绵羊BAD基因影响常年发情性状的功能研究

发布时间：2018-06-23 12:10

本文选题：BAD + siRNA　；参考：《中国农业科学院》2015年硕士论文

【摘要】：我国大部分绵羊为季节性发情品种,绵羊的季节性发情制约了养羊业生产效率。绵羊常年发情可以提高生产效率,促进养羊业发展,因此了解常年发情的分子机制,对改变绵羊季节性发情性状及新品种培育具有重要作用。本课题组前期通过RNA-Seq筛选发现BAD基因是常年发情及季节性发情绵羊卵巢组织中的差异表达基因。为了验证RNA-Seq结果,并探索BAD基因在绵羊发情启动中的作用机制,本研究对BAD基因进行了功能鉴定。本研究以小尾寒羊卵巢上成熟卵泡为试验材料,用抽吸法分离卵泡颗粒细胞,构建适合绵羊卵泡颗粒细胞生长的培养体系,并用细胞免疫荧光技术检测卵泡颗粒细胞上特异性表达的促卵泡刺激素受体(Follicle-stimulating hormone receptor,FSHR)蛋白,鉴定颗粒细胞纯度;设计合成有效的BAD-siRNA,转染原代绵羊卵泡颗粒细胞,以沉默BAD基因,用荧光定量PCR技术、Western-blot和细胞免疫荧光方法检测BAD基因在mRNA水平和蛋白水平的表达变化,用放射免疫方法检测颗粒细胞分泌孕酮和雌二醇的浓度变化。获得以下结果:荧光定量PCR检测表明BAD基因在绵羊不同情期卵巢中的表达与前期测序结果一致,在发情前期的表达量显著高于(P0.05)发情期、发情间期和休情期。我们所获得的原代绵羊卵泡颗粒细胞中FSHR阳性率高达98%,说明卵泡颗粒细胞纯度较高。BAD-siRNA转染颗粒细胞48小时后,荧光定量PCR检测结果表明BAD基因在mRNA水平表达下降68.82%,Western-blot和细胞免疫荧光检测结果表明BAD在蛋白水平表达下降明显。放射免疫结果显示颗粒细胞的孕酮分泌量随BAD基因的表达下降而极显著升高(P0.01);雌二醇的分泌量也有所下降但差异不显著(P0.05)。综合以上结果推测BAD基因可能通过调控孕酮的分泌来控制绵羊的发情启动和维持发情。
[Abstract]:Most sheep in China are seasonal estrous breeds. Seasonal estrus of sheep restricts the production efficiency of sheep industry. Perennial estrus of sheep can improve production efficiency and promote the development of sheep industry. Therefore, understanding the molecular mechanism of perennial estrus plays an important role in changing seasonal estrous traits and breeding new breeds of sheep. By RNA-Seq screening, we found that bad gene is a differential expression gene in ovaries of perennial estrus and seasonal estrus sheep. In order to verify the RNA-Seq results and explore the role of bad gene in estrus initiation in sheep, the function of bad gene was identified. In this study, mature follicles on ovaries of small tail Han sheep were used as experimental materials, follicular granulosa cells were isolated by suction method, and a culture system suitable for the growth of sheep follicular granulosa cells was constructed. The specific expression of Follicle-stimulating hormone receptor FSHR (FSHR) protein on follicular granulosa cells was detected by cellular immunofluorescence technique, and the purity of granulosa cells was identified, and the effective BAD-siRNAs were designed and synthesized and transfected into sheep follicular granulosa cells to silence the bad gene. Western-blot and immunofluorescence were used to detect the expression of bad gene at mRNA and protein levels, and the concentrations of progesterone and estradiol secreted by granulosa cells were detected by radioimmunoassay. The results showed that the expression of bad gene in ovaries of sheep was consistent with the results of early sequencing. The expression of bad gene in the early estrus was significantly higher than that in estrus, estrus and estrus. The positive rate of FSHR in primary sheep follicular granulosa cells was as high as 98, which indicated that the purity of follicular granulosa cells was high. BAD-siRNA was transfected into granulosa cells 48 hours after transfection. The results of fluorescence quantitative PCR showed that the expression of bad gene decreased at the mRNA level. The results of Western-blot and cellular immunofluorescence showed that the expression of bad gene decreased significantly at the protein level. Radioimmunoassay showed that the secretion of progesterone in granulosa cells increased significantly with the decrease of bad gene expression (P0.01), while the secretion of estradiol also decreased, but the difference was not significant (P0.05). These results suggest that the bad gene may regulate the secretion of progesterone to control the initiation and maintenance of estrus in sheep.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826

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