Construction of RNAi Vector Which Resist Cucumber Mosaic Virus and Transformation of Tobacco

[Objective] Aimed to construct RNAi vector resistant to cucumber mosaic virus and transferred this vector into tobacco. [Method] RT-PCR method was used to amplify cucumber mosaic virus NS04 and process RNA2 gene sequen of tomato isolates. The analysis results of phylogenetic tree demonstrated that the sequence in RNA2 encoded CMV-2a had 98.0% and 96.5% homology with nucleotide and amino acid of DQ412731 isolate of Zhejiang,China. The replicase fragment in CMV RAN2 gene was taken as target sequence to construct pBi35SCR2 eukaryotic expression vector,then the expression vector was identified. Through agrobacterium-mediated method,the expression vector was transferred into tabacco and PCR method was used to check the transfer. The PCR results demonstrated that the experiment had successfully construct eukaryotic expression vector of pBi35SCR2 and the expression vector was successfully transferred into tabacco. [Conclusion] The obtained transgenic tobacco could be used as challenge test material in following experiment and provided foundation for studying processing tomato resist cucumber mosaic virus.

Construction of RNAi Expression Vector Targeting FaVRN1 Gene in Tall Fescue

[Objective] This study was to construct RNAi expression vector targeting FaVRN1 gene. [Method] A 145 bp Arabidopsis actin intron was inserted into the expression vector to generate an intermediate vector pBI121-M-INT. And then two pairs of specific primers with enzyme restriction sites spanning a 351 bp cDNA conserved sequence fragment of FaVRN1 gene were designed for RT-PCR to construct RNAi expression cassette. The amplified fragment was inserted forwardly and reversely at two sides of the intron to construct an RNAi expression vector with hairpin structure. [Result] Double enzyme digestion（HindIII＋BamHI） showed the intron had been successfully into the vector pBI121. PCR amplification and double enzyme digestion indicated the success of forward and reverse ligation of target FaVRN1 fragment into the intermediate vector. [Conclusion] This study laid foundation for breeding novel flowering-inhibited tall fescue varieties.

Construction of RNAi Expression Vector Targeting Vernalizational Gene FaCONSTANS in Tall Fescue

[ Objective ] This study aimed to construct RNAi expression vector targeting vemalizational gene FaCONSTANS （GenBank accession number： GU214996） in tall rescue. [ Method] A 145 bp long Arabidopsis actin gene intron was inserted into the expression vector to construct an intermediate vector pBI121-M-INT. Two pairs of specific primers with restriction sites were designed to amplify a 351 bp long cDNA conserved sequence fragment of vemalizational gene FaCONSTANS for RT-PCR. After restriction enzyme digestion, the amplified fragment was inserted forwardly and reversely at two sides of the intron of intermediate vector to construct an RNAi expression vector with hairpin structure. [ Result ] Double digestion （HindIII ＋ BamHI） showed that the intron was successfully insert- ed into the vector pBI121. PCR amplification and double digestion indicated that target fragment FaCONSTANS was successfully inserted forwardly and reversely in- to the intermediate vector. [ Conclusion] This study laid foundation for breeding novel flowering-inhibited tall rescue cultivars.