公鸡弱精症相关候选基因的筛选及功能研究

发布时间：2018-05-13 16:27

本文选题：弱精症 + DGE　；参考：《扬州大学》2015年硕士论文

【摘要】：在家禽生产中,据不完全统计,每年有5-12%的种公鸡因产精能力低下遭到淘汰。中国的大部分地方鸡品种更是存在繁殖效率低下的显著弱点,其公母配比高于专门化高产品种一倍以上,是产业化程度不高,良种繁育体系不健全的重要原因之一。为了探究公鸡弱精症的发病机理,本课题前期应用数字基因表达谱技术(Digital Gene Expression,DGE)构建了无精症公鸡与正常公鸡的睾丸差异表达基因数据库。本研究用生物信息学方法重新分析DGE数据库,挑选了与公鸡弱精症相关的6个候选基因(COX7B.PTGDS, hPGDS、SPAG6、WNT2、CCNF),从300只42周龄的北京油鸡种公鸡的大群体中,进行了为期4周的精液品质检测,筛出弱精及正常个体各15只。采用RT-qPCR对这6个候选基因在两组个体的睾丸组织中的表达量进行了相对定量检测,来验证DGE结果的可靠性。从验证的差异表达基因中挑选了CCNF (cyclin F),同时以鸡精原干细胞-SSCs、睾丸支持细胞、DF1细胞为实验模型,进行了三种细胞CCNF基因表达量的检测；采用RNAi技术,构建的4条慢病毒靶向干扰载体在三种细胞上做了转染效率和干扰效率的检测,筛选出最佳转染效率时病毒滴度/MOI值和最优干扰序列,应用RT-qPC R技术检测转染后24 h、48 h、72 h、96 h模式细胞中CCNF基因的mRNA水平的表达量；用CCK8法检测慢病毒载体siR-1侵染后模式细胞在不同时间点细胞周期的变化,为探索公鸡弱精症的发病机理的深入研究和最终寻找到致病潜在标志物做了基础铺垫。结果表明：1. RT-qPCR对DGE中挑选的与弱精相关的6个候选基因的定量结果发现,COX7B、PTGDS在弱精组个体睾丸的表达量显著高于正常个体(P0.01), WNT2、hPGDS、CCNF在弱精组个体的表达量显著低于正常个体(P0.05), SPAG6在两组睾丸中的表达量没有显著差异,该结果与DGE的结果相吻合,说明DGE结果可靠,确立了COX7B、PTGDS, WNT2、hPGDS、CCNF这5个基因可作为公鸡弱精症的重要候选基因。2.从前期初步验证的候选基因中挑选了CCNF基因,SSCs、睾丸支持细胞、DF1细胞的定量检测结果显示CCNF在这三种细胞的表达量相当,说明CCNF基因具有广泛表达特性。3.在三种模式细胞上的转染效率检测和干扰载体的筛选结果发现,当病毒液与培养基以1：19即滴度为5×106TU/mL、MOI=5,此条件下的慢病毒液侵染DF1和支持细胞效率可达60%,而在SSCs上的转染效率只有20%,说明慢病毒载体对SSCs侵染效果不佳。进而在DF1和支持细胞上的CCNF靶向siRNA干扰的定量检测结果显示siR-1转染两种细胞后在72 h和96 hCCNF基因表达量极显著下调(P0.01),说明构建的干扰载体能有效沉默这两种细胞中CCNF基因的表达。4.在两种细胞上四个时间点CCK8检测细胞活力的结果发现,空白组和阴性对照组细胞活力均没有明显变化；与空白组相比,在siR-1干扰的支持细胞中,72 h和96 h这两个时间点的细胞活力极显著下降(P0.01),而在siR-1干扰的DF1细胞中,72 h和96 h这两个时间点的细胞活力显著下降(P0.05)；两种细胞在相同干扰条件下,支持细胞增值变化强于DF1细胞。结果说明CCNF基因参与了细胞的增殖的调控,且随着其表达量的降低,细胞增殖也会随之下降或停滞。
[Abstract]:In poultry production, according to incomplete statistics, 5-12% cocks are eliminated every year because of low sperm production capacity. Most of the local chicken breeds in China have a significant weakness in the low reproductive efficiency, and the proportion of their male and female parents is more than twice that of the specialized high-yield varieties, which is an important reason for the low industrialization and the imperfect breeding system. In order to explore the pathogenesis of cock asthenospermia, Digital Gene Expression (DGE) was used to construct a database of differentially expressed genes in testis of azoospermia cocks and normal cocks in the earlier period of this study. This study reanalyzed the DGE database by bioinformatics, and selected 6 related to the cock asthenospermia. The candidate genes (COX7B.PTGDS, hPGDS, SPAG6, WNT2, CCNF) were tested for 4 weeks of semen quality, sifting weak sperm and 15 normal individuals from 300 42 weeks old Beijing Chicken Rooster. RT-qPCR was used to detect the expression of the 6 candidate genes in the testis of two groups of individuals. The reliability of the DGE results was verified. CCNF (cyclin F) was selected from the verifying differentially expressed genes. At the same time, three kinds of cell CCNF gene expression were detected with Chicken Spermatogonial Stem Cells -SSCs, testis support cells and DF1 cells as experimental models. The 4 lentivirus targeting interference vectors constructed by RNAi Technology were transformed on three kinds of cells. Detection of infection efficiency and interference efficiency, the /MOI value of virus titer and the optimal interference sequence were screened out at the best transfection efficiency. RT-qPC R technique was used to detect the expression of mRNA level of CCNF gene in 24 h, 48 h, 72 h, 96 h mode cells; and CCK8 method was used to detect the cell cycle of the lentivirus carrier after the infected mode cells at different time points. In order to explore the pathogenesis of the cock asthenospermia and to find the underlying marker for the disease, the results showed that the quantitative results of 1. RT-qPCR on the 6 candidate genes associated with the weak spermatogenesis in DGE showed that the expression of COX7B and PTGDS in the spermatozoa of the asthenosin group was significantly higher than that of the normal individuals (P0.01). The expression of WNT2, hPGDS and CCNF in the asthenospermia group was significantly lower than that of the normal individuals (P0.05). There was no significant difference in the expression of SPAG6 in the two groups of testis. The results were consistent with the results of DGE, indicating that the results of DGE were reliable, and COX7B, PTGDS, WNT2, hPGDS, and CCNF these 5 genes could be used as an important candidate for the cock asthenospermia. CCNF gene, SSCs, testis support cells were selected in the preliminary candidate genes, and the quantitative detection results of DF1 cells showed that the expression of CCNF in these three cells was equal, indicating that the CCNF gene has extensive expression characteristics of.3. in the detection of transfection efficiency on the three mode cells and screening results of the stem disturbance carrier, when the virus fluid and the culture are cultured. At 1:19, the titer was 5 x 106TU/mL, MOI=5, and the lentivirus infecting DF1 and supporting cell efficiency under this condition could reach 60%, while the transfection efficiency on SSCs was only 20%, indicating that the lentivirus carrier was not effective to SSCs infection. Then the quantitative detection results of CCNF targeting siRNA on DF1 and support cells showed siR-1 transfection of two cells. The expression of 72 h and 96 hCCNF genes was significantly down (P0.01), indicating that the constructed interference carrier could effectively silence the expression of CCNF gene in the two cells, and.4. detected cell viability at four time point CCK8 on two cells, and found that the cell vitality of the blank group and the negative control group had no significant changes; compared with the blank group, the cell viability was in Si. In the support cells of R-1 interference, the cell viability of 72 h and 96 h time points decreased significantly (P0.01), while the cell vitality of 72 h and 96 h in siR-1 interfered DF1 cells decreased significantly (P0.05); the two cells were stronger than DF1 cells under the same interference conditions. The result indicated that CCNF gene was involved. The regulation of cell proliferation, and with the decrease of its expression, cell proliferation will also decline or stagnate.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31

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