拟南芥AtLCR67干扰质粒的构建及转基因植株的鉴定

The RNAi Plasmid Construction of At LCR67 and Identification of Transgenic Plants

种子发育过程是植物整个生长周期最重要的阶段,涉及到一系列功能物质的合成和转化,是由各种相互关联的基因共同控制完成的,因此,研究该阶段特异性表达的基因对研究种子发育过程非常重要。以种子特异性表达的基因At LCR67为研究对象,通过构建RNAi干扰质粒,借助根癌农杆菌介导的浸花法,将At LCR67基因的RNAi表达框转到拟南芥基因组中;经过PPT抗性筛选与PCR鉴定,成功获得了8株转基因植株。对其中的3株进一步作RT-PCR检测,显示有2株中的At LCR67基因表达被完全抑制,另一株的基因表达水平被抑制了大约70%,结果完全实现了设计目标;同时还发现T2代植株有晚花表型。这两株转基因植株为进一步研究At LCR67功能与作用机制提供了良好的遗传材料。

The processing of seed development is the most important stage for the whole life of plant growth,it depends on the synthesis and transformation of different kinds of metabolic controlled by corresponding genes. So study on the genes specifically expressed during seed developing stage is very important to understand the processing of seed development. AtLCR67 gene only expresses during the seed development stage was used as material, the RNAi expression box of AtLCR67 gene was transferred to Arabidopsis thaliana genome via construction of RNAi plasmid and agrobacterium - mediated transformations. Through resistance selection of PPT and PCR identification, eight transgenic plants were gained. The RT - PCR results of 3 plants selected from the transgenic plants showed that the AtLCR67 gene was silenced completely in 2 plants, and the expression level of AtLCR67 gene was inhibited by about 70% in another one, which sugges- ted the expression box of AtLCR67 gene RNAi worked. By the way, the late - flowering phynotype of T2 generation transgen- ic plants was found during the experiment. The two transgenic plants in which AtLCR67 gene was silenced completely were the good materials for further research on function and action mechanism of AtLCR67.