本文關(guān)鍵詞：不同發(fā)育期白牦牛睪丸蛋白質(zhì)組學(xué)分析及生殖相關(guān)候選基因HSP60生物學(xué)研究　出處：《甘肅農(nóng)業(yè)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 天祝白牦牛 睪丸蛋白質(zhì)組學(xué) 雙向電泳 HSP60 支持細(xì)胞

【摘要】：天祝白牦牛(Bos grunniens)是我國特有的牦牛品種,是當(dāng)?shù)啬撩裰饕纳a(chǎn)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(yán)重制約著白牦牛的生產(chǎn)能力。睪丸作為主要的生殖器官,影響著雄性動(dòng)物的繁殖性能,其主要功能是分泌激素、產(chǎn)生精子等,這些生理過程需要多種蛋白質(zhì)精確表達(dá)和調(diào)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(dá)蛋白質(zhì)將為解釋白牦牛睪丸發(fā)育和精子發(fā)生的分子機(jī)制提供參考,對提高白牦牛繁殖性能提供理論依據(jù)。本試驗(yàn)以1歲,2歲,4歲和8歲天祝白牦牛睪丸為實(shí)驗(yàn)材料,利用二維電泳和MALDI-TOF-TOF質(zhì)譜技術(shù)鑒定不同發(fā)育時(shí)期差異表達(dá)蛋白質(zhì),分析這些差異表達(dá)蛋白質(zhì)的功能及參與生物學(xué)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(jìn)行分子克隆及在下丘腦-垂體-睪丸軸表達(dá)定位研究,進(jìn)一步分析了HSP60對白牦牛睪丸支持細(xì)胞增殖的影響。本研究取得的主要結(jié)果如下:1、構(gòu)建了白牦牛睪丸蛋白質(zhì)表達(dá)圖譜,經(jīng)PDQuest 8.0.1分析發(fā)現(xiàn)白牦牛睪丸約有437個(gè)蛋白質(zhì)點(diǎn)。比較了不同發(fā)育階段差異蛋白質(zhì)表達(dá),結(jié)果顯示在4個(gè)年齡階段共有29個(gè)差異(p≤0.01)表達(dá)倍數(shù)在1.5倍以上的蛋白質(zhì)點(diǎn)。其中,2種蛋白質(zhì)隨年齡上調(diào),5種蛋白質(zhì)隨年齡下調(diào),3種蛋白質(zhì)在4歲前上調(diào)并隨后下調(diào),15種蛋白質(zhì)在2歲前上調(diào)而后下調(diào),4種蛋白質(zhì)隨年齡波動(dòng)。2、對鑒定出的差異蛋白進(jìn)行GO功能注釋,這些蛋白質(zhì)主要參與了“細(xì)胞過程、單有機(jī)體過程和新陳代謝過程”、“細(xì)胞、細(xì)胞器組成和胞外域”及“結(jié)合和催化活性”。亞細(xì)胞定位分析表明,鑒定蛋白主要位于細(xì)胞骨架(8個(gè)蛋白質(zhì)),細(xì)胞核(6個(gè)蛋白質(zhì)),線粒體(3個(gè)蛋白質(zhì))和細(xì)胞外基質(zhì)(2個(gè)蛋白質(zhì))。3、選擇2-DE檢測的2個(gè)差異表達(dá)蛋白質(zhì)(鈣結(jié)合蛋白、熱休克蛋白60)進(jìn)行免疫印跡分析,結(jié)果顯示所選蛋白的表達(dá)變化模式與2-DE結(jié)果基本一致,表明2-DE分析所得蛋白質(zhì)差異表達(dá)結(jié)果可靠。4、對篩選出的差異蛋白HSP60進(jìn)行分子克隆,發(fā)現(xiàn)白牦牛HSP60基因c DNA全長為2300bp,開放閱讀框?yàn)?722bp,編碼572個(gè)氨基酸。其理論分子量為60.977k Da、等電點(diǎn)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(jié)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(dá)及定位分析研究發(fā)現(xiàn),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(dá),其中下丘腦及垂體組織表達(dá)量高,睪丸組織表達(dá)量最低。免疫組化結(jié)果顯示,HSP60蛋白表達(dá)于白牦牛下丘腦組織的室旁核大細(xì)胞,室旁核小細(xì)胞和神經(jīng)角演網(wǎng),垂體組織的腺細(xì)胞,睪丸組織的精原細(xì)胞,精母細(xì)胞,支持細(xì)胞和間質(zhì)細(xì)胞,其中精子細(xì)胞中表達(dá)較弱。推斷HSP60參與雄性白牦牛生殖軸調(diào)控,并參與睪丸發(fā)育與精子發(fā)生。6、進(jìn)一步研究HSP60對原代培養(yǎng)的白牦牛睪丸支持細(xì)胞增殖的影響,通過構(gòu)建HSP60過表達(dá)載體p IRES2-EGFP-HSP60,合成靶向si RNA沉默HSP60,瞬時(shí)轉(zhuǎn)染支持細(xì)胞后,經(jīng)RT-q PCR檢測發(fā)現(xiàn)過表達(dá)組HSP60 m RNA在24、48和72h各時(shí)間點(diǎn)均上調(diào),沉默組HSP60 m RNA在24、48和72h各時(shí)間點(diǎn)均下調(diào)。四氮唑鹽法(MTS)檢測細(xì)胞的增殖情況,過表達(dá)HSP60組,支持細(xì)胞增殖率各時(shí)間點(diǎn)均顯著高于對照組。沉默HSP60組,支持細(xì)胞的增值率各時(shí)間點(diǎn)均低于對照組,但差異不顯著。RT-q PCR檢測細(xì)胞增殖標(biāo)志基因細(xì)胞周期蛋白D1(Cyclin D1)和增殖細(xì)胞核抗原(PCNA),發(fā)現(xiàn)過表達(dá)HSP60組Cyclin D1基因在48h時(shí)的表達(dá)顯著高于對照組,PCNA基因的表達(dá)在24、48和72h時(shí)均顯著高于對照組。沉默HSP60組Cyclin D1基因和PCNA基因的表達(dá)在24、48和72h時(shí)均低于對照組,但差異不顯著。表明HSP60在白牦牛睪丸支持細(xì)胞增殖調(diào)控中的作用是正向調(diào)控。本研究通過差異蛋白質(zhì)組學(xué)技術(shù)檢測不同發(fā)育階段睪丸蛋白質(zhì)表達(dá)差異變化,篩選出HSP60與生殖相關(guān),并對HSP60基因進(jìn)行分子克隆及表達(dá)定位研究,原核表達(dá)系統(tǒng)誘導(dǎo)表達(dá)出融合蛋白His-HSP60,同時(shí),對HSP60在睪丸支持細(xì)胞的增殖調(diào)控過程進(jìn)行了初步研究,結(jié)果表明HSP60在白牦牛性機(jī)能的旺盛期是通過下丘腦-垂體-性腺軸來調(diào)控睪丸支持細(xì)胞增殖,進(jìn)而影響睪丸發(fā)育,本實(shí)驗(yàn)為進(jìn)一步研究雄性白牦牛精子發(fā)生提供了基礎(chǔ)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(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S823.85

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