基于种群结构的新角度解析双生病毒卫星DNA快速变异及非专化复制的分子机理

Satellite DNAβ? molecule is an important pathogenic factor of Geminivirus/DNAβ? disease complex. It was proved that DNAβ has a higher level of mutation frequency and can be trans-replicated by non-cognate helper virus. But the mechanisms of the higher variability and trans-replication of DNAβ are still unclear. In the past research supported by National Natural Science Fund for Youth, the DNAβ molecules (TbCSB/TYLCCNB) which are associated with Tobacco curly shoot virus (TbCSV) and Tomato yellow leaf curl China virus (TYLCCNV) respectively, were proved as two different types of DNAβ with different population structures and mutation level. In this project, choosing TbCSB/TYLCCNB as the researching material and basing on the molecular clone with identical sequence, SSCP analysis and sequence comparison were used to reveal the main reasons, types and laws of variation and analyze the influence of host and helper virus on the variation and evolution of DNAβ. The infection clones of wild type, Chimera and mutation of TbCSB/TYLCCNB will be constructed to analyze the biological significance of mutation sites and to elucidate the roles of SCR and A-rich regions in the replication and sympton induction of helper virus and satellite as well as the influence of interaction of Rep and SCR on the population variation of satellite. Based on these researches, the project will try to make clear the molecular mechanism of rapid variability and non-specific trans-replication of satellite DNA. Completion of this project is expected to provide theoretical foundation to clarify the mechanism of population variation and evolution of satellite DNAβ, and non-specific interaction of DNAβ and helper virus.

卫星DNAβ是双生病毒病害复合体重要的致病因子，其突变频率高于辅助病毒，且可伴随异源病毒进行复制，但其机理仍是个谜。在前期国家青年科学基金项目的工作中我们已证实伴随TbCSV和TYLCCNV的卫星DNAβ（TbCSB/TYLCCNB）其种群结构和变异水平迥异，本项目拟以这两类卫星分子为材料，从序列一致的分子克隆出发，用SSCP及序列比较等方法，结合自然种群的大样本分析，弄清DNAβ变异的主要原因、类型和规律，明确寄主和辅助病毒对DNAβ变异及进化的影响；通过构建嵌合体及突变体DNAβ分子的侵染性克隆，明确卫星SCR和A-rich区在病毒及卫星复制和症状诱导中的作用；分析突变位点的生物学意义；基于种群结构的新角度探讨病毒Rep与卫星SCR的互作对卫星种群变异的影响；进而揭示DNAβ快速变异及非专化复制的分子机理。项目的完成可望为阐明卫星种群变异进化规律及与辅助病毒非专化互作机理提供理论依据。

本项目围绕辅助病毒和寄主对双生病毒卫星DNAβ变异及进化的影响开展了一年期的研究。利用PCR、克隆、测序、种群变异分析等技术，以中国番茄黄化曲叶病毒卫星DNAβ(TYLCCNB) 为实验材料，分别伴随同源辅助病毒TYLCCNV和异源辅助病毒TbCSV接种本氏烟，结果发现，伴随TbCSV的TYLCCNB种群均为杂合体，突变克隆百分比为100%，突变频率明显高于伴随TYLCCNV的TYLCCNB种群。伴随异源辅助病毒时，TYLCCNB在Airch-区发生突变的碱基数量有所减少，在βC1编码区碱基突变数量有所增加，此外在保守的SCR区检测到6个碱基发生了突变，在A-rich区与SCR区之间还检测到另一新的突变热点。且伴随异源辅助病毒时TYLCCNB的碱基突变类型具有明显的倾向性，碱基A缺失突变及T→G突变的克隆数量剧增。田间样本研究发现TYLCCNB与异源病毒PaLCuCNV和同源病毒TYLCCNV复合侵染番茄引起了更严重的番茄黄化曲叶病。这些结果表明辅助病毒对卫星DNA β的种群变异及其在致病性中的作用具有重要影响。以烟草曲茎病毒卫星(TbCSB)、赛葵黄脉病毒卫星（MYVB）及TYLCCNB为实验材料，分别伴随其同源辅助病毒接种本氏烟和心叶烟，对其种群进行分析表明，3种卫星在不同寄主中的种群变异水平存在差异，但其种群结构、变异分布及碱基突变类型具有相似的特点。且3种卫星中，MYVB和TYLCCNB的种群变异水平较高并具有相似的遗传结构和变异特点，TbCSB种群的变异水平最低。根据在卫星与辅助病毒的伴随关系及其对辅助病毒致病性的影响方面的已有报道，并结合本实验室已有研究结果，我们认为辅助病毒对卫星分子快速变异的作用机理及其与病害流行的相互关系值得进一步关注。.