本文選題：公山羊 + 生殖器官��； 參考：《山西農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：p-防御素家族蛋白是一種小的陽(yáng)離子抗菌肽,β-防御素家族中越來(lái)越多基因被證明在哺乳動(dòng)物的繁殖過(guò)程中也發(fā)揮著重要作用。公山羊β一防御素104a (goat beta-defensin 104a, gBD104a)是p-防御素家族的一員,目前關(guān)于gBD104a對(duì)公山羊生殖過(guò)程的影響鮮有報(bào)道。因此本試驗(yàn)旨在預(yù)測(cè)gBD104a蛋白質(zhì)特性,研究其在公山羊不同發(fā)育階段生殖器官及其他器官中的表達(dá)特性及其定位,以及在獲能前后精子表面的定位,探索gBD104a生理功能,為進(jìn)一步研究山羊繁殖調(diào)控提供理論基礎(chǔ)和數(shù)據(jù)支持。試驗(yàn)利用在線軟件對(duì)gBD104a蛋白序列進(jìn)行生物信息學(xué)分析;分別采集7日齡,2、3、4、6、9和18月齡公山羊的睪丸和附睪以及18月齡公山羊上皮組織,利用QRT-PCR技術(shù)、Western-blotting技術(shù)、免疫組織化學(xué)法對(duì)睪丸、附睪中g(shù)BD104a mRNA和蛋白的表達(dá)及定位進(jìn)行研究;并采集18月齡公山羊精液,體外獲能處理后,利用免疫熒光技術(shù)對(duì)在獲能前后的精子gBD104a的表達(dá)定位進(jìn)行研究。研究結(jié)果如下：(1)生物信息學(xué)分析結(jié)果表明gBD104a基因CDS區(qū)全長(zhǎng)324bp,編碼107個(gè)氨基酸,第1-19個(gè)氨基酸為信號(hào)肽,第27-55個(gè)氨基酸為潛在的p-防御素構(gòu)象區(qū)域,在C端有7個(gè)潛在的O-糖基化位點(diǎn)。(2) QRT-PCR結(jié)果表明BD104a在山羊機(jī)體內(nèi)廣泛表達(dá),在生殖器官中表達(dá)量較高,在氣管、皺胃、空腸、回腸等區(qū)域表達(dá)多于在其它器官中；附睪頭和附睪體是gBD104a基因表達(dá)的主要場(chǎng)所；從7日齡至18月齡,gBD104a基因在各階段附睪頭中的表達(dá)呈現(xiàn)逐漸降低的趨勢(shì),在附睪體中的表達(dá)呈現(xiàn)逐漸增加的趨勢(shì)。Western-blotting結(jié)果顯示gBD104a蛋白在附睪頭和附睪體中的表達(dá)趨勢(shì)與nRNA的表達(dá)趨勢(shì)相似。18月齡山羊睪丸附睪免疫熒光試驗(yàn)結(jié)果顯示在附睪頭和體部的假?gòu)?fù)層纖毛柱狀上皮細(xì)胞檢測(cè)到較強(qiáng)的gBD104a陽(yáng)性信號(hào),在附睪尾部的纖毛柱狀上皮細(xì)胞也檢測(cè)到較強(qiáng)的陽(yáng)性信號(hào),在睪丸器官內(nèi)沒(méi)有檢測(cè)到明顯的陽(yáng)性信號(hào)。gBD104a基因的這種時(shí)空差異性表達(dá),推測(cè)gBD104a對(duì)精子成熟有重要意義。(3)精子免疫熒光實(shí)驗(yàn)結(jié)果顯示gBD104a包裹在精子頭部頂體上和精子尾部中段線粒體上,精子尾部的主段和末端沒(méi)有g(shù)BD104a的包裹,獲能后gBD104a從精子頭部頂體部分脫落。表明gBD104a參與了精子獲能過(guò)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.
【學(xué)位授予單位】：山西農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S827

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