發(fā)布時(shí)間：2018-05-24 00:35

  本文選題：雞弱(無(wú))精癥 + 精液品質(zhì)��； 參考：《揚(yáng)州大學(xué)》2015年碩士論文

【摘要】：弱(無(wú))精種公雞因繁殖能力低下而被迫淘汰,據(jù)報(bào)道,在群體中無(wú)精癥種公雞約占10%-20%。而目前對(duì)于雞無(wú)精癥發(fā)生機(jī)制研究鮮有報(bào)道,致使此類問題未得到有效、根本的解決,這給我國(guó)正處在發(fā)展關(guān)鍵時(shí)刻的養(yǎng)禽業(yè)帶來(lái)一定損失。因此,對(duì)于影響種公雞弱(無(wú))精癥的作用機(jī)制探究已是勢(shì)在必行。本研究是以雪山雞為研究對(duì)象,分別從生理、基因表達(dá)、轉(zhuǎn)錄調(diào)控等3個(gè)方面對(duì)雞無(wú)精子癥的發(fā)生機(jī)制進(jìn)行較為系統(tǒng)的探究過(guò)程。首先利用生長(zhǎng)指標(biāo)測(cè)定、精液品質(zhì)測(cè)定及組織學(xué)觀察等方法將雞群分為弱(無(wú))精子雞群體和正常生精雞群體；Piwi基因作為睪丸特異性表達(dá)基因,在精子生成過(guò)程中具有重要作用,缺失導(dǎo)致不能形成成熟精子,因此通過(guò)實(shí)時(shí)熒光定量PCR(Real-Time qPCR, RT-qPCR)法,檢測(cè)Piwi-element induced wimpy testis)基因在正常、弱(無(wú))精癥雞中不同類型生精細(xì)胞中mRNA表達(dá)差異,探討Piwill基因表達(dá)水平與產(chǎn)精性能的關(guān)系；同時(shí)本研究利用PCR-SSCP的方法對(duì)Piwill基因的核心啟動(dòng)子區(qū)和第一外顯子區(qū)序列共531bp區(qū)域進(jìn)行遺變異位點(diǎn)檢測(cè),并分析無(wú)精癥雞群體中變異位點(diǎn)對(duì)基因轉(zhuǎn)錄水平和產(chǎn)精性能的影響,以期尋找有價(jià)值的分子遺傳標(biāo)記。最后采用BSP-克隆測(cè)序法對(duì)該區(qū)域CG二核苷酸位點(diǎn)分布情況進(jìn)行檢測(cè),進(jìn)一步確定其甲基化位點(diǎn),以期發(fā)現(xiàn)在精子發(fā)生過(guò)程中各級(jí)生精細(xì)胞中Piwill基因的表達(dá)與甲基化調(diào)控間的關(guān)系,進(jìn)而闡明Piwill基因在雞精子發(fā)生過(guò)程中的轉(zhuǎn)錄調(diào)控規(guī)律。實(shí)驗(yàn)結(jié)果如下：1.根據(jù)雞群對(duì)按摩法的反應(yīng)、產(chǎn)精情況及精液品質(zhì)將雞群分為正常、反應(yīng)強(qiáng)烈且有精子、反應(yīng)強(qiáng)烈且無(wú)精子、無(wú)反應(yīng)且無(wú)精液等四個(gè)組別,分別標(biāo)記為組1、組2、組3和組4。相比之下,組1的精子活力、精子活率顯著高于組2、組3(P0.05)；且組間精子畸形率差異顯著(P0.05)。在不同時(shí)間點(diǎn)不同組間的FSH、LH、To、TSHR血漿激素水平存在差異。2.在組1中雞睪丸中的曲細(xì)精管上皮結(jié)構(gòu)完整,在組2中雞的曲細(xì)精管結(jié)構(gòu)中,未見精子細(xì)胞；在組3中未見次級(jí)精母細(xì)胞及精子細(xì)胞；在組4中未見初級(jí)、次級(jí)精母細(xì)胞及精子細(xì)胞,僅在靠近基膜存在精原細(xì)胞,且深染的精原細(xì)胞極少。由此可見,曲細(xì)精管組織結(jié)構(gòu)損害較嚴(yán)重,僅有少量精原細(xì)胞,精子細(xì)胞和精母細(xì)胞缺失、稀少,致使睪丸組織生精功能喪失。3.檢測(cè)Piwill基因在正常、無(wú)精癥雞中不同類型生精細(xì)胞中mRNA表達(dá)差異,發(fā)現(xiàn)Piwill在正常雞無(wú)精癥雞群中的表達(dá)存在顯著差異,且從胚胎中分離的干細(xì)胞的Piwill表達(dá)水平較精原細(xì)胞低,且在成熟精子中幾乎不表達(dá),進(jìn)一步表明Piwill很可能參與精子發(fā)生的減數(shù)分裂過(guò)程。4.本實(shí)驗(yàn)擴(kuò)增出雞群Piwill基因5’調(diào)控區(qū)及第1外顯子部分序列總長(zhǎng)531bp區(qū)域,篩選出2個(gè)SNPs;同時(shí)檢測(cè)出雞Piwill基因啟動(dòng)子區(qū)-233～+298bp區(qū)域存在一個(gè)CpG島,該島有56個(gè)CG二核昔酸位點(diǎn)。第1～10個(gè)CG二核苷酸位點(diǎn)(-233～-129bp區(qū)域),精子細(xì)胞、四倍體細(xì)胞、PGCs、SSCs中處于未甲基化狀態(tài),而精原細(xì)胞中甲基化比例高達(dá)0.600；第39～55個(gè)CG二核苷酸位點(diǎn)(+105～+252bp區(qū)域),PGCs和SSCs中甲基化比例高于精子細(xì)胞、精原細(xì)胞、四倍體細(xì)胞。PGCs、SSCs中DNA高甲基化在一定程度上抑制了Piwill基因的表達(dá)。綜上,本研究從生理、基因表達(dá)、轉(zhuǎn)錄調(diào)控等3個(gè)方面進(jìn)行弱(無(wú))精癥公雞與正常雞差異研究,發(fā)現(xiàn)雞曲細(xì)精管上皮結(jié)構(gòu)受損程度與其精子發(fā)生呈負(fù)相關(guān)；Piwill基因在精子中表達(dá)量最低,在精原細(xì)胞的表達(dá)量顯著高于PGCs、SSCs和四倍體；同時(shí)在啟動(dòng)子區(qū)篩選出2個(gè)SNPs,且-151bp處的SNPs產(chǎn)生了1個(gè)新的轉(zhuǎn)錄因子結(jié)合位點(diǎn),但其偏離核心啟動(dòng)子區(qū),檢測(cè)目標(biāo)序列發(fā)現(xiàn)CpG島各位點(diǎn)的甲基化情況,預(yù)測(cè)存在該SNP位點(diǎn)通過(guò)影響DNA甲基化水平而調(diào)控Piwill基因表達(dá)的可能性。研究結(jié)果為進(jìn)一步分析雞弱(無(wú))精癥發(fā)生的遺傳機(jī)制奠定前期基礎(chǔ)。
[Abstract]:The weak (non) spermatosperm cock is forced to be eliminated because of the low reproductive capacity. It is reported that there are few reports on the mechanism of the azoospermia cocks in the population, and the mechanism of the chicken azoospermia is rarely reported. The problem is not effective, and the fundamental solution is to bring some loss to the poultry industry at the critical time of development in China. Therefore, the 10%-20%. It is imperative to explore the mechanism of the influence of the weak (non) spermatospermia of the cock. This study is based on the 3 aspects of the physiology, gene expression and transcriptional regulation of chicks. First, the growth index, the quality of semen and the histology are used to determine the mechanism of the chicken azoospermia. The chicken group is divided into weak (no) sperm chicken population and normal spermatogenic chicken population. Piwi gene, as a testicular specific expression gene, plays an important role in the process of spermatogenesis, and the deletion leads to the failure to form mature sperm. Therefore, Piwi-element induced wimpy tes is detected by real-time quantitative PCR (Real-Time qPCR, RT-qPCR) method. TIS) mRNA expression in different types of spermatogenic cells in normal and weak (non) spermatospermia chicken, the relationship between the expression level of Piwill gene and the performance of spermatogenesis is discussed. At the same time, this study uses the method of PCR-SSCP to detect the ectopic sites in the core promoter region of the Piwill gene and the sequence of the first exon region, and to analyze the inseminosinosis. The effect of variable ectopic spots on gene transcription and spermatogenesis in the group of chickens in order to find valuable molecular genetic markers. Finally, BSP- cloned sequencing was used to detect the distribution of CG dinucleotide loci in this region, and the methylation site was further determined in order to develop the Piwi in spermatogenesis of the present spermatogenesis. The relationship between the expression of ll gene and the regulation of methylation, and then clarifying the regulation of Piwill gene in the process of chicken spermatogenesis. The experimental results are as follows: 1. according to the reaction of the chicken group to the massage method, the production of spermatogenesis and the quality of the semen are divided into normal, strong reaction and spermatoson, strong reaction and no sperm, no reaction and no semen. Four groups were marked as group 1, group 2, group 3 and group 4., compared with group 1, sperm viability was significantly higher than group 2, and group 3 (P0.05), and the difference of sperm malformation rates between groups was significant (P0.05). The plasma levels of FSH, LH, To and TSHR in different groups of time points were different in.2. in the seminiferous tubule epithelium of chicken testis in group 1. In group 2, there was no spermatocyte and spermatocyte in group 3. No primary spermatocyte and spermatocyte were not found in group 4. There was no primary, secondary spermatocyte and spermatocyte in group 4. There were only spermatogonial cells near the basement membrane, and the deep stained spermatogonial cells were very few. Only a small amount of spermatogonial cells, spermatocytes and spermatocytes are missing and rare, resulting in the loss of spermatogenic function of the testis by.3. detection of the Piwill gene in normal, different types of spermatogenic cells in the azoospermia chicken and the difference in the expression of mRNA in the normal chicken azoospermia chicken group, and the P of the stem cells isolated from the embryo. The expression level of Iwill is lower than that of spermatogonial cells and is almost not expressed in mature spermatozoa. It further indicates that Piwill is very likely to participate in the meiosis process of spermatogenesis..4. experiment has amplified the 531bp region of the chicken group Piwill gene 5 'regulation region and the 1 exon part sequence, and screened 2 SNPs; meanwhile, the chicken Piwill gene promoter was detected. There is a CpG island in the region from -233 to +298bp, which has 56 CG two nucleotides. First to 10 CG dinucleotide loci (-233 ~ -129bp region), spermatids, tetraploid cells, PGCs, SSCs are in the non methylation state, and the methylation ratio in spermatogonial cells is up to 0.600; thirty-ninth to 55 CG dinucleotide loci (+105 ~ +252bp region) The proportion of methylation in GCs and SSCs was higher than that of sperm cells, spermatogonial cells, tetraploid cells.PGCs, and DNA hypermethylation in SSCs inhibited the expression of Piwill gene to a certain extent. In this study, the differences of the weak (non) spermatogonial cocks and normal chickens were studied from 3 aspects of physiology, gene expression and transcription regulation, and the epithelia of chicken fininal tubules was found. The degree of damage to the spermatogenesis was negatively correlated with the spermatogenesis, and the expression of Piwill gene was lowest in spermatozoa. The expression of spermatogonial cells was significantly higher than that of PGCs, SSCs and tetraploid. At the same time, 2 SNPs were screened in the promoter region, and the SNPs at -151bp produced 1 NEW transcription factor binding sites, but it deviated from the core promoter region and detected the target sequence. The methylation of CpG island sites was found to predict the possibility that the SNP locus could regulate the expression of Piwill gene by affecting the level of DNA methylation. The results provided a preliminary basis for further analysis of the genetic mechanism of the occurrence of chicken weak (no) spermatogenesis.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.31

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