非洲爪蛙Klf4基因新转录本的克隆及功能研究

Cloning and functional study of the new transcript of Xenopus laevis Klf4 gene

[目的]克隆非洲爪蛙Klf4基因的新转录本,并研究其在早期胚胎发育中的功能。[方法]提取胚胎总RNA,反转录构建c DNA文库。从Xenbase数据库中获得Klf4的基因组序列,分析序列发现新的ATG位点,设计引物、PCR扩增,将扩增的目的片段插入p CS2+,插入位点为EcoRⅠ和XhoⅠ。经双酶切和测序鉴定,发现载体中插入的目的片段为编码483个氨基酸的Klf4的新转录本。体外转录获得mRNA,显微注射入胚胎,进行表型分析以及原位杂交检测。[结果]成功克隆出了Klf4基因的新转录本;过表达该转录本,表型分析发现胚胎发育异常,原位杂交检测发现Xag2和Dkk1的表达上升。[结论]成功克隆编码483个氨基酸的Klf4基因的新转录本,过表达该转录本胚胎发育异常,Xag2和Dkk1的表达被激活。

[Objective]To clone the new transcript of Klf4 gene and study its function during Xenopus laevis early embryongenesis.[Method]The total RNA was extracted from the early embryos of Xenopus laevis,and the cDNA library was constructed by reverse transcription.The genome sequence of Klf4 gene was obtained from Xenbase database,and a new ATG translation initiation site was found through sequence analysis.Specific primers were designed and PCR amplification was performed to obtain the target fragment.Then the target fragment was inserted into the pCS2+to generate recombinant vector,the insertion site was EcoRⅠand XhoⅠ.Then the recombinant vector was identified by double enzyme digestion and sequencing.The identified new Klf4 transcript which encodes 483 amino acids.Its mRNAs were obtained by in vitro transcription,and were microinjected into embryos of Xenopus laevis.Phenotypic analysis and whole mount in situ hybridization were performed to study its effect on embryogenesis.[Result]A new transcript of Klf4 gene was successfully cloned,which encodes 483 amino acids.The phenotypic analysis shown that overexpression this new trancript led to abonormal embryonic development.Moreover,the expression of Xag2 and Dkk1 was promoted in overexpressed embryos.[Conclusion]A new transcript encoding 483 amino acids was successfully cloned.Overexpression of this transcript results in developmental abnormalities and activated the expression of Xag2 and Dkk1.