【作者】 熊伟；

【导师】 陈保善；

【作者基本信息】 广西大学 ， 生物化学与分子生物学， 2006， 硕士

【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AtTOM3序列具有部分同源的马铃薯EST序列，利用5’RACE和3’RACE方法，从马铃薯中克隆到与拟南芥AtTOM3基因具同源性的StTOM3基因。StTOM3全长cDNA有1283个碱基对，编码297个氨基酸，含有六个跨膜区结构，与已证明为拟南芥TMV寄主因子基因AtTOM3在核苷酸水平有61.2％的同源性，在氨基酸水平上有72.9％的同源性。为了研究StTOM3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：(1)正义及反义表达StTOM3的植物双元表达载体；(2)分别表达StTOM3 cDNA不同区域的RNA干涉植物表达载体。用根癌农杆菌介导的方法将上述表达载体导入马铃薯茎段及叶片无菌外植体，获得转StTOM3-RNAi和转反义StTOM3的转基因马铃薯共40株。PCR检测表明外源基因已整合入马铃薯染色体上。同时，对马铃薯品种PB04的遗传转化系统进行了优化，建立了高效的马铃薯转化系统，转化率达65％以上，可在6-9周之内得到抗性植株。更多还原

【Abstract】 The full-length cDNA of Solanum tuberosun L. StTOM3, a gene with high similarity to AtTOM3 of Arabidopsis thaliana, has been cloned by the methods of 5’RACE and 3’RACE, based on the partial cDNA sequence information of potato. The length of the cDNA was 1283 base pairs (bp) long, encoding a 297 amino acids putative protein with six predicted motifs of trans-membrane structure. StTOM3 was 61.2% and 72.9% identical to AtTOM3, an A. thaliana host factor gene supporting the replication of tabacco mosaic tobamovirus (TMV) at the level of the nucleotide acids and the deduced amino acids respectively.To identify the function of StTOM3 during the viral infection of the potato host, a series of plasmids were constructed: (1) sense and antisense StTOM3 gene fragments in pBI121 which derived from genomic potato DNA ; (2) StTOM3 cDNA-based RNAi fragments targeted against the coding,5’untranslated and 3’untraslated regions of the mRNA were construct in plant-transforming vector pBI121 respectively; These constructs were introduced into Agrobacterium tumefaciens via triparental conjugations and used to transform stem and leaf explants of potato varietis "PB04" . Twenty-eight transgcnic plants of "PB04"that were transformed with the RNAi construct were obtainted. Twelve plants of "PB04"were resistance to Kanamycin and derived from transformation by antisense StTOM3 construct were obtained.The results of PCR demonstrated that the target genes were integrated into the genome of potato plants. An efficient potato transformation system with transformation rate higher than 65%, and winthin a time frame of 6-9 weeks to obtain a transgenic potato plantlet was developped.更多还原