本文選題：牦牛 + 熱休克蛋白27��； 參考：《甘肅農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：為了克隆牦牛熱休克蛋白27(heat shock proteins27,HSP27)基因,分析其生物學(xué)特性,研究正常生理條件下HSP27基因在繁殖周期母牦牛主要生殖器官以及不同時期孤雌激活胚胎中的表達(dá)差異,本試驗采取卵泡期后期、黃體期后期同側(cè)的卵巢、輸卵管和子宮,以及妊娠期早期(胚胎約長2.3cm)妊娠側(cè)的卵巢、輸卵管和子宮組織,以其cDNA為模板,利用RT-PCR擴增牦牛HSP27基因,并將純化的PCR產(chǎn)物克隆到pMDTM18-T Vector載體中進(jìn)行測序;采用實時熒光定量RT-qPCR測定HSP27基因在繁殖周期母牦牛主要生殖器官和不同時期孤雌激活胚胎中的相對表達(dá)量;采用western-blot法研究HSP27蛋白在繁殖周期母牦牛主要生殖器官中的表達(dá)量;采用免疫組織化學(xué)SP法和間接免疫熒光染色分別研究HSP27蛋白在繁殖周期母牦牛主要生殖器官和不同時期孤雌激活胚胎中中的分布情況。研究結(jié)果表明:(1)成功克隆出包含完整編碼區(qū)的牦牛HSP27基因序列,其編碼區(qū)長450bp(GenBank登錄號:KP716832)。同源性分析和系統(tǒng)進(jìn)化樹表明,牦牛HSP27基因與家牛和印度水牛的進(jìn)化水平較為相近,同源性分別為99.8%和98.4%;與人類和猩猩的較遠(yuǎn),同源性分別為85.5%和85.3%。(2)牦牛HSP27基因在繁殖周期母牦牛主要生殖器官和不同時期孤雌激活胚胎中均有表達(dá),在不同繁殖周期母牦牛主要生殖器官中,牦牛HSP27基因在卵巢中的表達(dá)均極顯著大于在子宮中的表達(dá);在不同時期孤雌激活胚胎中,2~4細(xì)胞期胚胎中表達(dá)量最高,囊胚期胚胎中表達(dá)量最低,隨著胚胎的發(fā)育,HSP27的表達(dá)量逐漸降低。(3)HSP27蛋白在繁殖周期母牦牛主要生殖器官中也均有表達(dá),其中在卵泡期卵巢中的表達(dá)量最高,在黃體期子宮中的表達(dá)量最低;免疫組織化學(xué)結(jié)果顯示:牦牛卵泡膜內(nèi)層、顆粒層、卵丘、輸卵管黏膜上皮、子宮內(nèi)膜上皮和子宮腺中均有HSP27陽性表達(dá)。(4)間接免疫熒光染色顯示:牦牛HSP27蛋白主要存在于細(xì)胞核內(nèi)和細(xì)胞質(zhì)中。本研究通過與其他物種HSP27基因的同源性對比分析,發(fā)現(xiàn)HSP27基因在進(jìn)化中具有高度保守性,同時也存在種屬特異性;通過對HSP27在繁殖周期母牦牛主要生殖器官和不同時期孤雌激活胚胎中的表達(dá)情況分析,推測出HSP27可能參與了母牦牛妊娠的調(diào)控,為進(jìn)一步探討HSP27在牦牛生殖過程中發(fā)揮的作用提供了參考資料。
[Abstract]:In order to clone the heat shock protein 27 (heat shock proteins27 (HSP27) gene and analyze its biological characteristics, the expression of HSP27 gene in the main reproductive organs and parthenogenetic embryos of female yak during reproductive cycle was studied under normal physiological conditions. The ovary, fallopian tube and uterus, and the ovary, fallopian tube and uterus of early pregnancy (about the length of embryo) were used in this experiment. The HSP27 gene of yak was amplified by RT-PCR using the cDNA of the ovary, fallopian tube and uterus at the later stage of follicle and luteal phase. The purified PCR product was cloned into pMDTM18-T vector for sequencing, and the relative expression of HSP27 gene in the main reproductive organs of female yak and parthenogenetic activated embryos at different stages of reproductive cycle was determined by real-time fluorescence quantitative RT-qPCR. The expression of HSP27 protein in the main reproductive organs of female yak during reproductive cycle was studied by western-blot method. The distribution of HSP27 protein in the main reproductive organs and parthenogenetic activated embryos of female yaks during reproductive cycle was studied by immunohistochemical SP method and indirect immunofluorescence staining. The results showed that: (1) Yak HSP27 gene sequence containing complete coding region was cloned successfully and its coding region length 450bp (GenBank accession number: KP716832) was obtained. Homology analysis and phylogenetic tree showed that the phylogenetic level of HSP27 gene of yak was similar to that of domestic cattle and Indian buffalo, and the homology was 99.8% and 98.4%, respectively. The homology of HSP27 gene was 85.5% and 85.3% respectively. (2) the HSP27 gene was expressed in the main reproductive organs of female yak during reproductive cycle and in parthenogenetic activated embryos at different stages, and in the main reproductive organs of female yaks in different reproductive cycles. The expression of HSP27 gene in ovaries was significantly higher than that in uterus, and the highest expression level was found in the embryos at different stages of parthenogenetic activation, and the lowest in blastocyst embryos. With the development of embryo, the expression of HSP27 decreased gradually. (3) HSP27 protein was also expressed in the main reproductive organs of female yak during reproductive cycle, the highest in follicular ovary and the lowest in luteal uterus; Immunohistochemical results showed that the inner layer of follicular membrane, granular layer, cumulus, oviduct mucosal epithelium, (4) indirect immunofluorescence staining showed that HSP27 protein mainly existed in nucleus and cytoplasm of yak. By comparing the homology of HSP27 gene with other species, we found that HSP27 gene is highly conserved in evolution and species specific. By analyzing the expression of HSP27 in the main reproductive organs and parthenogenetic activated embryos of female yak during reproductive cycle, it is inferred that HSP27 may be involved in the regulation of female yak pregnancy. It provides reference for further study on the role of HSP27 in the reproductive process of yak.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S823.85

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 溫澤星;余四九;翟羽佳;楊琨;劉鵬剛;何俊峰;崔燕;;HSPA2在牦牛不同組織器官中的表達(dá)差異[J];中國農(nóng)業(yè)科學(xué);2014年17期

2 任碧瓊;余平;;eHSP的研究進(jìn)展[J];國際病理科學(xué)與臨床雜志;2011年05期

3 王汝;余四九;崔燕;;幼齡牦牛甲狀腺的顯微結(jié)構(gòu)和超微結(jié)構(gòu)觀察[J];中國獸醫(yī)科學(xué);2009年04期

4 郭憲;閻萍;梁春年;裴杰;曾玉峰;包鵬甲;;中國牦牛業(yè)發(fā)展現(xiàn)狀及對策分析[J];中國牛業(yè)科學(xué);2009年02期

5 字向東;鐘金城;;牦牛繁殖科學(xué)研究進(jìn)展[J];西南民族大學(xué)學(xué)報(自然科學(xué)版);2007年01期

6 陳秋生;馮霞;姜生成;;牦牛肺臟高原適應(yīng)性的結(jié)構(gòu)研究[J];中國農(nóng)業(yè)科學(xué);2006年10期

7 鐘金城;趙素君;陳智華;馬志杰;;牦牛品種的遺傳多樣性及其分類研究[J];中國農(nóng)業(yè)科學(xué);2006年02期

8 孫敏;馬月輝;葉紹輝;;生物信息學(xué)研究進(jìn)展[J];家畜生態(tài)學(xué)報;2006年01期

9 王海鴻,雷仲仁;昆蟲熱休克蛋白的研究進(jìn)展[J];中國農(nóng)業(yè)科學(xué);2005年10期

10 金歡勝,吳雄飛,金錫御,楊清浩,倪兵;HSP27基因的克隆與表達(dá)[J];第三軍醫(yī)大學(xué)學(xué)報;2005年09期

相關(guān)碩士學(xué)位論文 前3條

1 劉珊;Hsp27對小鼠植入前胚胎發(fā)育作用的研究[D];南京醫(yī)科大學(xué);2013年

2 王旭光;綿羊卵母細(xì)胞體外成熟和孤雌激活的研究[D];新疆農(nóng)業(yè)大學(xué);2003年

3 張t;牛卵母細(xì)胞孤雌激活與體外受精[D];西北農(nóng)林科技大學(xué);2002年

，

本文編號：2086498