甘蔗黄叶病毒P0蛋白基因细菌双杂交诱饵载体的构建和自激活鉴定

Bait Plasmid pBT-P0 Construction and Its Self-activation Identification in Bacterial Two-hybrid System

由甘蔗黄叶病毒所引起的甘蔗黄叶病是甘蔗生产中潜在的重要病毒病害之一。该病毒属黄症病毒科(Luteoviridae)中的马铃薯卷叶病毒属(Poleroviruses)。根据甘蔗黄叶病毒海南分离物(SCYLV-CHN-HN1)全基因组序列(Gen Bank No.HQ342888),设计P0蛋白基因的一对特异性引物P0-Bait F/P0-Bait R。以实验室保存的重组质粒p MD18T-P0为DNA模板,PCR扩增P0蛋白基因。然后将P0蛋白基因和细菌双杂交诱饵质粒p BT分别进行双酶切后,连接构建重组诱饵质粒p BT-P0。经PCR扩增、双酶切鉴定和序列分析,表明获得了细菌双杂交重组诱饵载体p BT-P0,且编码框正确。将重组载体p BT-P0转化细菌双杂交系统Bacterio Match RⅡScreening Reporter感受态细胞,进行自激活和毒性验证。结果显示p BT-P0重组载体在细菌双杂交系统中无自激活作用及毒性作用,表明本试验构建的p BT-P0可应用于该系统筛选与之互作的寄主蛋白。

Sugarcane yellow leaf disease(SCYLD), also known as sugarcane yellow leaf syndrome(SCYLS), is a significant viral disease of sugarcane production worldwide. The pathogen is Sugarcane yellow leaf virus(SCYLV),belonging to genus Luteoviridae, family Poleroviruses. In this study, the specific primers of P0-Bait F/P0-Bait R for SCYLV P0 protein gene were designed on the basis of SCYLV-CHN-HN1 complete genome sequence isolated from Hainan(Gen Bank No. HQ342888) and p BT bait plasmid sequences. P0 protein gene was amplified from the template of recombinant plasmid p MD18T-P0. The PCR product was double digested by Bam H玉/Bgl域, and ligated to linear bait plasmid p BT via the same digestion. In order to get the recombinant bait plasmid p BT-P0,single or double enzyme restrictions digestion and sequencing analysis was conducted. The results showed that p BT-P0 recombinant plasmid was obtained successfully. To verify the bait plasmid p BT-P0 can be used for proteinprotein interaction screening in bacterial two-hybrid system, the plasmid was transformed to Bacterio Match RⅡscreening reporter competent cell for self-activation and toxicity verification. The results showed that pBT-P0 did not have self-activation or toxicity in bacterial two-hybrid system. These outcomes indicate that the bait plasmid p BT-P0 can be further used for screening the interacted proteins from host c DNA library in the bacterial two-hybrid system.