HSP27基因的克隆及其在母牦牛生殖器官和孤雌激活胚胎的表达

发布时间：2018-06-30 18:50

本文选题：牦牛 + 热休克蛋白27　；参考：《甘肃农业大学》2016年硕士论文

【摘要】：为了克隆牦牛热休克蛋白27(heat shock proteins27,HSP27)基因,分析其生物学特性,研究正常生理条件下HSP27基因在繁殖周期母牦牛主要生殖器官以及不同时期孤雌激活胚胎中的表达差异,本试验采取卵泡期后期、黄体期后期同侧的卵巢、输卵管和子宫,以及妊娠期早期(胚胎约长2.3cm)妊娠侧的卵巢、输卵管和子宫组织,以其cDNA为模板,利用RT-PCR扩增牦牛HSP27基因,并将纯化的PCR产物克隆到pMDTM18-T Vector载体中进行测序;采用实时荧光定量RT-qPCR测定HSP27基因在繁殖周期母牦牛主要生殖器官和不同时期孤雌激活胚胎中的相对表达量;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达量;采用免疫组织化学SP法和间接免疫荧光染色分别研究HSP27蛋白在繁殖周期母牦牛主要生殖器官和不同时期孤雌激活胚胎中中的分布情况。研究结果表明:(1)成功克隆出包含完整编码区的牦牛HSP27基因序列,其编码区长450bp(GenBank登录号:KP716832)。同源性分析和系统进化树表明,牦牛HSP27基因与家牛和印度水牛的进化水平较为相近,同源性分别为99.8%和98.4%;与人类和猩猩的较远,同源性分别为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时期孤雌激活胚胎中均有表达,在不同繁殖周期母牦牛主要生殖器官中,牦牛HSP27基因在卵巢中的表达均极显著大于在子宫中的表达;在不同时期孤雌激活胚胎中,2~4细胞期胚胎中表达量最高,囊胚期胚胎中表达量最低,随着胚胎的发育,HSP27的表达量逐渐降低。(3)HSP27蛋白在繁殖周期母牦牛主要生殖器官中也均有表达,其中在卵泡期卵巢中的表达量最高,在黄体期子宫中的表达量最低;免疫组织化学结果显示:牦牛卵泡膜内层、颗粒层、卵丘、输卵管黏膜上皮、子宫内膜上皮和子宫腺中均有HSP27阳性表达。(4)间接免疫荧光染色显示:牦牛HSP27蛋白主要存在于细胞核内和细胞质中。本研究通过与其他物种HSP27基因的同源性对比分析,发现HSP27基因在进化中具有高度保守性,同时也存在种属特异性;通过对HSP27在繁殖周期母牦牛主要生殖器官和不同时期孤雌激活胚胎中的表达情况分析,推测出HSP27可能参与了母牦牛妊娠的调控,为进一步探讨HSP27在牦牛生殖过程中发挥的作用提供了参考资料。
[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.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S823.85

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