猪睾丸Dickkopf样顶体蛋白1基因的转录调控分析

Transcriptional regulation analysis of the gene for acrosomal protein Dickkopf-like 1 in porcine testis

【目的】Dickkopf样顶体蛋白1(DKKL1)是一种重要的顶体蛋白,为发掘其重要功能及潜在的临床价值,以版纳微型猪近交系(BMI)为对象,研究其睾丸组织中DKKL1的分子结构、转录调控特征和蛋白质功能。【方法】通过全转录组测序获得BMI DKKL1的表达量,使用逆转录聚合酶链反应(RT-PCR)获得DKKL1编码区序列并分析其基因结构和蛋白质特征,使用UniProt数据库对DKKL1进行功能注释并分析DKKL1与微小RNA(miRNA)、长链非编码RNA(lncRNA)间的竞争性内源RNA(ceRNA)转录调控。【结果】获得的BMI DKKL1编码区全长为702 bp,位于基因第6号染色体上,有5个外显子和4个内含子;蛋白质功能分析表明,DKKL1包含233个氨基酸,具有疏水性,含磷酸化位点、信号肽和较多的无规则卷曲亚结构;系统进化和同源性分析表明,DKKL1氨基酸序列在哺乳动物间高度保守;蛋白互作分析显示,DKKL1与含螺旋结构域的蛋白155(CCDC155)、转录增强缔合域蛋白2(TEAD2)、RAS癌基因家族成员11B(RAB11B)等多种蛋白互作;基因本体(GO)和京都基因与基因组百科全书(KEGG)富集分析表明,这些蛋白富集在典型Wnt信号等通路上;GO功能注释表明,DKKL1在睾酮生物合成过程的负向调控、透明带穿透、顶体泡生成等方面具有重要功能;ceRNA转录调控网络分析表明,DKKL1被miR-15a和miR-15b靶向调控。【结论】本研究获得了BMI睾丸全转录数据,阐明了DKKL1的分子特征、蛋白质功能并构建了转录调控网络。

【Objective】In order to explore the important function and potential clinical value of acrosomal protein Dickkopf-like 1(DKKL1),the molecular structure,transcriptional regulatory characteristics and function of DKKL1 in testicular tissue of Banna mini-pig inbred line(BMI)were studied.【Method】The expression level of DKKL1 in BMI was detected by whole transcriptome sequencing,and its gene structure and protein characteristics were analyzed with the coding sequences obtained by reverse transcription PCR(RT-PCR).The functional annotation via UniProt database was performed to analyze the competing endogenous RNA(ceRNA)regulatory relationship between DKKL1 and microRNA(miRNA)and long non-coding RNA(lncRNA).【Result】DKKL1 was located on the chromosome 6 of BMI genome,with its encoding region 702 bp in length and having 5 exons and 4 introns.Functional analysis implied that DKKL1 was a 233-amino acid protein with hydrophobic structure,phosphorylation sites,signal peptides and lots of random coils.Phylogenetic and homologous analysis showed that the amino acid sequence of DKKL1 was highly conserved among mammals.Protein interaction analysis indicated that DKKL1 interacted with many proteins like coiled-coil domain containing protein 155(CCDC155),transcriptional enhanced association domain protein-2(TEAD2)and member of RAS oncogene family 11B(RAB11B).Moreover,gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis revealed that these proteins were involved in canonical Wnt signaling pathway,and DKKL1 played pivotal roles in the negative regulation of testosterone biosynthesis process,zonapellucida penetration and acrosome vesicle formation by GO functional annotation.Lastly,ceRNA network analysis suggested that DKKL1 was targeted by miR-15a and miR-15b.【Conclusion】In this study,We claried the molecular characteristics and protein functions of BMI DKKL1 by whole transcriptome,and further constructed transcriptional regulatory network.