应用RD-PCR技术构建溶藻弧菌的cDNA文库

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

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 mRNA with poly（A） tracts was constructed and identified by restriction display PCR（ RD-PCR）, which can greatly decrease the redundancy of cDNA libraries and the polymorphic site of polyadenylation of bacterial mRNA. 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, secreted trypsin-like serine protease were identified. This indicated that polydenylation of the 3＇ end of mRNA could also be found in bacteria. The result showed the cDNA fragments library is of high quality, low reproducibility, which also have high efficiency. 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.