非洲爪蛙prmt5.L基因的克隆及时空表达谱分析

Cloning of prmt5.L gene of Xenopus laevis and analysis its spatio-temporal expression patterns

[目的]克隆非洲爪蛙prmt5.L基因并分析其在胚胎发育中的时空表达谱。[方法]收集胚胎,提取总RNA,反转录制备c DNA。设计特异引物,PCR扩增prmt5.L基因的ORF序列,连接到p CS2+载体构建p CS2+prmt5.L重组质粒,连接位点为EcoRⅠ和XhoⅠ。经双酶切和测序验证成功构建p CS2+prmt5.L重组质粒。将p CS2+prmt5.L重组质粒用EcoRⅠ酶切,T7 RNA聚合酶转录获得prmt5.L的反义探针。收集不同时期的爪蛙胚胎,固定并脱水后经原位杂交检测prmt5.L在胚胎发育中的表达情况。通过序列比对和进化树分析PRMT5在进化中的保守性。[结果]成功克隆非洲爪蛙prmt5.L基因,原位杂交检测了prmt5.L在早期胚胎中的时空表达谱。序列比对和进化树发现prmt5.L蛋白在进化中非常保守。[结论]成功构建了p CS2+prmt5.L重组质粒。prmt5.L基因在胚胎发育中呈现动态表达且prmt5.L蛋白在进化中非常保守。

[Objective]To clone the prmt5.L gene of Xenopus laevis,and analysis its spatio-temporal expression patterns during early embryogenesis.[Method]Embryos were collected to extract total RNA,and c DNAs were prepared by reverse transcription.Specific primers were designed,and the ORF of prmt5.L gene was amplified by PCR and ligated into p CS2+vector to generate the p CS2+prmt5.L recombinant plasmid.The ligation sites were EcoRⅠand XhoⅠ.Through double enzyme digestion and sequencing,the p CS2+prmt5.L recombinant plasmid was successfully constructed.Then the p CS2+prmt5.L plasmid was digested by EcoRⅠ,and the antisense probe of prmt5.L was prepared by T7 RNA polymerase.Different stages of embryos were collected,and the expression of prmt5.L during early embryogenesis was detected by antisense probes of prmt5.L via in situ hybridization.[Result]The prmt5.L gene of Xenopus laevis was successfully cloned,and the spatio-temporal expressions of prmt5.L were detected by in situ hybridization.Sequence alignment and phylogenetic tree suggested that prmt5.L was very conserved in evolution.[Conclusion]The p CS2+prmt5.L recombinant plasmid was successfully constructed.prmt5.L gene showed dynamic expression during embryogenesis and prmt5.L protein is very conserved in evolution.