畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (5): 972-982.doi: 10.11843/j.issn.0366-6964.2019.05.008

• 生物技术与繁殖 • 上一篇    下一篇

基于iTRAQ技术的卵巢静止奶牛血清差异蛋白分析

赵畅, 张江, 白云龙, 孙书函, 宋玉锡, 夏成*   

  1. 黑龙江八一农垦大学动物科技学院, 大庆 163319
  • 收稿日期:2018-05-08 出版日期:2019-05-23 发布日期:2019-05-23
  • 通讯作者: 夏成,主要从事奶牛营养代谢病研究,E-mail:Xcwlxyf2014@163.com
  • 作者简介:赵畅(1994-),女,黑龙江大庆人,博士生,主要从事临床兽医学实践和科研,E-mail:277863347@qq.com
  • 基金资助:

    国家自然科学基金面上项目(31372488);奶牛重要群发生产性疾病群防群控技术及产品研发与示范(GA16B202);畜禽群发普通病防控技术研究(2017YFD0502200)

Analysis of Serum Differential Proteins in Cows with Inactive Ovaries Based on iTRAQ Technology

ZHAO Chang, ZHANG Jiang, BAI Yunlong, SUN Shuhan, SONG Yuxi, XIA Cheng*   

  1. College of Animal Science and Veterinary Medicine, Heilongjiang Bayi Agricultural University, Daqing 163319, China
  • Received:2018-05-08 Online:2019-05-23 Published:2019-05-23

摘要:

旨在利用iTRAQ技术探究卵巢静止奶牛血清差异蛋白。本试验在奶牛产后60~90 d,根据发情表现、直肠检查和B超检查,按判定标准将试验奶牛分为卵巢静止组(IO)和正常发情对照组(CON),每组50头,采集血清。应用同位素标记绝对定量(iTRAQ)/质谱(MS)联用技术筛选差异蛋白,并应用免疫印迹以及ELISA方法鉴定奶牛卵巢静止血清差异蛋白。结果表明,试验共获得61种血清差异表达蛋白。GO分析表明,生物学过程包括34个注释,分子功能包括15个注释,细胞组成包括13个注释。蛋白质网络互作分析共得到57个节点,68条蛋白互作注释,蛋白质互作富集P<10-16,得到11个信号代谢通路。经过筛选发现,糖酵解/糖异生、氨基酸的生物合成、胰高血糖素信号通路,维生素的消化与吸收可能与卵巢静止的发生存在相关性。与卵巢静止相关差异表达蛋白共有14种:其中GPX3、SCGB1D和PKM2表达下调,ADIPOQ、AHSG、APOA4、FETUB、ALDOB、SPAM1、LDHB、RBP4、IGFBP2、ITIH3及GLYCAM1表达上调。ADIPOQ、IGFBP2和RBP4通过生殖激素生物过程影响卵泡发育,GPX3通过氧化应激而影响卵泡发育。

Abstract:

This study aimed to explore serum differential proteins in cows with inactive ovaries using iTRAQ technology. The Holstein cows in a intensive dairy farm were determined for energy balance (EB) status by blood β-hydroxybutyric acid (BHBA), and were assigned into inactive ovaries groups (IO, n=50), normal estrus control group (CON, n=50) at 60 to 90 day postpartum by the estrus manifestation, rectal examination and B-ultrasound examination.The isotope-labeled absolute quantification (iTRAQ)/mass spectrometry (MS) combined technique were used to screen differentially expressed proteins,Western blot and ELISA methods were used to identify differentially expressed proteins in serum of dairy cows with inactive ovaries. The results showed that 61 differentially expressed proteins were obtained,GO analysis showed that the biological process consisted of 34 annotations, the molecular function consisted of 15 annotations, the cellular composition consisted of 13 annotations. A total of 57 nodes were obtained from the analysis of protein network interaction. 68 proteins interpreted each other, protein interactions were enriched P<10-16, 11 signal metabolic pathways were obtained. After screening, it were found that glycolysis/gluconeogenesis, amino acid biosynthesis,glucagon signaling pathway, vitamin digestion and absorption might be related to the occurrence of ovarian quiescence.A total of 14 differentially expressed proteins verified by Western blot and ELISA were associated with inactive ovaries:GPX3, SCGB1D and PKM2 were down-regulated;ADIPOQ, AHSG, APOA4, FETUB, ALDOB, SPAM1, LDHB, RBP4, IGFBP2, ITIH3 and GLYCAM1 were up-regulated.ADIPOQ, IGFBP2 and RBP4 affect follicular development by affecting reproductive hormone biological processes, GPX3 affects follicular development through oxidative stress.

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