本文選題：豬 + ST細胞��； 參考：《華中農(nóng)業(yè)大學》2016年博士論文

【摘要】：課題組前期通過芯片技術檢測到180日齡大白豬(性成熟,睪丸中有各種類型生精細胞)和60日齡大白豬(睪丸中出現(xiàn)精母細胞)睪丸組織中有129個差異表達的microRNA(miRNA),其中miR-762差異極顯著(P0.01)。ST細胞系為豬睪丸細胞系,是從80到90天胚胎期公豬睪丸中分離獲得的一種細胞系,本課題在鑒定ST細胞的基礎上,以mi R-762為主要研究對象,探究其在ST細胞增殖和精子發(fā)生過程中的功能。此外,為了探究公豬精液品質(zhì)的改良方法,對相關基因中的分子標記位點在杜洛克豬、大白豬和長白豬公豬群體中進行精液品質(zhì)性狀關聯(lián)分析,探尋有效的分子標記進行輔助選種。本課題主要研究結(jié)果如下:1、通過光學顯微鏡觀察到ST細胞核中存在類似支持細胞核中特有的衛(wèi)星核小體,推測ST細胞可能為豬睪丸支持細胞;經(jīng)Feulgen染色和電鏡觀察,鑒定出ST細胞核中的確存在衛(wèi)星核小體,表明ST細胞為支持細胞;免疫熒光檢測到100%的ST細胞表達支持細胞的標記蛋白ABP和FASL。通過檢測60日齡、180日齡大白豬睪丸組織和ST細胞中未成熟支持細胞標記基因KRT18和成熟支持細胞標記基因FSHR的mRNA表達,發(fā)現(xiàn)ST細胞只表達KRT18 mRNA,不表達FSHR mRNA,表明ST細胞為未成熟的支持細胞;免疫熒光檢測到100%的ST細胞表達未成熟支持細胞的標記蛋白AMH,表明ST細胞為豬未成熟支持細胞。2、通過雙熒光素酶報告系統(tǒng)驗證了miR-762可與RNF4基因3‘UTR的651bp-677bp位點結(jié)合;通過熒光定量PCR和Western blotting檢測發(fā)現(xiàn)miR-762可抑制ST細胞中RNF4基因的mRNA和蛋白質(zhì)表達,表明RNF4為miR-762的靶基因。通過RNA22在線網(wǎng)站預測發(fā)現(xiàn)RNF4基因3‘UTR內(nèi)的一個SNP的不同等位基因型使得miR-762與RNF4基因3‘UTR的結(jié)合位點數(shù)量發(fā)生改變,這一預測通過雙熒光素酶報告系統(tǒng)得以驗證,該SNP與公豬精液品質(zhì)性狀間存在顯著關聯(lián)。通過RTCA xCELLigence系統(tǒng)檢測和流式細胞儀檢測miR-762對ST細胞增殖、周期和凋亡的影響,結(jié)果顯示miR-762可以促進ST細胞的增殖,促進ST細胞由DNA合成前期(G1期)向DNA合成期(S期)轉(zhuǎn)變,抑制ST細胞的凋亡。利用相同的技術檢測RNF4對ST細胞的影響,結(jié)果顯示RNF4與miR-762的作用效果相似,說明miR-762不是直接通過RNF4對ST細胞生長產(chǎn)生影響。進一步檢測發(fā)現(xiàn)mi R-762能夠減少ST細胞核中雄激素受體(AR)的水平,且可以降低AR的轉(zhuǎn)錄調(diào)控活性,而RNF4對AR的作用與miR-762相反。免疫熒光結(jié)果顯示AR與RNF4蛋白質(zhì)在ST細胞核內(nèi)共定位,表明miR-762可能通過作用于細胞核中的RNF4來調(diào)控AR的轉(zhuǎn)錄調(diào)控活性,從而影響ST細胞的生長。通過檢測miR-762對DNA損傷標記蛋白γ-H2AX和DNA損傷修復相關基因的作用發(fā)現(xiàn)miR-762可以增強ST細胞的DNA損傷修復的能力。3、在杜洛克豬、大白豬和長白豬三個公豬群體中對DAZL、H2AFZ、RNF4、SPAG1、MMP9和NR4A1基因中的7個分子標記位點進行基因型分析,并與公豬精液品質(zhì)性狀進行關聯(lián)分析,結(jié)果顯示除了MMP9基因的SNP外,其他6個分子標記均與公豬精液品質(zhì)性狀存在顯著關聯(lián)(P0.05)。
[Abstract]:In the earlier period, we detected 129 differentially expressed microRNA (miRNA) in the testicular tissue of 180 day old white pigs (sexually mature, all kinds of spermatogenic cells in the testis) and 60 day old white pigs (spermatocytes appearing in the testis) by chip technology, of which the miR-762 difference (P0.01).ST cell line was a pig's testis cell line, from 80 to 90 days. A cell line obtained from the testicle of the boar during the embryonic period. On the basis of identification of ST cells, the main object of this study is to explore the function of MI R-762 in the proliferation and spermatogenesis of ST cells. In addition, in order to explore the improvement method of the quality of the semen of the boar, the molecular marker loci in the related genes are in the Duroc pig. The correlation analysis of semen quality traits in white and white pig population was carried out to explore effective molecular markers for auxiliary selection. The main results of this study were as follows: 1, through optical microscopy, it was observed that there was a similar satellite nucleus in the nucleus of ST by optical microscopy, and that ST cells might be porcine testis support cells; Feulgen staining and electron microscopy showed that there was a satellite nucleosome in the nucleus of ST, indicating that ST cells were supporting cells, and that the labeled protein ABP and FASL. expressing support cells of 100% ST cells were detected at 60 days of age, and the unmature supporting cell marker gene KRT18 and formation in the testis and ST cells of 180 day old white pigs and ST cells. The mRNA expression of the mature support cell marker gene FSHR showed that ST cells only expressed KRT18 mRNA and did not express FSHR mRNA, which indicated that ST cells were immature supporting cells, and that 100% of ST cells were detected by immunofluorescence to express the marker protein AMH of immature support cells, indicating that ST cells were pigs immature supporting cells.2, and the double luciferase reporter system was used. It was verified that miR-762 could bind to the 651bp-677bp locus of RNF4 gene 3 'UTR, and the mRNA and protein expression of RNF4 gene in ST cells was detected by fluorescence quantitative PCR and Western blotting, indicating that RNF4 is the target gene. The number of binding sites of miR-762 and RNF4 gene 3 'UTR was altered, and this prediction was verified by the dual luciferase reporter system. There was a significant association between the SNP and the quality traits of the boar semen. The effects of miR-762 on the proliferation, cycle and apoptosis of ST cells were detected by RTCA xCELLigence system detection and flow cytometry. The results showed that miR-762 could promote the proliferation of ST cells, promote the transformation of ST cells from the early stage of DNA synthesis (G1 phase) to the DNA synthesis phase (S phase) and inhibit the apoptosis of ST cells. The same technique was used to detect the effect of RNF4 on ST cells. Further detection found that MI R-762 can reduce the level of androgen receptor (AR) in the nucleus of ST and reduce the transcriptional regulation activity of AR, and the effect of RNF4 on AR is contrary to miR-762. The results of immunofluorescence show that AR and RNF4 proteins are Co located in the ST nucleus, suggesting that miR-762 may regulate the regulation by acting on the nuclei in the nucleus. Transcriptional regulation activity, thus affecting the growth of ST cells. By detecting the effect of miR-762 on DNA damage marker protein gamma -H2AX and DNA damage repair related genes, miR-762 can enhance the ability to improve the DNA damage repair of ST cells. In the three Duroc pigs, the big white and the long white pigs, DAZL, H2AFZ, RNF4, societies, etc. 7 molecular marker loci in the gene were genotyped and associated with the quality traits of the boar semen. The results showed that there was a significant association between the other 6 molecular markers and the quality traits of the boar semen (P0.05) except the SNP of the MMP9 gene.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S828

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本文編號：2082252