[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 seque...[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.展开更多
[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....[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.展开更多
[ 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...[ 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.展开更多
基于DNA载体的RNA i技术可以较长时间高效地抑制基因的活性。利用家蚕核型多角体病毒ie-1基因启动子,构建了在细胞内能转录形成与ie-1基因+19^+446区域互补同源的发夹状dsRNA的RNA i DNA载体。研究结果表明,该载体DNA在体外、体内均能...基于DNA载体的RNA i技术可以较长时间高效地抑制基因的活性。利用家蚕核型多角体病毒ie-1基因启动子,构建了在细胞内能转录形成与ie-1基因+19^+446区域互补同源的发夹状dsRNA的RNA i DNA载体。研究结果表明,该载体DNA在体外、体内均能较好地抑制家蚕核型多角体病毒在细胞中的复制。展开更多
基金Supported by International Science and Technology Cooperation Program (2008DFA30560)Preliminary Research Special Foundation of 973 Program (2008CB117018)Scientific Research Project for High Level of Talents of Shihezi University (RCZX200732)~~
文摘[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.
基金Research Fund from Guizhou Academy of Agricultural Sciences([2009]038)Programs for Science and Technology Development in Guizhou Province([2009]3067)~~
文摘[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.
基金Supported by Outstanding Young Scientific and Technological Talent Training Fund of Guizhou Province"Study on Innovation and Application of Tall Fescue Germplasms"
文摘[ 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.
文摘基于DNA载体的RNA i技术可以较长时间高效地抑制基因的活性。利用家蚕核型多角体病毒ie-1基因启动子,构建了在细胞内能转录形成与ie-1基因+19^+446区域互补同源的发夹状dsRNA的RNA i DNA载体。研究结果表明,该载体DNA在体外、体内均能较好地抑制家蚕核型多角体病毒在细胞中的复制。
