水稻细胞色素P450基因家族成员CYP81A6基因RNAi载体构建

Construct of RNAi Vector for Cytochrome P450 Gene CYP81A6 in Rice

水稻P450基因家族成员CYP81A6参与稻瘟病抗性反应,为了深入研究该基因的功能,本研究利用3次克隆的策略构建了该基因的RNAi载体。首先设计带有酶切位点的基因特异性引物,以c DNA为模板扩增3'非翻译区的特异片断,将目的产物连接到T载体上,经菌落PCR和酶切鉴定后进行测序。测序结果表示,目的片断已正确克隆到T载体上。然后利用双酶切通过2次亚克隆将目的片断以反向重复克隆到RNAi载体ds1301,通过分子检测表明,CYP81A6的RNAi载体构建成功,有助于后续的基因功能和作用机理的研究。

A member of P450 gene family CYP81A6 is involved in resistance response of rice blast disease. To study the function of CYP81A6 gene, we used three times cloning strategy to construct the RNAi vector of CYP81A6. Firstly, the gene-specific primers with restriction enzyme site were designed. And, c DNA were used as the template to amplify the specific fragment of 3' untranslated region, and cloned into the T vector. After colony PCR and digestion identified, it began to sequence. The result showed that the objective fragments were correctly cloned into the T vector. Then, the sequenced positive clone was subcloned into RNAi vector ds1301 by double enzyme digestions in reverse repeat. The results of molecular detection indicated that the RNAi vector of CYP81A6 was successfully constructed. This study will help the subsequent study of CYP81A6 gene function and mechanism.