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

【摘要】：我國(guó)大部分綿羊?yàn)榧竟?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(jìn)養(yǎng)羊業(yè)發(fā)展,因此了解常年發(fā)情的分子機(jī)制,對(duì)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(guò)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(dá)基因。為了驗(yàn)證RNA-Seq結(jié)果,并探索BAD基因在綿羊發(fā)情啟動(dòng)中的作用機(jī)制,本研究對(duì)BAD基因進(jìn)行了功能鑒定。本研究以小尾寒羊卵巢上成熟卵泡為試驗(yàn)材料,用抽吸法分離卵泡顆粒細(xì)胞,構(gòu)建適合綿羊卵泡顆粒細(xì)胞生長(zhǎng)的培養(yǎng)體系,并用細(xì)胞免疫熒光技術(shù)檢測(cè)卵泡顆粒細(xì)胞上特異性表達(dá)的促卵泡刺激素受體(Follicle-stimulating hormone receptor,FSHR)蛋白,鑒定顆粒細(xì)胞純度;設(shè)計(jì)合成有效的BAD-siRNA,轉(zhuǎn)染原代綿羊卵泡顆粒細(xì)胞,以沉默BAD基因,用熒光定量PCR技術(shù)、Western-blot和細(xì)胞免疫熒光方法檢測(cè)BAD基因在mRNA水平和蛋白水平的表達(dá)變化,用放射免疫方法檢測(cè)顆粒細(xì)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(cè)表明BAD基因在綿羊不同情期卵巢中的表達(dá)與前期測(cè)序結(jié)果一致,在發(fā)情前期的表達(dá)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(xì)胞中FSHR陽(yáng)性率高達(dá)98%,說(shuō)明卵泡顆粒細(xì)胞純度較高。BAD-siRNA轉(zhuǎn)染顆粒細(xì)胞48小時(shí)后,熒光定量PCR檢測(cè)結(jié)果表明BAD基因在mRNA水平表達(dá)下降68.82%,Western-blot和細(xì)胞免疫熒光檢測(cè)結(jié)果表明BAD在蛋白水平表達(dá)下降明顯。放射免疫結(jié)果顯示顆粒細(xì)胞的孕酮分泌量隨BAD基因的表達(dá)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(cè)BAD基因可能通過(guò)調(diào)控孕酮的分泌來(lái)控制綿羊的發(fā)情啟動(dòng)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(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S826

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