版纳微型猪近交系ASB17基因克隆及睾丸组织特异表达研究

Cloning of ASB17 gene and testis specific expression in Banna mini-pig inbred line

ASB17基因与睾丸形成和精子发生密切相关。为研究ASB17基因在猪睾丸功能及精子发生过程中的作用,本研究从GenBank下载猪及近缘物种ASB17序列作为参考序列,设计特异引物从版纳微型猪近交系(BMI)睾丸组织中扩增ASB17基因完全编码序列及部分侧翼序列,对ASB17翻译的蛋白质序列进行多种功能生物信息学分析并构建多物种系统进化树,最后用实时荧光定量PCR技术检测BMI 15个重要组织的ASB17 mRNA转录表达水平。结果表明,扩增得到BMI ASB17编码区序列长888bp,编码295个氨基酸。功能生物信息学分析表明ASB17存在2个保守结构域ANK和SOCS_box,无跨膜螺旋结构,无N端信号肽序列,二级结构以α螺旋为主,N末端和C末端均亲水,有4类功能活性位点,位于细胞质的概率70.6%。系统进化分析表明,ASB17基因在进化中高度保守,且与牛、羊的亲缘关系最近,符合物种的系统分类学。多组织相对荧光定量表达分析表明ASB17基因在睾丸中特异高表达,在心、肝等其他14个组织中不表达。研究结果为探索BMI ASB17基因在睾丸形成和精子发生过程中的作用及功能奠定基础。

ASB17 gene is closely related to the testicular development and the spermatogenesis.In order to study the role of ASB17 gene in pig testis function and spermatogenesis,the specific primers were designed based on GneBank ASB17 mRNA sequences of pig related spe-cies,and the coding sequences and flanking sequence of ASB17 gene were amplified from Banna mini-pig inbred line（BMI）testis tissues.ASB17 protein sequence was used to carry out functional bioinformatics analysis and construct multiple species phylogenetic tree.Real-time quantitative PCR was applied to analyze the ASB17 gene expression profiles of 15 important tissues.The results obtained an 888 bp coding sequence of BMI ASB17 gene,which encodes a protein of 295 amino acids.Functional bioinformatics analysis indicated that ASB17 protein contained two conserved domains of ANK and SOCS_box without transmembrane region and signal peptide sequences.Its secondary structure is predominantly alpha helices and its N-terminal and C-terminal are hydrophilic.The ASB17 protein has four different modification sites.According to prediction 70.6% mature proteins locate in cytoplasm.Overall BMI ASB17 had the closest relationship with that of cattle and sheep by phylogenetic analysis.This demonstrated that sequences are coincident with classical taxonomy.Real-time quantitative PCR in multi-tissues indicated that ASB17 gene was expressed exclusively in the testis tissue and expressions in the heart,liver and other 14 tissues were not detected.Our study will lay a foundation for further study of the ASB17 gene functions in pig testicular development and spermatogenesis.