利用RNA干扰技术抑制水稻淀粉极限糊精酶基因表达

构建了含极限糊精酶基因片段反向重复结构的RNAi载体,抑制水稻籽粒中极限糊精酶基因表达获得了极限糊精酶基因功能缺失突变体。经酶切及序列测定证实,成功地构建了胚乳特异表达启动子启动的水稻极限糊精酶基因反向重复结构RNAi载体;通过根癌农杆菌(Agrobacterium tumefaciens)介导水稻(Oryza sativa sp.japonica)转化,经PCR及Southern杂交检测,获得28个水稻转基因再生株系;以极限糊精酶抗体对转基因株系胚乳发育期籽粒蛋白质进行Western杂交,并经SDS-PAGE极限糊精酶活性检测,结果显示,该反向重复结构在胚乳发育期表达,减少了极限糊精酶的积累,获得了不同沉默效果的转基因株系。转化株系012的极限糊精酶活性只有野生型的10%,表明该载体可有效地抑制水稻胚乳中极限糊精酶的活性。

Down-Regulation of Rice Pullulanase Gene Expression by RNA Interference

The accumulation of pullulanase in rice grains was down regulated by RNA interference(RNAi). An RNAi plasmid,which contained an inverted repeat sequences of a pullulanase gene fragment spliced by an intron fragment and driven by an endosperm-specific promoter, was generated. Agrobacterium-mediated rice (Oryza sativa sp. japonica) transformation was conducted and 28 transgenic lines were confirmed containing the designed RNAi construct by PCR and Southern blot. Western analysis of the protein from developing endosperms with antibody against rice pullulanase showed that the accumulation of pullulanase was affected. Rice lines with varying amounts of pullulanase accumulation were obtained. This observation was confirmed by in-gel activity assays of pullulanase. Transgenic line 012 contained approximately 10% pullulanase protein comparing to wildtype, suggesting the RNAi construct from this study can be used to silence pullulanase activity in rice effectively.