不同发育期白牦牛睾丸蛋白质组学分析及生殖相关候选基因HSP60生物学研究

发布时间：2018-01-10 00:03

本文关键词：不同发育期白牦牛睾丸蛋白质组学分析及生殖相关候选基因HSP60生物学研究　出处：《甘肃农业大学》2017年博士论文　论文类型：学位论文

【摘要】：天祝白牦牛(Bos grunniens)是我国特有的牦牛品种,是当地牧民主要的生产生活资料,但由于该品种原始,繁殖力低下,自然繁育为2年1胎或3年2胎,严重制约着白牦牛的生产能力。睾丸作为主要的生殖器官,影响着雄性动物的繁殖性能,其主要功能是分泌激素、产生精子等,这些生理过程需要多种蛋白质精确表达和调控。因此,以天祝白牦牛睾丸为材料,研究不同发育阶段睾丸中的差异表达蛋白质将为解释白牦牛睾丸发育和精子发生的分子机制提供参考,对提高白牦牛繁殖性能提供理论依据。本试验以1岁,2岁,4岁和8岁天祝白牦牛睾丸为实验材料,利用二维电泳和MALDI-TOF-TOF质谱技术鉴定不同发育时期差异表达蛋白质,分析这些差异表达蛋白质的功能及参与生物学过程或代谢途径。对筛选出的生殖候选基因热休克蛋白60(HSP60)进行分子克隆及在下丘脑-垂体-睾丸轴表达定位研究,进一步分析了HSP60对白牦牛睾丸支持细胞增殖的影响。本研究取得的主要结果如下:1、构建了白牦牛睾丸蛋白质表达图谱,经PDQuest 8.0.1分析发现白牦牛睾丸约有437个蛋白质点。比较了不同发育阶段差异蛋白质表达,结果显示在4个年龄阶段共有29个差异(p≤0.01)表达倍数在1.5倍以上的蛋白质点。其中,2种蛋白质随年龄上调,5种蛋白质随年龄下调,3种蛋白质在4岁前上调并随后下调,15种蛋白质在2岁前上调而后下调,4种蛋白质随年龄波动。2、对鉴定出的差异蛋白进行GO功能注释,这些蛋白质主要参与了“细胞过程、单有机体过程和新陈代谢过程”、“细胞、细胞器组成和胞外域”及“结合和催化活性”。亚细胞定位分析表明,鉴定蛋白主要位于细胞骨架(8个蛋白质),细胞核(6个蛋白质),线粒体(3个蛋白质)和细胞外基质(2个蛋白质)。3、选择2-DE检测的2个差异表达蛋白质(钙结合蛋白、热休克蛋白60)进行免疫印迹分析,结果显示所选蛋白的表达变化模式与2-DE结果基本一致,表明2-DE分析所得蛋白质差异表达结果可靠。4、对筛选出的差异蛋白HSP60进行分子克隆,发现白牦牛HSP60基因c DNA全长为2300bp,开放阅读框为1722bp,编码572个氨基酸。其理论分子量为60.977k Da、等电点为5.69,HSP60编码蛋白为非跨膜可溶性蛋白。氨基酸序列比对结果显示,白牦牛HSP60基因编码氨基酸序列与黄牛、瘤牛、绵羊、藏羚羊、骆驼、白犀牛、兔和黑猩猩的氨基酸序列同源性分别为100%、99%、99%、99%、99%、99%、98%和98%,说明HSP60在物种间高度保守。5、对HSP60表达及定位分析研究发现,HSP60基因及蛋白在白牦牛的下丘脑、垂体及睾丸组织中均有表达,其中下丘脑及垂体组织表达量高,睾丸组织表达量最低。免疫组化结果显示,HSP60蛋白表达于白牦牛下丘脑组织的室旁核大细胞,室旁核小细胞和神经角演网,垂体组织的腺细胞,睾丸组织的精原细胞,精母细胞,支持细胞和间质细胞,其中精子细胞中表达较弱。推断HSP60参与雄性白牦牛生殖轴调控,并参与睾丸发育与精子发生。6、进一步研究HSP60对原代培养的白牦牛睾丸支持细胞增殖的影响,通过构建HSP60过表达载体p IRES2-EGFP-HSP60,合成靶向si RNA沉默HSP60,瞬时转染支持细胞后,经RT-q PCR检测发现过表达组HSP60 m RNA在24、48和72h各时间点均上调,沉默组HSP60 m RNA在24、48和72h各时间点均下调。四氮唑盐法(MTS)检测细胞的增殖情况,过表达HSP60组,支持细胞增殖率各时间点均显著高于对照组。沉默HSP60组,支持细胞的增值率各时间点均低于对照组,但差异不显著。RT-q PCR检测细胞增殖标志基因细胞周期蛋白D1(Cyclin D1)和增殖细胞核抗原(PCNA),发现过表达HSP60组Cyclin D1基因在48h时的表达显著高于对照组,PCNA基因的表达在24、48和72h时均显著高于对照组。沉默HSP60组Cyclin D1基因和PCNA基因的表达在24、48和72h时均低于对照组,但差异不显著。表明HSP60在白牦牛睾丸支持细胞增殖调控中的作用是正向调控。本研究通过差异蛋白质组学技术检测不同发育阶段睾丸蛋白质表达差异变化,筛选出HSP60与生殖相关,并对HSP60基因进行分子克隆及表达定位研究,原核表达系统诱导表达出融合蛋白His-HSP60,同时,对HSP60在睾丸支持细胞的增殖调控过程进行了初步研究,结果表明HSP60在白牦牛性机能的旺盛期是通过下丘脑-垂体-性腺轴来调控睾丸支持细胞增殖,进而影响睾丸发育,本实验为进一步研究雄性白牦牛精子发生提供了基础资料。
[Abstract]:Tianzhu White Yak (Bos grunniens) is a unique Chinese yak breeds, is the main local herdsmen's production and life, but because of the original varieties, low fecundity, natural breeding for 2 years or 2 fetal fetal 1 3 years, seriously restricting the production capacity of white yak testis. As the main reproductive organs, affect the reproductive performance of male animal, its main function is to secrete hormones and sperm production, these processes require precise expression of multiple proteins and regulation. Therefore, the Tianzhu White Yak testis as material, the protein will provide reference for explaining the molecular mechanism of white yak testis development and spermatogenesis in the study of differential expression in different developmental stages in the testis. To provide a theoretical basis for improving reproductive performance of white yak. In this experiment, 1 years old, 2 years old, 4 years old and 8 years old Tianzhu White Yak testis as experimental materials, using two-dimensional electrophoresis and mass spectrometry identification technology MALDI-TOF-TOF With the development of differentially expressed proteins, analysis of these differentially expressed proteins involved in the biological process or function and metabolism. The screened reproductive candidate gene of heat shock protein 60 (HSP60) was cloned and expressed in the localization of the hypothalamic pituitary testicular axis, in order to analyze the influence of HSP60 on Yak Sertoli cell proliferation. The main results are as follows: 1, the construction of white yak testis protein expression profiles, PDQuest 8.0.1 analysis showed that the white yak testis about 437 protein spots. The differentially expressed proteins in different developmental stages, the results shown in the 4 age there are 29 differences (P < 0.01) expression in 1.5 times the protein spots multiples. Among them, 2 proteins up-regulated and 5 proteins with age, with age reduction, 3 proteins at the age of 4 and subsequently raised down, 15 egg white matter at the age of 2 Increase then down, 4 proteins with age fluctuation.2, GO functional annotation of the differentially proteins, these proteins are mainly involved in the cellular process, single organism process and The new supersedes the old. process "," cell, organelle composition and extracellular domain "and" binding and catalytic activity. Subcellular localization analysis showed that identification of protein mainly located in cell skeleton (8 protein), nucleus (6 proteins), mitochondria (3 protein) and extracellular matrix (2 protein).3, expression of 2 proteins detected by 2-DE selection (calcium binding protein, heat shock protein 60) for Western blot analysis showed that the selected. The expression patterns of variation and 2-DE results showed that expression of.4 2-DE proteins obtained reliable results, molecular cloning of differentially expressed protein HSP60, length of the white yak HSP60 gene C DNA 2300bp, an open reading frame of 1722bp, encoding 572 amino acids. The theoretical molecular weight of Da is 60.977k, its isoelectric point is 5.69. HSP60 encoding protein is a non transmembrane protein. The amino acid sequence comparison showed that the HSP60 gene encoding the amino acid sequence of white yak and cattle, zebu, sheep, antelope, camels, white rhino chimpanzee, rabbit and the homology of amino acid sequences were 100%, 99%, 99%, 99%, 99%, 99%, 98% and 98%, indicating that HSP60 is highly conserved among species of.5, found on the study of HSP60 expression and localization of hypothalamic HSP60 gene and protein in white yak, expressed in pituitary and testicular tissues, which the hypothalamus and pituitary tissue high expression level was the lowest in testicular tissue. Immunohistochemistry showed that the expression of HSP60 protein in white yak hypothalamus paraventricular nucleus of the cell, small cell and nerve nucleus paraventricularis angle play, pituitary gland cells, Testicular spermatogonia, spermatocytes and Sertoli cells and Leydig cells, weak expression in sperm cells. Conclude that HSP60 participates in white yak male reproductive axis control, and participate in the development of testis and spermatogenesis of.6, further study the influence of HSP60 on primary cultured Sertoli cell proliferation of white yak, by building HSP60 over expression vector p IRES2-EGFP-HSP60, Si RNA HSP60 to silence target synthesis, transient transfection of Sertoli cells, by RT-q PCR detection found over expression of group HSP60 m RNA in 24,48 and 72h at different time points were raised, were down HSP60 m RNA silent group in 24,48 and 72h at different time points. The tetrazolium salt (MTS) proliferation the detection of cells, overexpression of HSP60 group support cell proliferation rate at each time point was significantly higher than the control group. The silencing of HSP60 group, Sertoli cell increment rate at each time point was lower than the control group, but the difference was not significant.RT-q PCR fine detection The cell proliferation marker gene cyclin D1 (Cyclin D1) and proliferating cell nuclear antigen (PCNA), found that overexpression of HSP60 gene in 48h D1 Cyclin group was significantly higher than the control group, the expression of PCNA gene in 24,48 and 72h were significantly higher than the control group. The expression of Cyclin D1 gene silencing HSP60 group and PCNA gene were lower than the control group in 24,48 and 72h, but the difference was not significant. The results indicated that HSP60 in white yak Sertoli cell proliferation is the role of positive regulation. Through the study of differential proteomics expression changes at different developmental stages of testis protein detection, HSP60 screening and reproductive related research and the expression and molecular orientation cloning of HSP60 gene, prokaryotic expression system for expression of His-HSP60 fusion protein and HSP60 was studied in the regulation of proliferation process of Sertoli cells, the result showed that HSP60 In the vigorous period of white yak, the hypothalamic pituitary gonadal axis regulates the proliferation of Testis Sertoli cells and affects testicular development. This experiment provides basic data for further research on spermatogenesis of male white yak.

【学位授予单位】：甘肃农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S823.85

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