中国农业科学 ›› 2022, Vol. 55 ›› Issue (23): 4743-4752.doi: 10.3864/j.issn.0578-1752.2022.23.014

• 畜牧·兽医 • 上一篇    下一篇

二花脸公猪不同耐冻性精子的蛋白质组学分析

童世锋1(),任智彬1,林斐2,葛雨竹1,陶景丽1,刘杨1,*()   

  1. 1南京农业大学动物科技学院动物遗传育种系,南京 210095
    2江苏省常熟市牧工商有限公司,江苏常熟 215500
  • 收稿日期:2021-09-18 接受日期:2021-12-28 出版日期:2022-12-01 发布日期:2022-12-06
  • 基金资助:
    江苏省种业振兴揭榜挂帅项目(JBGS(2021)026);农业农村部地方猪遗传材料采集制作专项课题(19190632);农业农村部都市农业重点实验室开放课题(UA201909)

Proteomic Analysis of Sperm with Different Freezing Tolerance in Erhualian Boar

TONG ShiFeng1(),REN ZhiBin1,LIN Fei2,GE YuZhu1,TAO JingLi1,LIU Yang1,*()   

  1. 1Department of Animal Genetics and Breeding, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095
    2Jiangsu Changshu Animal Husbandry Industry and Commerce Co., Ltd., Changshu 215500, Jiangsu
  • Received:2021-09-18 Accepted:2021-12-28 Published:2022-12-01 Online:2022-12-06

摘要: 目的 二花脸猪是我国优良的地方猪种之一,冷冻精液可提高二花脸公猪的利用率,并加强其种质资源保护,但目前生产的二花脸猪冷冻精液基本不能满足生产需要。研究通过分析二花脸公猪不同耐冻性精子的蛋白质组学,以促进二花脸公猪精子耐冻性蛋白质标志物的筛选,从遗传水平分析精子耐冻性的影响因素,为提高精子耐冻性提供参考依据。方法 通过冻存14头二花脸种公猪的精液,再对冷冻精液的冻后质量进行分析,根据精子冻后活率和冻后活力的高低筛选出耐冻性好和差(good freezability ejaculates, GFE和poor freezability ejaculates, PFE)的公猪各3头,利用串联质谱标记(tandem mass tag, TMT)技术对GFE组和PFE组的精子蛋白进行定量蛋白质组学分析,鉴定出GFE组和PFE组精子中的差异蛋白(differentially abundant proteins, DAPs)。利用GO富集分析对差异蛋白的功能进行注释,利用 KEGG富集分析对差异蛋白的生物通路进行注释,通过STRING蛋白质互作数据库进行差异蛋白的互作网络分析并利用Cytoscape软件构建蛋白质-蛋白质相互作用(PPI)网络。结果 研究发现GFE组和PFE组间共存在138个差异蛋白,PFE组与GFE组相比,有109个DAPs上调,29个DAPs下调。GO富集分析表明,共124个DAPs富集到47个GO term,主要富集到“细胞进程”“单有机体过程”“分子结合”“细胞”“细胞组分”等GO term。KEGG富集分析表明,DAPs主要富集到“肾素-血管紧张素系统”“补体和凝血级联”“血小板活化”等通路。从PPI网络可见53个节点和69 条连接边线,有8个具有强互作关系的蛋白ALB、ACRBP、ACR、ZAN、ZPBP2、HSPA5、FGB、FGG,这些蛋白质的功能主要富集于“单受精”“有性生殖”“精细胞发育”等与动物生殖生理以及精子发生密切相关的功能。进一步分析表明,GFE组和PFE组间的DAPs主要包括氧化还原相关蛋白(GSTM3、PRDX5、ALB、PDIA1、PDIA4等)、精子发生与功能相关蛋白(HSPA5、MFGE8、DNAH1、DNAH7、CUL3等)、能量相关蛋白(HK1、PGM1等)以及凋亡与炎症相关蛋白(THBS1、ELSPBP1、CP等);PPI网络图分析可知3个DAPs(MME、ANPEP和PRCP)在肾素-血管紧张素系统(RAS)中相互作用。结论 二花脸公猪不同耐冻性精子的蛋白质组分存在明显差异,可能对精子的冷冻性能产生影响,这些DAPs可作为精子耐冻性的候选蛋白标志物。

关键词: 二花脸公猪, 精子耐冻性, TMT, 差异蛋白

Abstract:

【Objective】 Erhualian pig is one of the excellent indigenous pig breeds in China. Frozen semen can improve the utilization rate of Erhualian boar and strengthen the protection of germplasm resources. However, the frozen semen of Erhualian pigs can not meet the production demand. This study analyzed the proteomics of Erhualian boar sperm with different freezing tolerance to promote the screening of protein markers of Erhualian boar sperm freezing tolerance, analyze the influencing factors of sperm freezing tolerance from the genetic level, and provide reference basis for improving sperm freezing tolerance. 【Method】 In this study, semen from14 Erhualian boars were frozen and their quality was analyzed. Boars with good and poor freezability ejaculates (GFE and PFE, n=3) were selected respectively, according to the motility and progressive motility of frozen-thawed spermatozoa. Using tandem mass tag (TMT) analyzed the sperm proteomics of GFE and PFE, and the differentially abundant proteins (DAPs) of spermatozoa of GFE and PFE were identified. The functions of DAPs were annotated by GO enrichment analysis, and the biological pathways of DAPs were annotated by KEGG enrichment analysis. The interaction network of DAPs was analyzed by STRING protein interaction database, and the protein-protein interaction (PPI) network was constructed by Cytoscape software..【Result】 The study found that there were 138 DAPs between GFE and PFE. Compared with GFE, 109 DAPs were up-regulated and 29 DAPs were down-regulated in PFE. GO enrichment analysis showed that a total of 124 DAPs were enriched to 47 GO terms, mainly enriched in “cellular process” “single-organism process” “binding” “cell” “cell part” and so on. KEGG enrichment analysis showed that DAPs were mainly enriched in “Renin-angiotensin system” “Complement and coagulation cascades” “Platelet activation” and so on. There were 53 nodes and 69 edges in the PPI network, among which 8 proteins showed high combine_score, including ALB, ACRBP, ACR, ZAN, ZPBP2, HSPA5, FGB and FGG. The functions of these proteins were mainly enriched in GO terms closely related to animal reproductive physiology and spermatogenesis, such as “single fertilization” “sexual reproduction” and “spermatid development”. Further analysis shows that DAPs in GFE and PFE mainly included redox related proteins (GSTM3, PRDX5, ALB, PDIA1, PDIA4), spermatogenesis and sperm function related proteins (HSPA5, MFGE8, DNAH1, DNAH7, CUL3), energy related proteins (HK1, PGM1), apoptosis and inflammation related proteins (THBS1, ELSPBP1, CP), and PPI network analysis shows that three DAPs (MME, ANPEP and PRCP) interact in renin-angiotensin system (RAS)..【Conclusion】 There are significant differences in sperm protein components of Erhualian boar with different freezing tolerance, which may affect the freezing performance of sperm. These DAPs can be used as candidate protein markers of sperm freezing tolerance.

Key words: Erhualian boar, sperm freezing tolerance, TMT, differentially abundant proteins