卡地腐霉Pr1蛋白酶miniDNA文库的构建及基因片段的克隆

Cloning of the Unknown Sequence of Subtilisin-like Protease Gene in Pythium carolinianum by Constructing Mini Genomic DNA Library

目的: 在本实验室已克隆得到的灭蚊真菌卡地腐霉类枯草杆菌蛋白酶基因的部分序列的基础上,继续分离该基因的全基因组序列。方法:采用构建卡地腐霉mini基因组文库的方法,首先利用已知序列设计引物,克隆得到该蛋白酶基因的部分基因组序列;然后采用限制性内切酶XhoⅠ消化卡地腐霉基因组DNA,同时用该内切酶消化质粒pBluescriptSK( -),经去磷酸化后,将基因组酶切产物克隆到载体上并转化到大肠杆菌JM109中,构建mini基因组文库。利用载体上的通用引物和根据已知序列设计的引物,进行嵌套式PCR。结果:获得一780bp的PCR产物,经克隆、测序和分析为卡地腐霉类枯草杆菌蛋白酶基因已知序列一端301bp未知序列。结论:这种方法操作简便,实验周期短,能够特异地扩增与已知位点相邻的基因组DNA。

Objective:To obtain the full-long genomic sequence of the subtilisin-like protease (Pr1) gene of Pythium carolinianum. Methods: A mini genomic DNA library was constructed. Firstly, the genomic DNA was digested with Xho I and then inserted into the pBluescript SK(-) plasmid which was digested with the same enzyme and treated with calf intestinal alkaline phosphatase. The recombinant plasmid was transferred into Ecoli JM109 and the mini genomic DNA library was constructed. Results: A 780bp PCR product was subsequently subcloned with nested PCR method with T3 promoter primer and nested primers S1 and S2. Sequence analysis indicated that it contains the 301bp unknown sequences adjacent to the known sequence of Pr1 gene of P. carolinianum. Conclusion: Constructing mini genomic DNA library is an efficient and expeditious strategy of searching for unknown sequence adjacent to the known.