论文部分内容阅读
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。
Sugar cane yellow leaf disease caused by sugarcane yellow leaf virus is one of the potential important virus diseases in sugar cane production. The virus belongs to the genera Poleroviruses in the Luteoviridae family. A pair of specific primers P0-Bait F / P0-Bait R was designed according to the whole genome sequence of the Hainan Yellow Seaweed (SCYLV-CHN-HN1) of Sugarcane Yellow Leaf Virus (GenBank No.HQ342888). The P0 protein gene was amplified by PCR using the recombinant plasmid pMD18T-P0 in the laboratory. Then the P0 protein gene and the bacterial two-hybrid bait plasmid p BT were double-digested respectively and ligated to construct the recombinant bait plasmid p BT-P0. PCR amplification, double enzyme digestion and sequence analysis showed that the bacterial two-hybrid recombinant bait vector p BT-P0 was obtained, and the coding frame was correct. The recombinant vector p BT-P0 was transformed into Bacterio Match R II Screening Reporter competent cells for self-activation and toxicity verification. The results showed that the p BT-P0 recombinant vector had no self-activation and virulence in the bacterial two-hybrid system, indicating that p BT-P0 constructed in this study can be applied to the screening of the host protein with which the interaction is carried out.