马铃薯PTOM1基因全长cDNA的克隆及其RNAi转基因植株的构建-微生物学专业毕业论文.docxVIP

马耀华 马耀华 马铃著PTOMI基因cDNA的克隆及其RNAi转基因植株的构建 马铃薯PTOMI基因全长cDNA的克隆 及其RNA i转基因植株的构建 摘要 在Genebank中搜索到与拟南芥中支持烟草花叶病毒复制的基因 TOMI序列具部分同源的马铃薯EST序列。用特异性引物和RACE方法，从 马铃薯(F-28-1)中克隆到与拟南芥AtTOMI基因具同源。性的马铃薯PTOMI 基因，PTOMI全长cDNA有1257个碱基对，编码245个氨基酸，是一个 含有五个跨膜区结构的蛋白。PTOMIcDNA与拟南芥AtTOMIcDNA有59．1％ 的同源性。二者的编码产物有70．3％的同源性。为了研究PTOMI基因在 RNA病毒侵染马铃薯过程中的作用，构建了以下一系列的植物表达载体 和病毒表达载体：(1)将PTOMIcDNA反义构建于表达载体；(2)将PTOMI cDNA分为两段分别构建RNA干涉质粒；(3)用基因组PTOMI基因片段构 建反义表达载体；(4)将PTOMI cDNA反义构建于病毒表达载体PTV00。 将用上述方法得到的表达载体导入农杆菌并用于侵染马铃薯得到转基 因马铃薯植株，PCR检测表明外源基因已整合入马铃薯染色体上。同 时，我们建立了高效快速的马铃薯转化系统，转化率达65％以上，可 在5-7周之内得到抗性植株。 关键词：PTOMl基因马铃薯RNA干扰RNA病毒寄主因子 广嚣大学鞴士学位论文Cloning 广嚣大学鞴士学位论文 Cloning of Potato PTOMI Full—length cDNA and Construction of Trangenic Potato Plants expressing varied forms of PTOMl Abstract RNA viruses encompass about two-third of all virus genera and include numerous important pathogens。Host factors play important roles in the viral RNA replication and transportation in host cells．Here，we report the cloning of a host factor PTOMl gene from potato with high homology with Arabdopsis thaliania TOMl that supports the replication of tobacco mosaic virus．The full—length cDNA of PTOMl，1257 bp in length，encodes a deduced 256 tein。To identify the function of PTOM I during the viral infection of the potato host，a series of plasmids were constructed：≤1)antisense PTOMI eDNA in plant-transforming vector pBll21；(2)PTOMl cDNA-based RNAi fragments in plant-transforming vector pBll21；3)antisense PTOMl gene fragments in pBll21 which derived from genomic potato DNA and(4)Antisense PTOMl eDNA into virus—transforming vector PTV00．These constructs were used to generate transgenic potato plants．The results of PCR demonstrated that the target genes were integrated into the genome of 马耀华 马耀华 马铃薯PTOM．f基艘eDNA豹壳建及羹RNAi转基雹檀拣抟橱建 potato plants。An efficient potato transformation system with transformation rate higher than 65％，and winthin a time frame of 5-7 weeks to obtain a transgenic potato plantlet was developped． Key Words：PTOMl，pota