摘要： 利用酵母双杂合体系统，检测与植物线状病毒组中热休克蛋白HSP70相结合的其它相关的BYV蛋白。在体外构建两种编码融合蛋白的酵母表达质粒：一组为将编码转录激活区域的基因与HSP70r基因融合并克隆到pACT质粒中（pACT·HSP70r）,它编码转录激活区域与HSP70蛋白的融合蛋白；另一组为将编码DNA结合区域的基因分别与甜菜黄化病毒基因组其它7个基因融合并克隆到pAS质粒中（pAS·-）,分别编码7个融合蛋白。经两质粒共转化酵母细胞后，通过双报告基因（Lacz基因和组氨酸His3基因）筛选获得阳性菌落，以阳性菌落总DNA为模板，经体外特异性引物的PCR扩增，检测出HSP70蛋白与p20蛋白之间的相互作用，并证实这一相互作用是特异的。
关键词:
酵母双杂合体系统,
热休克蛋白HSP70,
甜菜黄化病毒,
蛋白质间相互作用
Abstract: This thesis analyzed the beet yellow virus’ proteins which are possible to interact with Heat shock protein 70 of plant closteroviruses in the host cells,using the highly specific yeast two hybrid system.In vitro,two yeast expression plasmids containing hybrids were constructed:one was the plasmid pACT?HSP70r containing the fused genes between the gene encoded for activation domain and HSP70r gene,which codes their fused protein;the other one was the plasmid pAS?BYV gene(s) containing the fused gene(s) betwwen the gene encoded for DNA binding domain and BYV gene(s),which respectively codes seven fused protein(s).Yeast cells were cotransformed by both of the two plasmids,and the transformed cells were detected by two report genes( Lacz gene and His3 gene).Positive colonies were obtained.Total DNA extracting from the positive colonies was used as the template and PCR was done in vitro.The interaction between HSP70 and p20 was found,and it was proved that this interaction was specific.This result will give us the new way to study the function of HSP70.
Key words:
Yeast two hybrid system,
Heat shock protein 70,
Beet yellow virus(BYV),
Proteinprotein interaction
中图分类号:
金红, V.V.Doljia, V.V.Peremyslov. 利用酵母表达质粒检测甜菜黄化病毒蛋白质间相互作用的遗传学研究[J]. 华北农学报, 1998, 13(2): 57-63. doi: 10.3321/j.issn:1000-7091.1998.02.011.
Jin Hong, V.V.Doljia V.V.Peremyslov. Genetic Studies on the Interactions of BYV ProteinsUsing Yeast Expression Plasmid Vectors[J]. ACTA AGRICULTURAE BOREALI-SINICA, 1998, 13(2): 57-63. doi: 10.3321/j.issn:1000-7091.1998.02.011.