抗番茄花叶病毒RNAi载体转化加工番茄

加工番茄是普通番茄中的一种栽培类型,主要特点是矮化,果实比普通栽培番茄略小,果皮比普通栽培番茄厚,耐贮藏运输,目前已逐渐成为新疆的特色产业和出口创汇的支柱产业.ToMV(番茄花叶病毒)是影响新疆加工番茄产量的重要病害,发病时可造成番茄严重减产.药剂、疫苗等均起不到有效的防治作用.抗病毒育种是最经济、有效的防治方法.     

抗黄瓜花叶病毒RNAi载体的构建及烟草的转化

[目的]构建抗黄瓜花叶病毒RNAi载体,并将载体转入烟草.[方法]采用RT-PCR方法,扩增黄瓜花叶病毒NS04加工番茄分离物的RNA2基因组的序列选取CMV RAN2基因组中的复制酶片段作为靶序列,构建pBi35SCR2真核表达载体,并对表达载体时行鉴定;通过农杆菌介导的方法将表达载体转入烟草,用PCR的方法检测载体是否转入.[结果]系统进化树分析结果表明,RNA2中编码CMV-2a的序列与中国浙江的DQ412731 分离物有较高核苷本乡酸及氨基酸同源性,分别达到98.0%和96.5%;RCR结果表明,试验成功构建了pBi35SCR2真核表达载体,并成功将表达载体转入烟草[结论]试验获得的转基因烟草可作为后期攻毒试验的材料,并为研究加工番茄抗黄瓜花叶病毒奠定了基础. Abstract： [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 NSO4 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.     

抗黄瓜花叶病毒RNAi载体的构建及烟草的转化

[目的]构建抗黄瓜花叶病毒RNAi载体,并将载体转入烟草。[方法]采用RT-PCR方法,扩增黄瓜花叶病毒NS04加工番茄分离物的RNA2基因组的序列。选取CMVRAN2基因组中的复制酶片段作为靶序列,构建pBi35SCR2真核表达载体,并对表达载体进行鉴定;通过农杆菌介导的方法将表达载体转入烟草,用PCR的方法检测载体是否转入。[结果]系统进化树分析结果表明,RNA2中编码CMV-2a的序列与中国浙江的DQ412731分离物有较高核苷酸及氨基酸同源性,分别达到98.0%和96.5%;PCR结果表明,试验成功构建了pBi35SCR2真核表达载体,并成功将表达载体转入烟草。[结论]试验获得的转基因烟草可作为后期攻毒试验的材料,并为研究加工番茄抗黄瓜花叶病毒奠定了基础。