eIF4E gene.