RNA介导的百合双抗CMV和LMoV病毒研究

Synergism of plant virus, infections with two viruses mixed, makes much severer symptom to plants than separate infection. The synergism of plant virus can break the RNA-mediated single resistance. In this study, the most typical synergism of plant virus of Cucumber mosaic virus (CMV) and Lily mosettle virus (LMoV), which affecting lily, was investigated. The CMV 2b-LMoV cp fusion gene fragment with a length of 300~600bp, will be obtained by overlapping PCR technique. It will be cloned into pWMB006 vector with one intron, and then subcloned into the plant expression vector- pZP211 vector so as to build a plant expression vector containing inverted repeats sequence of CMV-LMoV fusion gene. After that, it will be transferred into lilies, and double-resistance virus transgenic plants which are stable and efficient on expression of the RNA silencing suppression function of breaking of CMV and LMoV Synergism are expected to be obtained..Based on this，the mechanism of double-virus resistant transferred plant on the pathogen and the host will be studied using virus attack test and growth duration test. The resistant inheritance and stability of the descendants of the transformed plants derived from inbreeding and crossing between hybrids, will be observed. In this study, we will obtain the multi-virus resistant new germplasm and explore the interactional response of transgenic lily plants with virus. It is significant works to relieve lily production from virus harm.

两种病毒的混合侵染可以产生比一种病毒分别单独侵染更严重症状的现象称为病毒间的协生作用。这种协生作用可以攻破RNA介导的单抗抗性。.本研究拟以百合上协生作用最典型、危害最严重的黄瓜花叶病毒(CMV)和百合斑驳病毒（LMoV)为对象。应用重叠延伸PCR，得到CMV -2b和 LMoV -CP约300~600bp的融合基因片段，与含有内含子的载体pWMB006连接，再导入含有抗性标记的载体pZP211，构建CMV-LMoV融合基因反向重复序列的植物表达载体，转化百合，获得破解CMV与LMoV协生作用的具RNA沉默抑制机制的双抗转化植株。在此基础上，设置攻毒试验，研究双抗转化植株对病原及寄主本身的生长发育影响作用机制，并通过对转化植株获得的自交及与杂种系内杂交等后代抗性遗传稳定性观测，创制打破病毒协生作用的百合多抗种质，探索其抗性作用机理，为解决或减缓病毒对百合生产的危害这一重要科学难题积累基础。

本研究在进一步建立与优化百合愈伤组织再生体系基础上，以得到CMV 2b基因的295bp片段和LMoV cp基因的428bp片段，首先构建单抗CMV、LMoV的RNAi植物表达载体pFGC5941-C2和pFGC5941-L2单抗病毒载体转化东方百合‘索邦’无菌苗鳞片。经抗性初步筛选得到转化pFGC5941-C2载体的抗性苗8株，转化pFGC5941-L2载体的抗性苗11株，Southern blot分析得到2株抗LMoV转基因植株、1株抗CMV转基因植株。.然后，构建了CL融合片段双抗载体，按优化好的遗传转化体系，用含有双抗载体pFGC5941-CL的农杆菌EHA105转化东方百合‘索邦’的愈伤组织，通过抗生素和PCR筛选已获得92株抗性苗，正在进行RT-PCR检测、Southern blot验证和攻毒试验及数据分析。.基于本项目试验，还开展了百合基因密码子偏性分析及其表达受体选择，百合内参基因（lilyActin）克隆与表达等相关试验研究。