公山羊β-防御素104a表达特性及功能研究

发布时间：2018-05-29 07:26

  本文选题：公山羊 + 生殖器官　； 参考：《山西农业大学》2015年硕士论文

【摘要】：p-防御素家族蛋白是一种小的阳离子抗菌肽,β-防御素家族中越来越多基因被证明在哺乳动物的繁殖过程中也发挥着重要作用。公山羊β一防御素104a (goat beta-defensin 104a, gBD104a)是p-防御素家族的一员,目前关于gBD104a对公山羊生殖过程的影响鲜有报道。因此本试验旨在预测gBD104a蛋白质特性,研究其在公山羊不同发育阶段生殖器官及其他器官中的表达特性及其定位,以及在获能前后精子表面的定位,探索gBD104a生理功能,为进一步研究山羊繁殖调控提供理论基础和数据支持。试验利用在线软件对gBD104a蛋白序列进行生物信息学分析;分别采集7日龄,2、3、4、6、9和18月龄公山羊的睾丸和附睾以及18月龄公山羊上皮组织,利用QRT-PCR技术、Western-blotting技术、免疫组织化学法对睾丸、附睾中gBD104a mRNA和蛋白的表达及定位进行研究;并采集18月龄公山羊精液,体外获能处理后,利用免疫荧光技术对在获能前后的精子gBD104a的表达定位进行研究。研究结果如下：(1)生物信息学分析结果表明gBD104a基因CDS区全长324bp,编码107个氨基酸,第1-19个氨基酸为信号肽,第27-55个氨基酸为潜在的p-防御素构象区域,在C端有7个潜在的O-糖基化位点。(2) QRT-PCR结果表明BD104a在山羊机体内广泛表达,在生殖器官中表达量较高,在气管、皱胃、空肠、回肠等区域表达多于在其它器官中；附睾头和附睾体是gBD104a基因表达的主要场所；从7日龄至18月龄,gBD104a基因在各阶段附睾头中的表达呈现逐渐降低的趋势,在附睾体中的表达呈现逐渐增加的趋势。Western-blotting结果显示gBD104a蛋白在附睾头和附睾体中的表达趋势与nRNA的表达趋势相似。18月龄山羊睾丸附睾免疫荧光试验结果显示在附睾头和体部的假复层纤毛柱状上皮细胞检测到较强的gBD104a阳性信号,在附睾尾部的纤毛柱状上皮细胞也检测到较强的阳性信号,在睾丸器官内没有检测到明显的阳性信号。gBD104a基因的这种时空差异性表达,推测gBD104a对精子成熟有重要意义。(3)精子免疫荧光实验结果显示gBD104a包裹在精子头部顶体上和精子尾部中段线粒体上,精子尾部的主段和末端没有gBD104a的包裹,获能后gBD104a从精子头部顶体部分脱落。表明gBD104a参与了精子获能过程。
[Abstract]:Pdefensin family protein is a small cationic antimicrobial peptide. More and more genes in 尾 -defensin family have been proved to play an important role in mammalian reproduction. Goat 尾 -defensin 104a / goat beta-defensin 104a, gBD104a) is a member of pdefensin family. There are few reports on the effect of gBD104a on the reproductive process of male goat. Therefore, the purpose of this study was to predict the characteristics of gBD104a protein, to study its expression and localization in male goat reproductive organs and other organs at different stages of development, and to explore the physiological function of gBD104a on the surface of spermatozoa before and after capacitation. It provides theoretical basis and data support for further study on goat reproduction regulation. The gBD104a protein sequence was analyzed by online software, the testis and epididymis of 7-day-old male goats were collected by Western-blotting, and the epididymis and epididymis of 18-month-old male goats were collected by Western-blotting. Immunohistochemical method was used to study the expression and localization of gBD104a mRNA and protein in testis and epididymis. The expression localization of gBD104a in spermatozoa before and after capacitation was studied by immunofluorescence technique. The results are as follows: (1) Bioinformatics analysis shows that the CDS region of gBD104a gene is 324bpp, encoding 107 amino acids, the 1-19 amino acids are signal peptides, and the 27-55 amino acids are potential P-defensin conformation regions. There were 7 potential O-glycosylation sites in C-terminal. The results of QRT-PCR showed that BD104a was widely expressed in goat body and higher in reproductive organs, and expressed more in trachea, abomasum, jejunum and ileum than in other organs. The head of epididymis and the body of epididymis were the main sites for the expression of gBD104a gene, and the expression of gBD104a gene in the head of epididymis gradually decreased from 7 days to 18 months of age. The results of Western-blotting showed that the expression trend of gBD104a protein in epididymal head and epididymis was similar to that of nRNA. 18 months old goat testicular epididymis immunofluorescence assay showed that the expression trend was similar to that in epididymal epididymis. A strong gBD104a positive signal was detected in the pseudostratified ciliated columnar epithelial cells of the head and body. Strong positive signals were also detected in the ciliated columnar epithelial cells of the tail of the epididymis, and there was no significant positive signal in the testis. The spatiotemporal difference of the expression of the gBD104a gene was found in the testis. It is speculated that gBD104a plays an important role in sperm maturation. The results of sperm immunofluorescence assay showed that gBD104a was encapsulated on the acrosome of the head and the mitochondria of the middle part of the sperm tail, and there was no gBD104a in the main segment and the end of the tail of the sperm. After capacitation, gBD104a was partially removed from the acrosome of the sperm head. The results showed that gBD104a was involved in sperm capacitation.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S827

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