中国番茄黄化曲叶病毒编码的RNA沉默抑制子AC4蛋白的核酸结合特性  

Nucleic acid binding property of RNA silencing suppressor AC4 protein encoded by Tomato yellow leaf curl China virus

作　　者：张馨月[1] 章颉[1] 邓凤林[1] 吴建祥[1] 

机构地区：[1]浙江大学农业与生物技术学院/生物技术研究所,浙江杭州310029

出　　处：《南京农业大学学报》2011年第2期85-90,共6页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(30670087);国家自然科学基金重点项目(30530520)

摘　　要：农杆菌共浸润试验结果表明:中国番茄黄化曲叶病毒(Tomato yellow leaf curl China virus,TYLCCNV)Y10分离物编码AC4蛋白是RNA沉默抑制子。为了研究RNA沉默抑制子AC4蛋白的作用机制,将TYLCCNV-Y10的AC4基因插入到原核表达载体pET-32a的多克隆位点上,构建重组原核表达载体pET32a-Y10AC4。将重组载体导入BL21(DE3)pLysS进行AC4蛋白表达,并在自然条件下纯化蛋白。利用原核表达的AC4蛋白进行电泳迁移率变动试验(electrophoretic mobilityshift assay,EMSA),分析AC4蛋白分别与单、双链siRNA和单、双链长RNA的结合情况。试验结果表明:TYLCCNV-Y10编码的AC4蛋白具有结合单链siRNA和单链长RNA特性,而不与双链siRNA和双链长RNA结合。The result of Agrobacterium-mediated co-infiltration assay indicated that AC4 protein encoded by Tomato yellow leaf curl China virus（TYLCCNV）isolate Y10 was a RNA silencing suppressor.In order to analyse the mechanism of RNA silencing suppressor AC4,the AC4 gene of TYLCCNV-Y10 was obtained by PCR and was linked to multiple cloning sites of a prokaryotic expression vector pET-32a to construct the recombinant vector,pET32a-Y10AC4.The recombinant vector was transformed into Escherichia coli BL21（DE3）pLysS and TYLCCNV-Y10 AC4 was expressed and purified under natural conditions.The binding property of the expression protein Y10 AC4 with ss/ds-siRNA and long ss/dsRNA were analyzed by electrophoretic mobility shift assay（EMSA）.The results indicated that the fusion protein Y10 AC4 can bind ss-siRNA and long ssRNA,but not ds-siRNA and long dsRNA.

关 键 词：中国番茄黄化曲叶病毒 AC4蛋白 原核表达 电泳迁移率变动试验 

分 类 号：Q78[生物学—分子生物学] S432.4[农业科学—植物病理学]