To mutagenize two conserved CCCT and PTK motifs in the central domain of Chinese strain of potato Y potyvirus (PVY-C) helper component proteinase (HC-Pro), four mutants of HC-Pro gene were obtained by PCR and site-directed mutagenesis, and then were inserted into the constitutive expression vector pBin438. Leaves from tobacco ( Nicotiana tabacum L. cv. K326) were transformed with these four plant expression plasmids by Agrobacterium -mediated transformation, respectively. Southern and Western blotting analyses showed that these four mutants were integrated into tobacco genomic DNA and could express the corresponding proteins in most of the transgenic plants. The challenge of transgenic plants with potato X potexvirus (PVX) revealed that the expression products of PVY-C HC-Pro mutants in transgenic plants greatly abolished functions of HC-Pro in enhancing the accumulation and pathogenicity of PVX, indicating that CCCT and PTK motifs of HC-Pro were required for PVX/PVY synergism. Meanwhile, the results demonstrated that PVY-C HC-Pro had a function in accelerating the long-distance movement of PVX in these transgenic plants for the first time.
马铃薯Y 病毒蚜传辅助因子促进马铃薯X病毒长距离运输
李为民1,2 鲁瑞芳1* 郭明1 陈毓荃2 彭学贤1
（1. 中国科学院微生物研究所植物生物技术开放实验室，北京100080；"# 西北农林科技大学基础科学系，陕西杨凌712100）
摘要：采用PCR和定点突变法,对马铃薯Y病毒中国株系(Chinese strain of potato Y potyvirus, PVY_C) 蚜传辅助成分(helper component proteinase, HC_Pro)基因中心区域的CCCT 基序和PTK基序进行了定点改造,获得了4种突变体.然后将突变体克隆到植物表达载体pBin438 中, 所得到的重组体通过根癌土壤杆菌(Agrobacterium tumefaciens (Smith et Townsend) Conn)介导法转化了烟草(Nicotiana tabacum L. cv. K326). Southern blotting和Western blotting分析表明4种突变体已经成功地整合到烟草的基因组中, 并在蛋白水平上得到了表达.马铃薯X病毒(potato X potexvirus, PVX)对转基因烟草的攻毒实验表明, 4种突变体均使PVY_C HC_Pro严重丧失了促进PVX病毒粒子在寄主体内积累和提高 PVX致病性的功能,说明CCCT、PTK基序为PVY_C HC_Pro介导PVX/PVY协生作用所必需.同时证明了HC_Pro具有增强PVX在寄主体内长距离运输的功能.
关键词： 马铃薯Y病毒；马铃薯X 病毒；蚜传辅助成分基因；突变；协生作用
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