中国番茄黄曲叶病毒和烟草曲茎病毒Rep蛋白介导卫星DNAβ复制选择性的分子机制研究

Tomato yellow leaf curl China virus (TYLCCNV) and Tobacco curly shoot virus (TbCSV) were each monopartite begomoviruses found to be associated with a distant species of betasatellite molecule. When either TYLCCNV or TbCSV was associated with both DNAβ molecules simultaneously, the cognate DNAβ became dominant and the noncognate DNAβ became undetectable at late stages of infection. To further understand the molecular mechanism of DNAβ replication specificity, in this project, we plan to demonstrate that the interaction between Rep protein of begomovirus and DNAβ will determine the replication specificity, in addition, to identify the key region of DNAβ involved in the replication specificity. Furthermore, utilizing biochemistry assays in vitro, we try to find the Rep binding motif in the above region and proved that the binding activity of Rep protein to its cognate motif and that of its noncognate. Further infectivity tests using infectious DNAβ clones of binding motif mutant will show whether the motif is involved in DNAβ infection and replication specificity. Finally analysis of the DNAβ and its mutants replication in the tabacco protoplast will give evidence that the accumulation level of DNAβ has influence on DNAβ replication specificity. The research work mentioned above will expanding our understanding of the molecular mechanism between betasatellite and its heper virus, providing valuable knowledge that may be useful in controlling disease.

在前期研究已经发现中国番茄黄曲叶病毒（简称Y10A）和烟草曲茎病毒（简称Y35A）对其同源和异源的DNAβ（简称Y10β和Y35β）存在复制选择性的基础上，本项目拟通过基因置换和侵染性分析，明确Y10A和Y35A编码的Rep蛋白和卫星DNAβ复制选择性的关系，并确定DNAβ基因组上与其复制选择性相关的关键区域；利用Rep蛋白结合DNAβ分子的体外生化试验，分析Rep蛋白与同源和异源卫星复制选择性关键区域中特异性结合元件的亲和性；将Y10β和Y35β的Rep蛋白结合元件置换或缺失后进行侵染性分析，验证Rep蛋白对卫星上特异性结合元件的识别对卫星DNAβ复制选择性的影响；同时利用烟草原生质体进行Rep蛋白支持下野生型卫星DNAβ及其突变体的瞬时复制，明确受Rep介导的卫星复制效率与卫星复制选择性的关系。本项目取得的研究结果有助于系统地解析双生病毒卫星DNAβ复制选择性的分子机制。

Begomoviruses可反式复制同源或异源的卫星DNAβ，其复制缺乏显著的特异性；但当同源及异源的DNAβ共同存在时，前期研究发现中国番茄黄曲叶病毒（简称Y10）和烟草曲茎病毒（简称Y35）对其同源的DNAβ（简称Y10β或Y35β）存在复制选择性，但其机理尚不清楚。本项目利用生物学、分子生物学和生物化学相结合的手段，以Rep蛋白和卫星DNAβ的互作作为切入点，通过体外和体内试验相结合的方法研究双生病毒卫星DNAβ复制选择性的分子机制。取得的研究结果表明辅助病毒对同源卫星的复制选择性是由Rep蛋白特异性地识别卫星DNA分子上保守颈环结构上游200 bp序列中的RBM序列元件（Rep binding motif，RBM）介导的。体外竞争性EMSA试验以及卫星DNβ的突变和置换分析表明， Y10β和Y35β RBM序列中存在的两个串联重复子（iteron）样的顺式元件决定了其与同源辅助病毒Rep亲和结合的特异性和强度。进一步的本氏烟侵染试验及叶盘复制试验揭示iterons对于卫星的反式复制也具有重要作用，且3’-重复子对于复制起关键作用，而5’-重复子能够增强卫星的复制水平。通过构建Y10β和Y35β的iterons互换突变体进行侵染性分析，发现卫星DNAβ内的iterons序列是决定辅助病毒对同源卫星选择性复制的关键元件。