梅花鹿Ghrelin cDNA全长序列的克

发布时间：2018-06-14 11:32

本文选题：Ghrelin + 梅花鹿　；参考：《内蒙古农业大学》2017年硕士论文

【摘要】：1999年Ghrelin作为一个内在生长激素促分泌素受体(Growth hormone secretagogue receptor,GHS-R)的配体被发现,当 Ghrelin 与GHS-R结合后可以刺激生长激素(Growth hormone,GH)的分泌。研究表明Ghrelin在生物体内除能调节GH分泌外还有增加食欲、调节能量平衡和促进胃酸分泌等作用。当前国内外对Ghrelin的研究主要集中在人、啮齿动物和家畜上,极少有梅花鹿方面的相关报道,只有本课题组获得过梅花鹿Ghrelin cDNA的部分序列。为了得到梅花鹿Ghrelin基因cDNA的全长序列,同时检测Ghrelin在动物体内的表达与分布情况。本试验利用cDNA末端快速扩增技术(RACE)、反转录-聚合酶链式反应(RT-PCR)、实时荧光定量PCR(RT-qPCR)及免疫组织化学等技术来进行研究。试验在本课题组已获取的梅花鹿Ghrelin cDNA部分片段的基础上进行3'和5' RACE。结果成功克隆出了梅花鹿Ghrelin cDNA的3'及5'末端序列,通过与已知序列的拼接首次得到了 539 bp的梅花鹿Ghrelin cDNA全长序列,其中3'非翻译区(3' UTR)为128 bp,5' UTR为46 bp,开放阅读框(ORF)为351 bp,该ORF编码116个氨基酸残基;RT-PCR和RT-qPCR结果显示:GhrelinmRNA在梅花鹿食管、瘤胃、网胃、瓣胃、皱胃、十二指肠、空肠、回肠、盲肠、结肠、肝、胰、肺、脾、肾、卵巢、膀胱、心、输尿管、子宫、胸腺、淋巴结、心内膜、垂体前叶、肾上腺、下丘脑、舌和甲状腺等组织内均有表达,其中在皱胃的表达量显著高于其他器官(P0.05);免疫组织化学结果发现:Ghrelin的免疫阳性细胞在消化管壁内分布较多,且主要定位于黏膜层和黏膜下层,在食管、皱胃、十二指肠和盲肠的肌层也可见免疫阳性细胞;在实质器官(肝、胰、肺、脾、肾、垂体前叶、甲状腺和卵巢)内也均有Ghrelin免疫阳性细胞的分布,但相对较少。以上研究结果的获得丰富了 Ghrelin的相关生物学信息,为深入研究Ghrelin在梅花鹿体内的生理功能奠定了基础。
[Abstract]:In 1999, ghrelin was found as a ligand of growth hormone secretagogue receptor GHS-R, which can stimulate the secretion of growth hormone (GH) when ghrelin binds to GHS-R. It has been shown that ghrelin can not only regulate GH secretion, but also increase appetite, regulate energy balance and promote gastric acid secretion in vivo. At present, researches on ghrelin mainly focus on human, rodent and domestic animals. There are few reports on sika deer. Only our team has obtained partial sequence of ghrelin cDNA of sika deer. In order to obtain the full-length cDNA of ghrelin gene of sika deer, the expression and distribution of ghrelin in animals were detected. In this study, rapid amplification technique of cDNA terminal, reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative PCR (RT-qPCR) and immunohistochemistry were used to study the results. The experiments were carried out on the basis of the partial cDNA fragments of ghrelin from sika deer obtained by our research group for 3 'and 5' RACEs. Results the 3 'and 5' terminal sequences of ghrelin cDNA of sika deer were cloned successfully. The full length of ghrelin cDNA of sika deer was obtained by splicing with known sequence for the first time. The ORF encodes 116 amino acid residues: RT-PCR and RT-qPCR. The results of RT-PCR and RT-qPCR showed that the expression of 10 ghrelin mRNA was detected in esophagus, rumen, reticulum, valve, rumen, duodenum, jejunum, ileum, cecum of sika deer. Colon, liver, pancreas, lung, spleen, kidney, ovary, bladder, heart, ureter, uterus, thymus, lymph node, endocardium, anterior pituitary lobe, adrenal gland, hypothalamus, tongue and thyroid were all expressed. The expression level in the abomasum was significantly higher than that in the other organs (P0.05A). The immunohistochemical results showed that the immunoreactive cells of 1% ghrelin were mainly located in the mucosal layer and submucous layer, and in the esophagus and abomasum. Immunoreactive cells were also found in the muscular layer of duodenum and cecum, and ghrelin immunoreactive cells were also found in parenchymal organs (liver, pancreas, lung, spleen, kidney, anterior pituitary lobe, thyroid gland and ovary). The above results enrich the biological information of ghrelin and lay a foundation for further study of ghrelin's physiological function in sika deer.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S825

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