应用RD-PCR技术构建溶藻弧菌的cDNA文库

应用限制性显示PCR(RD-PCR)技术构建溶藻弧菌poly(A)化mRNA的cDNA文库,筛选,收集巧妙地解决了由于细菌mRNA poly(A)化位点的高度多态性、用常规方法构建原核cDNA文库所遇到的文库大量重复冗余的难题。并进行筛选、收集cDNA片段进行测序,根据测序结果进行生物信息学分析和结果验证。实验结果表明,我们成功地构建了溶藻弧菌poly (A)化mRNA的cDNA文库,获得一百多个基因片段并对其中53个基因片段进行测序分析,获得了如细菌Ⅲ型分泌系统易位蛋白、趋药性传感器、类丝氨酸蛋白酶等毒力相关因子的基因片断。并且在一定程度上证实了poly(A)化在细菌中不是个别和偶然的现象。实验结果说明了我们所构建的溶藻弧菌poly(A)化mRNA的cDNA文库质量高,文库基因片段的重复性低,筛选基因片段效率高、目的性强。本文并探讨了限制性显示PCR技术在细菌poly(A)化mRNA的cDNA1文库构建中的应用价值。

A cDNA library of Vibrio alginolyticus constructed by restriction display-PCR

Abstract: A cDNA library of Vibrio alginolyticus mRNA with poly(A) tracts was constructed and identified by restriction display- polymerase chain reaction (RD-PCR). After being selected and screened, bioinformatics of 53 clones were analyzed. The results showed that some genes such as translocation protein in type III secretion, type III secretory pathway component EscU, putative chemotaxis transducer and secreted trypsin-like serine protease were identified, which indicated a high quality cDNA. The applied value of RD-PCR in construction of cDNA library of Vibrio alginolyticus mRNA with poly(A) tracts was also analyzed in this research. Key words:cDNA library; restriction display-PCR(RD-PCR); Vibrio alginolyticus