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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。
The RNAi vector containing the reverse repeat structure of the limit dextrinase gene fragment was constructed and the limit dextrinase gene deletion mutant was obtained by inhibiting the expression of limit dextrinase gene in rice grain. The restriction endonuclease digestion and sequence analysis confirmed that RNAi vector of rice endodextrin gene inverted repeat structure was successfully constructed, and Agrobacterium tumefaciens mediated Oryza sativa sp.japonica ), And 28 transgenic lines of transgenic rice were obtained by PCR and Southern blotting. Western blotting was carried out with the limit dextrinase antibody against the grain protein of the endosperm during the development of transgenic lines. SDS- The results showed that the inverted repeat structure expressed in the endosperm development, reducing the limit of dextrinase accumulation, obtained a different silencing effect of transgenic lines. The limit dextrinase activity of transformant 012 was only 10% of the wild type, indicating that the vector can effectively inhibit the activity of limit dextrinase in rice endosperm.