OsPUT1基因三个不同位置的RNA干扰效果

The Effect of RNAi on Three Different Positions of OsPUT1 Gene

RNA干扰是一种双链RNA引发的转录后基因沉默技术,具有特异性和高效性,已成为一种基因功能研究的有效手段。在RNA干扰技术中,干扰片段的选取方式会影响基因沉默的效果。但是,目前并无选取干扰片段的固定标准。本研究以水稻多胺转运蛋白基因OsPUT1为对象,在CDS前部、中部和后部分别设计了三种不同干扰片段,构建基因三个RNAi载体,借助农杆菌EHA105介导水稻遗传转化,得到三种转基因苗;通过RT-PCR和Q-PCR技术检测目的基因的表达,结果显示三个干扰部位都具有一定的沉默效果,且在CDS的中间位置干扰效果最好。说明OsPUT1在进行RNAi载体构建时,应考虑在CDS的中间位置进行干扰。

RNA interference is a gene silencing technology induced by ds RNA and has become an effective means of gene function research for its specificity and efficiency. The selection of target region for RNAi in the gene body will affect the effectiveness of the RNA interference technology. However, there is no criterion for the selection of interfering fragments of the gene. Rice polyamine transporter gene OsPUT1 as research objective, three different interfering fragments were selected in the front, middle and back of CDS and then three RNAi vectors were constructed, finally three transgenic plants were obtained by Agrobacterium EHA105 mediated gene transformation. The expression of OsPUT1 was examined by RT-PCR and q RT-PCR, and the results indicated the effectiveness of RNAi knock-down based on the three sites, among which the most efficient one was in the middle of CDS. Our results indicated that the middle position of the gene should be taken into account to interfering when constructing the RNAi vector of OsPUT1.