本文選題：BAD + siRNA��； 參考：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文

【摘要】：我國大部分綿羊為季節(jié)性發(fā)情品種,綿羊的季節(jié)性發(fā)情制約了養(yǎng)羊業(yè)生產(chǎn)效率。綿羊常年發(fā)情可以提高生產(chǎn)效率,促進養(yǎng)羊業(yè)發(fā)展,因此了解常年發(fā)情的分子機制,對改變綿羊季節(jié)性發(fā)情性狀及新品種培育具有重要作用。本課題組前期通過RNA-Seq篩選發(fā)現(xiàn)BAD基因是常年發(fā)情及季節(jié)性發(fā)情綿羊卵巢組織中的差異表達基因。為了驗證RNA-Seq結(jié)果,并探索BAD基因在綿羊發(fā)情啟動中的作用機制,本研究對BAD基因進行了功能鑒定。本研究以小尾寒羊卵巢上成熟卵泡為試驗材料,用抽吸法分離卵泡顆粒細胞,構(gòu)建適合綿羊卵泡顆粒細胞生長的培養(yǎng)體系,并用細胞免疫熒光技術(shù)檢測卵泡顆粒細胞上特異性表達的促卵泡刺激素受體(Follicle-stimulating hormone receptor,FSHR)蛋白,鑒定顆粒細胞純度;設(shè)計合成有效的BAD-siRNA,轉(zhuǎn)染原代綿羊卵泡顆粒細胞,以沉默BAD基因,用熒光定量PCR技術(shù)、Western-blot和細胞免疫熒光方法檢測BAD基因在mRNA水平和蛋白水平的表達變化,用放射免疫方法檢測顆粒細胞分泌孕酮和雌二醇的濃度變化。獲得以下結(jié)果:熒光定量PCR檢測表明BAD基因在綿羊不同情期卵巢中的表達與前期測序結(jié)果一致,在發(fā)情前期的表達量顯著高于(P0.05)發(fā)情期、發(fā)情間期和休情期。我們所獲得的原代綿羊卵泡顆粒細胞中FSHR陽性率高達98%,說明卵泡顆粒細胞純度較高。BAD-siRNA轉(zhuǎn)染顆粒細胞48小時后,熒光定量PCR檢測結(jié)果表明BAD基因在mRNA水平表達下降68.82%,Western-blot和細胞免疫熒光檢測結(jié)果表明BAD在蛋白水平表達下降明顯。放射免疫結(jié)果顯示顆粒細胞的孕酮分泌量隨BAD基因的表達下降而極顯著升高(P0.01);雌二醇的分泌量也有所下降但差異不顯著(P0.05)。綜合以上結(jié)果推測BAD基因可能通過調(diào)控孕酮的分泌來控制綿羊的發(fā)情啟動和維持發(fā)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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S826

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本文編號：2057079