猪DAZL启动子克隆、转录表达及蛋白功能特性分析  

Promoter Cloning,Transcriptional Expression and Functional Characterization of DAZL in Pig

作　　者：许静 张霞 刘志朋 王淑燕[1] 李洪林 李小伟 王配[1] 霍金龙[1] XU Jing;ZHANG Xia;LIU Zhipeng;WANG Shuyan;LI Honglin;LI Xiaowei;WANG Pei;HUO Jinlong(College of Animal Science and Technology,Yunnan Agricultural University,Kunming 650201,China;Department of Life Science,Lyuliang University,Lyuliang 033001,Shanxi,China)

机构地区：[1]云南农业大学动物科学技术学院,昆明650201 [2]吕梁学院生命科学系,山西吕梁033001

出　　处：《四川农业大学学报》2023年第6期1098-1106,共9页Journal of Sichuan Agricultural University

基　　金：国家自然科学基金(32060733、31660637、31460580、31660650);云南农业大学第十六届学生科技创新创业行动基金(2023YA0257)。

摘　　要：【目的】研究猪类无精症缺失基因DAZL在版纳微型猪近交系(Banna mini-pig inbred line,BMI)中的启动子特征、转录表达及蛋白功能。【方法】使用PCR技术克隆DAZL启动子序列,并分析其结构;使用RNA测序技术获得BMI睾丸组织中DAZL的表达量,并构建转录调控网络;分析DAZL的氨基酸同源性与蛋白功能特性,并构建互作蛋白网络。【结果】成功扩增了DAZL长度为2378 bp的启动子序列,含1个CpG岛、3个核心启动子以及Oct-1、Sp1、Pit-1a等10种转录因子结合位点;DAZL在BMI睾丸组织中有较高的正表达;ceRNA调控网络分析表明DAZL被sscmiR-199a-5p、ssc-miR-199b-5p、ssc-miR-17-5p、ssc-miR-103、ssc-miR-24-3p和ssc-miR-107等6个miRNAs靶向调控;功能注释发现其主要在精子发生、生殖细胞发育、翻译调控中发挥重要功能。DAZL及其邻近基因分析发现其在各物种中高度保守,且具有较为保守的邻近基因;蛋白互作网络、GO和KEGG富集分析显示DAZL与PUM2、DAZAP2和SYCE1等多个蛋白互作,主要参与精子发生、卵子发生、有性生殖等重要生殖相关通路;转录组数据与蛋白编码基因之间的表达量相关性显示DAZL的表达量与SOHLH1显著相关。【结论】本研究从DNA、RNA和蛋白质角度全面分析了猪DAZL的启动子结构、转录表达和蛋白功能,结果为更进一步研究DAZL在精子发生中的作用奠定了基础。【Objective】The aim of this study was to investigate the characteristics of promoter,transcriptional expression,protein function of the porcine azoospermia deletion gene DAZL in Banna mini-pig inbred line(BMI).【Method】The DAZL promoter sequence was cloned by PCR and its structure was analyzed.The DAZL expression data in BMI testis tissues were obtained by RNA sequencing technique and the transcriptional regulatory network was constructed.Amino acid homology and functional characteristics of DAZL protein were analyzed and the interacting protein network was constructed.【Result】The DAZL promoter sequence of 2378 bp length was amplified successfully,including 1 CpG island,3 core promoters and 10 transcription factor binding sites such as Oct-1,Sp1 and Pit-1a.DAZL was positively expressed in BMI testicular tissue.The ceRNA regulatory network analysis showed that DAZL was targeted and regulated by six miRNAs,including ssc-miR-199a-5p,ssc-miR-199b-5p,ssc-miR-17-5p,ssc-miR-103,ssc-miR-24-3p and ssc-miR-107.Functional annotation indicated that DAZL mainly plays important roles in spermatogenesis,germ cell development,and translation regulation.Analysis of DAZL and its neighboring genes indicated that DAZL was highly conserved and had relatively conserved neighboring genes in species.The analysis of protein interaction network,GO and KEGG enrichment suggested that DAZL interacted with several proteins,including PUM2,DAZAP2,SYCE1,etc,which is mainly involved in important pathways associated with reproduction such as spermatogenesis,oogenesis,and sexual reproduction.The correlation between transcriptome data and protein-coding genes implied that the expression of DAZL was significantly correlated with SOHLH1.【Conclusion】This study comprehensively analyzed the promoter structure,transcriptional expression and protein function of porcine DAZL from the levels of DNA,RNA and protein,laying a foundation for further study on the role of DAZL in porcine spermatogenesis.

关 键 词：版纳微型猪近交系 DAZL 启动子 转录组测序 蛋白互作 

分 类 号：S828.3[农业科学—畜牧学]