非洲爪蟾β-synuclein基因的克隆、亚细胞定位及原位表达检测

Cloning,Subcellular Localization and in situ Detection of Xenopus Laevis β-synuclein Gene

目的克隆非洲爪蟾β突触核蛋白基因(xenopus-β-synuclein,xSYNB)并研究其亚细胞定位。方法以成年爪蟾脑部的总RNA为模板,RT-PCR扩增获得xSYNB基因,并克隆入pGEM-TEasy载体而获得TA-xSYNB重组质粒,亚克隆到pEGFPN1载体上获得pEGFPN1-xSYNB真核表达载体。将pEGFPN1-xSYNB重组质粒转染HEK293细胞,用TA-xSYNB重组质粒为模板合成的正义与反义探针对非洲爪蟾胚胎进行原位杂交。结果成功构建了pEGFPN1-xSYNB重组质粒,绿色荧光检测结果发现xSYNB蛋白在细胞浆中表达。原位杂交实验表明xSYNB基因主要表达于胚胎的头部。结论首次鉴定了非洲爪蟾β-synuclein基因的表达,并发现xSYNB基因可能在爪蟾胚胎神经系统发育中起一定作用。

Objective To clone Xenopus laevis β-synuclein gene（xSYNB） and study the subcellular localization of xSYNB protein.Methods According to the xSYNB cDNA sequence published in GenBank,a pair of primers were designed.The encoding region of xSYNB gene from the adult Xenopus laevis brain was amplified by RT-PCR,and then was cloned into pGEM-T Easy vector.The resulting recombinant plasmid was named as TA-xSYNB.The xSYNB cDNA was further subcloned into the pEGFP-N1 vector,and the resulting recombinant expression plasmid pEGFPN1-xSYNB was transfected into HEK293 cells to analyze the subcellular localization of xSYNB.The whole-mount in situ hybridization of embryos were used to identify the expression localization of xSYNB.Results The recombinant expression plasmid pEGFPN1-xSYNB was successfully constructed.The results of green fluorescence detection suggested that xSYNB gene was mainly expressed in the cytoplasm,and the results of in situ hybridization suggested that xSYNB gene was mainly expressed in the brain of embryo.Conclusion The xSYNB gene may play a role in the development of the nervous system of Xenopus laevis.