猪DUXA基因克隆及过表达载体的构建

Cloning of porcine DUXA gene and construction of overexpression vector

收集猪体外受精(IVF)和体细胞核移植(SCNT)的2-细胞、4-细胞、8-细胞和16-细胞时期的胚胎,对各细胞阶段DUXA基因的表达进行比较分析;提取猪卵巢的RNA后通过RT-PCR克隆得到DUXA目的基因,对DUXA基因进行生物信息学分析;将目的基因片段连接至pCDNA3.1-EGFP载体中,再将载体转染至293T细胞中。结果显示,与IVF胚胎相比,DUXA基因在SCNT胚胎中的表达滞后;克隆的DUXA基因编码区全长678bp,与预期相符;核苷酸序列比对分析显示,猪DUXA和马DUXA基因同源性最高;各物种间DUXA蛋白氨基酸保守性较高;进化树结果显示,猪DUXA基因与骆驼相应序列聚为一支,与山羊、水牛、长江江豚等哺乳动物的遗传距离相对较近;利用在线分析工具对猪DUXA编码蛋白的理化性质进行预测,结果显示猪DUXA的化学分子式C1082H1732N344O354S7,相对分子质量为25448.17,理论等电点为9.4;猪DUXA二级结构预测包含11个α-螺旋、3个β-折叠、19个T-转角和20个无规则卷曲,具有2个HOX结构域,预测其为细胞膜外蛋白,其三级结构和黄牛、山羊、人相似。构建的pCDNA3.1-EGF-DUXA质粒载体长度为6797bp,转染结果显示构建的过表达载体可以在293T细胞中表达。结果表明,本试验成功克隆了猪DUXA基因,并成功构建了过表达载体pCDNA3.1-EGFP-DUXA。

The latest studies found that the DUX family genes play a crucial role during the(zygotic gene activation,ZGA)period of embryo development.However,our understanding about the function of the DUXA remains limited.In this study,DUXA will be cloned,conducted bioinformatics analysis and constructed overexpression vector of DUXA,which aim to lay the foundation for exploring the function of DUXA in embryonic development and improving the development rate of pig cloned embryos.2-cell,4-cell,8-cell,and 16-cell pig embryos derived fromin vitro fertilization(IVF)and somatic cell nuclear transfer(SCNT)were collected,then the expression level of DUXA in embryos at each embryonic stage was analyzed.RNA was extracted from pig ovaries,and the DUXA was cloned using RT-PCR,and then bioinformatic analysis conducted.Subsequently,the fragment of DUXA were inserted into the PCDNA3.1-EGFP vector and transfected the 293Tcells.The results demonstrated that the expression of the DUXA was delayed in SCNT embryos compared to IVF embryos.The cloned DUXA was found to have a full-length coding region of 678bp,consistent with expectations.Nucleotide sequence alignment analysis revealed that the pig DUXA exhibited the highest homology with the horse DUXA.The DUXA showed a high level of amino acid conservation across different species.Moreover,the evolutionary tree analysis suggested that the pig DUXA formed a branch with the camel sequence and exhibited a relatively close genetic distance to goats,water buffalo,and Yangtze finless porpoises.Furthermore,the physicochemical properties of the pig DUXA coding protein were predicted using online analysis tools,the results indicated that pig DUXA had a chemical formula of C1082H1732N344O354S7,a molecular weight of 25448.17,and a theoretical isoelectric point of 9.4.The secondary structure of pig DUXA was predicted to include 11α-helices,3β-folds,19T-turns,and 20irregular coils.It contained 2HOX domains and was predicted to be an extracellular membrane protein.Its tertiary structure exhibited similarities to that of cattle,goats,and humans.Finally,the constructed pcDNA3.1-EGF-DUXA plasmid vector had a length of 6797bp,and the transfection results showed that the constructed overexpression vector could be expressed in 293Tcells.In conclusion,this study successfully cloned the pig DUXA and constructed the overexpression vector PCDNA3.1-EGFP-DUXA.