马铃薯卷叶病毒双链RNA干涉载体构建-作物遗传育种专业毕业论文.docxVIP

万方数据 万方数据 摘 要 本研究从感染马铃薯卷叶病毒(PLRV)的马铃薯叶片中提取出病毒 RNA 进行 cDNA 合成并运用 RT-PCR 技术进行体外扩增，得到了符合理论设计的 PCR 扩增产物，将这 些产物正反向插入到经改造的适合转化马铃薯的双元载体 RNA 干涉区，成功构建了马 铃薯卷叶病毒 RNA 干涉载体。主要研究结果如下： （1） 利用 Trizol 法直接从感 PLRV 的马铃薯叶片中提取了 PLRV 总 RNA； （2） 通过分析马铃薯卷叶病毒基因组序列，设计了 6 对引物。RT-PCR 扩增出 5 对引物的产物，大小分别为 240bp、270bp、360bp、380bp、400bp； （3） 对质粒载体 pFGC5941 和 pCAMBIA1300 重组。重组质粒 pCADS1341 用 EcoRI/PstI 酶切鉴定，能够切出大小为 3.5kb 和 8.9kb 的两条特异性的条 带，证明了适合马铃薯转化的 RNA 干涉诱导载体构建的正确性； （4） 扩增出的片段正反向两次插入质粒载体 pCADS1341，经酶切鉴定，得到 3 个 RNA 干涉载体，分别命名为 pCADS1341-PLRV1、pCADS1341-PLRV2、 pCADS1341-PLRV3。 关键词：马铃薯卷叶病毒 RNA 干涉 载体 构建 I Construction of Double-stranded RNA interference vector against potato leafroll virus ZHOU Yun(Crop heredity and breeding) Directed by Professor WANG Jian YANG Yongzhi Abstract In this research，we extracted the potato leafroll virus(PLRV) RNA which was used for cDNA synthesis from virus-infected potato leaves，some specific PCR fragments which we had expected were obtained by reverse transcription and polymerase chain reaction(RT-PCR) amplification. With the insert these fragments in RNA interference zone on binary vector plasmid use two-step cloning method ， we successfully construct double-strinded RNA interference vector for PLRV. The main results were showed as following： The PLRV RNA were extracted form virus-infected potato leaves use the method of Trizol. Six pair of DNA primer were disigned by analyzed the nucleotide sequence of potato leafroll virus. Five specific fragments about 240bp、270bp、360bp、380bp and 400bp were obtained by reverse transcription and polymerase chain reaction(RT-PCR) amplification. Recombined the plasmid vector pFGC5941and pCAMBIA1300.Two specific fragments about 3.5kb and 8.9kb were obtained by cuting up the new vector with restriction endonucleases EcoRI and PstI， showed the proper construction of RNA mediate plasmid vector for potato transformation. Insert amplified fragments to the pCADS1341 use two-step cloning method.After cut u