猪类无精症缺失基因DAZL的cDNA全长克隆、组织表达和亚细胞定位

cDNA Full-length Cloning,Tissue Expression and Subcellular Location of DAZL Gene in Pig

旨在获得猪类无精症缺失基因DAZL cDNA全长序列,阐明该基因的序列、mRNA组织表达模式、蛋白质结构特征及亚细胞定位情况。本研究以版纳微型猪近交系(Banna mini-pig inbred line,BMI)10月龄成年公猪为研究对象,屠宰收集组织样,利用RACE和RT-PCR技术获得DAZL基因cDNA全长序列;使用实时荧光定量PCR(qPCR)技术检测其mRNA多组织表达谱;在线分析蛋白质的结构特点和保守结构域;用体外细胞转染技术鉴定其在ST猪睾丸细胞中的定位。结果表明,BMI DAZL cDNA全长2985 bp(KU705632),包含888 bp的CDS区,编码295个氨基酸(AOC89050);该基因位于猪13号染色体,含11个外显子。qPCR结果显示,DAZL mRNA在睾丸中特异高表达。生物信息学分析表明,猪DAZL蛋白含有哺乳动物RMP和DAZ同源区,无规则卷曲在二级结构中超过50%。进化分析表明,BMI DAZL氨基酸序列高度保守,与牛科的亲缘关系最为接近。pEGFP-C1-DAZL转染ST细胞后的荧光共定位结果显示,DAZL蛋白主要分布在细胞核中。本研究分别从DNA、mRNA和蛋白质层面阐明了BMI DAZL基因的序列特征、表达、蛋白质结构和定位,为进一步研究DAZL在BMI精子发生方面的功能奠定基础。

The purpose of this study was to clone the full-length cDNA sequence of deleted in azoospermia like(DAZL)from Banna mini-pig inbred line(BMI),obtain its multi-tissue mRNA expression patterns,characterize the protein structure,and acquire the subcellular localization of DAZL.The tissue samples were collected from 10-month-old adult BMI boar and the full-length cDNA of DAZL gene was cloned from BMI using RACE and RT-PCR technologies.The multi-tissue mRNA transcription levels of DAZL were analyzed using qPCR.The structural characteristics and conserved domains of DAZL protein were analyzed using online tools.The subcellular localization of DAZL protein in the ST cell was detected by in vitro cell transfection technology.The results showed that the full-length cDNA sequence of DAZL was 2985 bp(KU705632)with an 888 bp CDS encoding 295 amino acids(AOC89050).The BMI DAZL gene was located on chromosome 13 of BMI genome and contained 11 exons.Multi-tissue qPCR results showed that DAZL mRNA was exclusively and highly expressed in the testis.Further bioinformatics analysis showed that DAZL protein contained two conserved domains,RMP and DAZ,that were homologous with other mammals,with random coil structures more than 50%.Evolutionary analysis showed that the amino acid sequence of BMI DAZL was highly conserved and shared the closest genetic relationship with Bovidae.Fluorescence co-localization results of pEGFP-C1-DAZL transfected ST cells showed that DAZL protein was localized in the nucleus.The sequence characterization,mRNA expression,protein structure and location of BMI DAZL gene were clarified at DNA,mRNA and protein levels,respectively.This study will lay the foundation for further functional studies of DAZL during spermatogenesis in BMI.