miR-762调控猪未成熟支持细胞生长的分子机制探究及猪精液性状的遗传改良

发布时间：2018-06-29 13:22

本文选题：猪 + ST细胞　；参考：《华中农业大学》2016年博士论文

【摘要】：课题组前期通过芯片技术检测到180日龄大白猪(性成熟,睾丸中有各种类型生精细胞)和60日龄大白猪(睾丸中出现精母细胞)睾丸组织中有129个差异表达的microRNA(miRNA),其中miR-762差异极显著(P0.01)。ST细胞系为猪睾丸细胞系,是从80到90天胚胎期公猪睾丸中分离获得的一种细胞系,本课题在鉴定ST细胞的基础上,以mi R-762为主要研究对象,探究其在ST细胞增殖和精子发生过程中的功能。此外,为了探究公猪精液品质的改良方法,对相关基因中的分子标记位点在杜洛克猪、大白猪和长白猪公猪群体中进行精液品质性状关联分析,探寻有效的分子标记进行辅助选种。本课题主要研究结果如下:1、通过光学显微镜观察到ST细胞核中存在类似支持细胞核中特有的卫星核小体,推测ST细胞可能为猪睾丸支持细胞;经Feulgen染色和电镜观察,鉴定出ST细胞核中的确存在卫星核小体,表明ST细胞为支持细胞;免疫荧光检测到100%的ST细胞表达支持细胞的标记蛋白ABP和FASL。通过检测60日龄、180日龄大白猪睾丸组织和ST细胞中未成熟支持细胞标记基因KRT18和成熟支持细胞标记基因FSHR的mRNA表达,发现ST细胞只表达KRT18 mRNA,不表达FSHR mRNA,表明ST细胞为未成熟的支持细胞;免疫荧光检测到100%的ST细胞表达未成熟支持细胞的标记蛋白AMH,表明ST细胞为猪未成熟支持细胞。2、通过双荧光素酶报告系统验证了miR-762可与RNF4基因3‘UTR的651bp-677bp位点结合;通过荧光定量PCR和Western blotting检测发现miR-762可抑制ST细胞中RNF4基因的mRNA和蛋白质表达,表明RNF4为miR-762的靶基因。通过RNA22在线网站预测发现RNF4基因3‘UTR内的一个SNP的不同等位基因型使得miR-762与RNF4基因3‘UTR的结合位点数量发生改变,这一预测通过双荧光素酶报告系统得以验证,该SNP与公猪精液品质性状间存在显著关联。通过RTCA xCELLigence系统检测和流式细胞仪检测miR-762对ST细胞增殖、周期和凋亡的影响,结果显示miR-762可以促进ST细胞的增殖,促进ST细胞由DNA合成前期(G1期)向DNA合成期(S期)转变,抑制ST细胞的凋亡。利用相同的技术检测RNF4对ST细胞的影响,结果显示RNF4与miR-762的作用效果相似,说明miR-762不是直接通过RNF4对ST细胞生长产生影响。进一步检测发现mi R-762能够减少ST细胞核中雄激素受体(AR)的水平,且可以降低AR的转录调控活性,而RNF4对AR的作用与miR-762相反。免疫荧光结果显示AR与RNF4蛋白质在ST细胞核内共定位,表明miR-762可能通过作用于细胞核中的RNF4来调控AR的转录调控活性,从而影响ST细胞的生长。通过检测miR-762对DNA损伤标记蛋白γ-H2AX和DNA损伤修复相关基因的作用发现miR-762可以增强ST细胞的DNA损伤修复的能力。3、在杜洛克猪、大白猪和长白猪三个公猪群体中对DAZL、H2AFZ、RNF4、SPAG1、MMP9和NR4A1基因中的7个分子标记位点进行基因型分析,并与公猪精液品质性状进行关联分析,结果显示除了MMP9基因的SNP外,其他6个分子标记均与公猪精液品质性状存在显著关联(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.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S828

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