猪精子发生候选基因PYGO2的分离、表达和亚细胞定位研究

旨在分离猪PYGO2编码区序列，获悉该基因mRNA组织表达模式、蛋白质结构特征、细胞中的分布和定位，并构建蛋白相互作用网络。本研究首先利用RT-PCR从成年版纳微型猪近交系（BMI）睾丸组织中克隆PYGO2编码区序列；利用生物信息学解析其基因结构并对蛋白质进行多种功能分析，比较多个哺乳动物PYGO2的氨基酸序列同源性，构建系统进化树和蛋白质相互作用网络；然后利用qPCR技术检测PYGO2在15个组织中的mRNA表达情况；最后通过构建pEGFP-C1-PYGO2融合表达载体，转染猪睾丸细胞（ST），确定PYGO2的亚细胞定位。结果表明，PYGO2基因CDS长1 221 bp，编码406个氨基酸（基因和氨基酸号分别为KY644518和AVB77243.1），定位在猪4号染色体；PYGO2蛋白二级结构以无规则卷曲为主，N端和C端均疏水；氨基酸序列同源比对分析表明，猪PYGO2与其他哺乳动物的相似度均大于97%，在进化上高度保守；蛋白互作网络分析显示，猪PYGO2与9个蛋白可能存在相互作用，其中与BCL9蛋白作用最为紧密；qPCR表达分析表明，猪PYGO2在被检的15个组织中均有不同程度表达，在生殖腺中表达相对较高；ST细胞中的亚细胞定位结果表明，PYGO2 mRNA主要分布在细胞核。本研究获得了PYGO2基因的编码序列，蛋白质结构和定位，蛋白质相互作用网络，mRNA多组织表达特征，可为进一步解析该基因在猪精子生成中的分子机制提供参考。

Isolation,Expression and Subcellular Localization of Spermatogenesis Candidate Gene PYGO2 in Pig

The purpose of this study was to isolate coding sequence of PYGO2 gene in pig, obtain its multi-tissue mRNA expression patterns, structural characteristics of protein and localization in cell, and construct the protein-protein interaction network. The coding sequence of PYGO2 was cloned from testis tissue of adult BMI using RT-PCR. The bioinformatics methods was performed to dissect its gene structure and protein functions, compare amino acid homology in mammals, construct phylogenetic tree and protein-protein interaction network. PYGO2 mRNA expression profiles in 15 tissues were detected using qPCR technology. The subcellular location of PYGO2 protein was conducted by constructing the expression vector of pEGFP-C1-PYGO2 and transfecting into ST cell. The results showed that the CDS sequence of PYGO2 was 1 221 bp, encoding 406 amino acids, and located on the chromosome 4 of pig genome. The accession numbers of gene and amino acid in GenBank were KY644518 and AVB77243.1, respectively. The secondary structure of PYGO2 protein contained mainly random coil with hydrophodic N and C terminal. The homology analysis of amino acid sequences implied that the similarities were more than 97% between pig and other mammals, suggesting high conservation of PYGO2 in evolution. The analysis of interaction network of proteins represented that pig PYGO2 had interactions with 9 proteins, and interacted most intensively with BCL9 protein. Further, multi-tissue qPCR results suggested that PYGO2 mRNA were extensively expressed in the 15 detected tissues and with higher expression in gonads. The result of subcellular location in transfected ST cells indicated that PYGO2 protein was predominantly localized in the nucleus. This study obtained the coding sequence of PYGO2 gene, protein structure and location, interaction network of protein, expression characteristics of mRNA in multiple tissues, which will provide a reference for further explanation of molecular mechanisms on pig PYGO2 during spermatogenesis.