中文摘要：

【目的】构建农杆菌介导的黄瓜花叶病毒（CMV）侵染性克隆，测试缺失CMV2b基因后的侵染表型。【方法】首先将pHST40上的1x35S-MCS-HDVRZ-NOS转移到中间载体pB luescript SK II上，用pRTL2上的2x35S替换中闻载体上的1×35S增强子序列，然后将2×35S-MCS-HDVRZ-NOS转移到微型植物表达载体pCB301，构建适用于病毒侵染性克隆和农杆菌浸润的植物表达载体，在此基础上将CMVFny株系的全长eDNA基因组构建到该植物表达载体的2×35S启动子和HDV核酶之间，通过农杆菌浸润在本氏烟中瞬时表达产生具有侵染活性的CMV基因组RNA，进一步缺失励来测试该缺失突变体侵染植物的表型。【结果】构建了一个用于构建RNA病毒侵染性克隆的植物表达载体pCB301—2x35S—MCS-HDVRz-NOS，将CMVFny的3条基因组分别构建在这个植物表达载体上后，通过农杆菌浸润本氏烟可诱导产生曲叶、花叶和矮化等症状，进一步对2b进行缺失，表明该缺失突变体接种本氏烟早期具有微弱曲叶症状，中后期表现无症，RT-PCR检测表明CMVFny2b缺失突变体能够系统侵染本氏烟。【结论】在改造的微型植物表达载体pCB301—2x35S-MCS—HDVRZ-NOS上构建了CMV的侵染性克隆，构建的侵染性克隆通过农杆菌介导可快速高效侵染植物,26对CMVFny在本氏烟中的症状发展具有重要作用，但乃对其在本氏烟上的复制和系统侵染并不是必需。

英文摘要：

[Objective] The objective of this study is to generate the Agrobacterium-mediated infectious cDNA clones of Cucumber mosaic virus and investigate the phenotype of 2b deletion mutant in Nicotiana benthamiana. [ Method ] 1×35S-MCS-HDVp, z-NOS cassette was transferred from pHST40 into pBluescrpt SK II and 2x35S enhancer from pRTL2 was used to replace lx35S on pBluescrpt SK II, then the whole 2×35S-MCS-HDVRZ-NOS cassette was transferred into mini binary vector pCB301 to generate pCB301-2x35S-MCS-HDVRZ-NOS. The full length cDNAs of CMV Fny were inserted between 2×35S promoter and HDV Rybozyme on pCB301-2x35S-MCS-HDVRZ-NOS, then they were introduced into agrobacterium and inoculated N. benthamiana by agro-infiltration. Based on those infectious clones, 2b deletion mutant was constructed to investigate its phenotype in N. benthamiana. [Result] A mini binary vector pCB301-2×35S-MCS-HDVRZ-NOS was constructed for making infectious clones of plant RNA virus. The full lengths of three genomic cDNAs of CMV Fny were inserted into this binary vector, then they were introduced into agrobacterium, respectively, and infiltrated into N. benthamiana. The inoculated plant could produce severe symptoms including leaf curl, mosaic and dwarf etc. Further deletion of 2b demonstrate that 2b deletion mutant could induce mild leaf curl symptoms on N. benthamiana in earlier time, but the symptoms disappear in later stage, RT-PCR shows that 2b deletion mutant still could infect the N. benthamiana systemically. [ Conclusion ] The infectious clones of CMV were constructed based on a modified mini binary vector pCB301-2×35S-MCS-HDVRZ-NOS. The Agrobacterium-mediated infection of N. benthamiana by those infectious eDNA clones of CMV was successful artd efficient. It is a simple, fast and low cost approach to make viral infectious clones. The phenotype of CMV Fny 2b mutant on N. benthamiana suggest that although 2b deletion mutant induce mild leaf curl symptoms at earlier time, 2b is a key determinant for symptom development for CMV in N, bent