农杆菌介导的CMV侵染性克隆及2b缺失突变体构建

Construction of Agrobacterium-mediated Cucumber mosaic virus Infectious cDNA Clones and 2b Deletion Viral Vector

【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】1x35S-MCS-HDVRZ-NOS cassette was transferred from pHST40 into pBluescrpt SK II and 2x35S enhancer from pRTL2 was used to replace 1x35S on pBluescrpt SK II, then the whole 2x35S-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 2x35S 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 pCB301-2x35S-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 pCB301-2x35S-MCS-HDVRZ-NOS. The Agrobacterium-mediated infection of N. benthamiana by those infectious cDNA clones of CMV was successful and 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. benthamiana. 2b is not required for viral replication and systemic infection in this plant.