本文關鍵詞：不同發(fā)育期白牦牛睪丸蛋白質組學分析及生殖相關候選基因HSP60生物學研究　出處：《甘肅農業(yè)大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 天祝白牦牛 睪丸蛋白質組學 雙向電泳 HSP60 支持細胞

【摘要】：天祝白牦牛(Bos grunniens)是我國特有的牦牛品種,是當地牧民主要的生產生活資料,但由于該品種原始,繁殖力低下,自然繁育為2年1胎或3年2胎,嚴重制約著白牦牛的生產能力。睪丸作為主要的生殖器官,影響著雄性動物的繁殖性能,其主要功能是分泌激素、產生精子等,這些生理過程需要多種蛋白質精確表達和調控。因此,以天祝白牦牛睪丸為材料,研究不同發(fā)育階段睪丸中的差異表達蛋白質將為解釋白牦牛睪丸發(fā)育和精子發(fā)生的分子機制提供參考,對提高白牦牛繁殖性能提供理論依據。本試驗以1歲,2歲,4歲和8歲天祝白牦牛睪丸為實驗材料,利用二維電泳和MALDI-TOF-TOF質譜技術鑒定不同發(fā)育時期差異表達蛋白質,分析這些差異表達蛋白質的功能及參與生物學過程或代謝途徑。對篩選出的生殖候選基因熱休克蛋白60(HSP60)進行分子克隆及在下丘腦-垂體-睪丸軸表達定位研究,進一步分析了HSP60對白牦牛睪丸支持細胞增殖的影響。本研究取得的主要結果如下:1、構建了白牦牛睪丸蛋白質表達圖譜,經PDQuest 8.0.1分析發(fā)現白牦牛睪丸約有437個蛋白質點。比較了不同發(fā)育階段差異蛋白質表達,結果顯示在4個年齡階段共有29個差異(p≤0.01)表達倍數在1.5倍以上的蛋白質點。其中,2種蛋白質隨年齡上調,5種蛋白質隨年齡下調,3種蛋白質在4歲前上調并隨后下調,15種蛋白質在2歲前上調而后下調,4種蛋白質隨年齡波動。2、對鑒定出的差異蛋白進行GO功能注釋,這些蛋白質主要參與了“細胞過程、單有機體過程和新陳代謝過程”、“細胞、細胞器組成和胞外域”及“結合和催化活性”。亞細胞定位分析表明,鑒定蛋白主要位于細胞骨架(8個蛋白質),細胞核(6個蛋白質),線粒體(3個蛋白質)和細胞外基質(2個蛋白質)。3、選擇2-DE檢測的2個差異表達蛋白質(鈣結合蛋白、熱休克蛋白60)進行免疫印跡分析,結果顯示所選蛋白的表達變化模式與2-DE結果基本一致,表明2-DE分析所得蛋白質差異表達結果可靠。4、對篩選出的差異蛋白HSP60進行分子克隆,發(fā)現白牦牛HSP60基因c DNA全長為2300bp,開放閱讀框為1722bp,編碼572個氨基酸。其理論分子量為60.977k Da、等電點為5.69,HSP60編碼蛋白為非跨膜可溶性蛋白。氨基酸序列比對結果顯示,白牦牛HSP60基因編碼氨基酸序列與黃牛、瘤牛、綿羊、藏羚羊、駱駝、白犀牛、兔和黑猩猩的氨基酸序列同源性分別為100%、99%、99%、99%、99%、99%、98%和98%,說明HSP60在物種間高度保守。5、對HSP60表達及定位分析研究發(fā)現,HSP60基因及蛋白在白牦牛的下丘腦、垂體及睪丸組織中均有表達,其中下丘腦及垂體組織表達量高,睪丸組織表達量最低。免疫組化結果顯示,HSP60蛋白表達于白牦牛下丘腦組織的室旁核大細胞,室旁核小細胞和神經角演網,垂體組織的腺細胞,睪丸組織的精原細胞,精母細胞,支持細胞和間質細胞,其中精子細胞中表達較弱。推斷HSP60參與雄性白牦牛生殖軸調控,并參與睪丸發(fā)育與精子發(fā)生。6、進一步研究HSP60對原代培養(yǎng)的白牦牛睪丸支持細胞增殖的影響,通過構建HSP60過表達載體p IRES2-EGFP-HSP60,合成靶向si RNA沉默HSP60,瞬時轉染支持細胞后,經RT-q PCR檢測發(fā)現過表達組HSP60 m RNA在24、48和72h各時間點均上調,沉默組HSP60 m RNA在24、48和72h各時間點均下調。四氮唑鹽法(MTS)檢測細胞的增殖情況,過表達HSP60組,支持細胞增殖率各時間點均顯著高于對照組。沉默HSP60組,支持細胞的增值率各時間點均低于對照組,但差異不顯著。RT-q PCR檢測細胞增殖標志基因細胞周期蛋白D1(Cyclin D1)和增殖細胞核抗原(PCNA),發(fā)現過表達HSP60組Cyclin D1基因在48h時的表達顯著高于對照組,PCNA基因的表達在24、48和72h時均顯著高于對照組。沉默HSP60組Cyclin D1基因和PCNA基因的表達在24、48和72h時均低于對照組,但差異不顯著。表明HSP60在白牦牛睪丸支持細胞增殖調控中的作用是正向調控。本研究通過差異蛋白質組學技術檢測不同發(fā)育階段睪丸蛋白質表達差異變化,篩選出HSP60與生殖相關,并對HSP60基因進行分子克隆及表達定位研究,原核表達系統(tǒng)誘導表達出融合蛋白His-HSP60,同時,對HSP60在睪丸支持細胞的增殖調控過程進行了初步研究,結果表明HSP60在白牦牛性機能的旺盛期是通過下丘腦-垂體-性腺軸來調控睪丸支持細胞增殖,進而影響睪丸發(fā)育,本實驗為進一步研究雄性白牦牛精子發(fā)生提供了基礎資料。
[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.

【學位授予單位】：甘肅農業(yè)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S823.85

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