【作者】 高鹏；

【导师】 张恒木；

【作者基本信息】 浙江师范大学 ， 植物学， 2010， 硕士

【摘要】 水稻条纹病毒(Rice stripe virus, RSV)引起的水稻条纹叶枯病是最重要的水稻病毒病之一,给我国水稻生产安全造成了巨大威胁。为了创制高抗RSV的水稻新种质,本研究首先根据天然microRNA (miRNA)的结构特点和作用原理,针对水稻条纹叶枯病毒基因组设计了人工miRNA(artificial microRNA, amiRNA),同时克隆了miRNA528基因的前体(pre-miRNA)。应用重叠PCR (over-lapping)技术并以这个水稻天然miRNA前体作为骨架,构建2个水稻转化载体(pAmiR-1和pAmiR-2),其中载体pAmiR-1是针对RSV p3基因；载体pAmiR-2是针对RSV pc3基因。通过农杆菌介导法将2个人工miRNA表达载体导入水稻品种武育粳3号,分子鉴定表明共获得219株转基因水稻植株。利用携带RSV的介体灰飞虱(Laodelphax striatellus, Fallen)对部分转基因株系进行抗病毒鉴定,结果表明共有22个转基因水稻株系表现为高抗。进一步通过实时定量RT-PCR(real-time RT-PCR)分析表明所设计构建的人工miRNA高效大量表达。更多还原

【Abstract】 The disease caused by rice stripe virus is one of the most important viral diseases, and it is a threat to rice yield in our country. According to the theory of miRNA, several artificial microRNAs (amiRNAs) were designed for down-regulating the gene expression of RSV. The precursor of miRNA528 was cloned and used for construction of 2 amiRNA genes, amiR-1 and amiR-2, by over-lapping PCR. These genes were ligated into plasmid pBI121 respectively, The pAmiR-1 and pAmiR-2 are specific for interfering the expression of p3 and pc3 genes encoded by RSV RNA3. The 2 vectors were transferred into the japonica rice cultivar Wuyujing 3 by Agrobacterium-mediated transformation.219 transgenic rice plants (To) harbouring the amiRNAs were identified by PCR. The T1 transgenic plants were used to evaluate the resistance to rice stripe virus by artificial inoculation. The results showed that 22 plants are highly resistant to RSV. Real-time RT-PCR analysis suggested the transgenic amiRNA could be expressed at high level expressed in transgenic plants.更多还原