摘要
为了方便利用RNA干涉(RNAinterference,RNAi)研究植物基因的功能,本研究构建了2个用于RNAi的植物转化载体pRNAi-35S和pRNAi-Ubi。这两个载体以pCAMBIA双元载体为骨架,含有2个多克隆位点区进行目的基因片段的正向和反向克隆,并分别由CAMV35S启动子和玉米泛素基因启动子控制发夹结构RNA的产生。为了验证该载体的有效性,用PCR扩增了1个水稻转录因子(ArabidopsisAP2/EREBP同源基因)基因片段组装入pRNAi-Ubi,转化水稻获得转化体。在所检测的7株转化株中,5株的内源目标基因的mRNA被降低至RNAblot检测不到的水平。这些结果说明本研究构建的RNAi载体对基因表达沉默是有效的。
In order to efficiently identify the functions of plant genes via RNA interference （RNAi）, two plant-transformation vectors, pRNAi-35S and pRNAi-Ubi, suitable for generation ofhairpin-RNA constructs were prepared. The vectors have two multi-cloning site regions to facilitate the cloning of target gene fragments in the sense and antisense orientations, and hairpin RNA constructs are driven by the maize Ubiquitin promoter and CAMV35S promoter, respectively. To test the efficiency of the vectors for gene silencing in rice, a gene encoding a transcription factor homologous to Arabidopsis AP2/EREBP was isolated from rice by PCR and cloned into pRNAi-Ubi. The construct was introduced into rice varieties by Agrobacterium-mediated transformation. RNA blot analysis showed that the targeted mRNA in five of the seven tested transgenic plants was decreased to undetectable levels. The results demonstrated that the vectors are effective for gene silencing in plants.
出处
《分子植物育种》
CAS
CSCD
2006年第5期621-626,共6页
Molecular Plant Breeding
基金
国家重大基础研究计划（2005CB12080）资助.
