本文選題：綿羊 + 產(chǎn)羔性狀��； 參考：《甘肅農(nóng)業(yè)大學(xué)》2015年博士論文

【摘要】：產(chǎn)羔性狀是綿羊重要的經(jīng)濟(jì)性狀之一,直接影響到養(yǎng)羊業(yè)生產(chǎn)的效率和成本,對(duì)養(yǎng)羊業(yè)的發(fā)展起到了關(guān)鍵性的作用。綿羊的產(chǎn)羔性狀分為單羔性狀、雙羔性狀和多羔性狀三種,不同品種綿羊的產(chǎn)羔率差異較大。選育綿羊理想的產(chǎn)羔性狀最有效的方法是掌握綿羊產(chǎn)羔性狀的分子遺傳機(jī)制和調(diào)控機(jī)理,從DNA分子角度尋找到產(chǎn)羔性狀的主效基因和分子標(biāo)記,進(jìn)行分子選育。綿羊的產(chǎn)羔數(shù)是受排卵數(shù)控制的,在卵泡發(fā)育和排卵過程中,存在一系列的細(xì)胞調(diào)控機(jī)制,涉及到多種類型蛋白質(zhì)相互作用。因此,要對(duì)綿羊產(chǎn)羔性狀的調(diào)控機(jī)理有全面和深入的認(rèn)識(shí),就必須從整體、動(dòng)態(tài)、調(diào)控途徑上對(duì)作用于產(chǎn)羔性狀的蛋白質(zhì)進(jìn)行研究,確定綿羊不同產(chǎn)羔性狀的主效信號(hào)分子及其傳遞通路,揭示綿羊不同產(chǎn)羔性狀的分子遺傳機(jī)制和調(diào)控機(jī)理,為綿羊產(chǎn)羔性狀分子標(biāo)記選育和分子育種提供理論依據(jù)。本研究開展了用TCA/丙酮沉淀法和直接裂解法提取綿羊卵巢組織全蛋白方法的比較、篩選與優(yōu)化研究,利用雙向凝膠電泳和質(zhì)譜技術(shù),對(duì)乏情期(蒙古羊乏情期,無角陶賽特羊和小尾寒羊間情期,下同)、發(fā)情周期第1天和發(fā)情周期第11天的蒙古羊繁殖母羊(包括產(chǎn)單羔和雙羔的母羊,簡稱單羔蒙古羊、雙羔蒙古羊)、無角陶賽特羊繁殖母羊(包括產(chǎn)單羔和雙羔的母羊,簡稱單羔無角陶賽特羊、雙羔無角陶賽特羊)和產(chǎn)多羔小尾寒羊(簡稱多羔小尾寒羊)繁殖母羊的卵巢蛋白質(zhì)組成及含量的動(dòng)態(tài)變化進(jìn)行分析,篩選不同產(chǎn)羔性狀綿羊卵巢組織中差異表達(dá)的蛋白質(zhì),利用生物信息學(xué)方法對(duì)差異蛋白質(zhì)進(jìn)行亞細(xì)胞定位、GO分析(細(xì)胞組件、分子功能和生物過程)以及KEGG通路分析,構(gòu)建差異蛋白質(zhì)相互作用網(wǎng)絡(luò),以期探索綿羊不同產(chǎn)羔性狀的分子作用機(jī)理及調(diào)控機(jī)制。⑴在相同電泳過程和操作步驟情況下獲得的綿羊卵巢全蛋白2-DE圖譜分析發(fā)現(xiàn),直接裂解法對(duì)綿羊卵巢全蛋白的提取效果優(yōu)于TCA/丙酮沉淀法,其中裂解液配方為7M尿素,2M硫脲,4%CHAPS,0.5%兩性電解質(zhì)pH 3-10,100mmol/L DTT,60 mmol/L Tris,20μL/mL蛋白酶抑制劑混合物時(shí)適合綿羊卵巢全蛋白質(zhì)的提取。⑵運(yùn)用雙向凝膠電泳技術(shù)、生物質(zhì)譜技術(shù)和蛋白質(zhì)生物信息學(xué)對(duì)單羔蒙古羊、雙羔蒙古羊、單羔無角陶賽特羊、雙羔無角陶賽特羊和多羔小尾寒羊在乏情期、發(fā)情周期第1天和發(fā)情周期第11天卵巢組織差異蛋白進(jìn)行篩選和分析,結(jié)果表明,共篩選到19種差異蛋白質(zhì),其中乏情期15種,發(fā)情周期第一天10種,發(fā)情周期第11天15種,差異蛋白質(zhì)亞細(xì)胞定位預(yù)測結(jié)果顯示19種差異蛋白質(zhì)2種蛋白質(zhì)位于分泌通路中(GDF9和glutathione S-transferase Mu 1-like isoform3),3種蛋白質(zhì)位于線粒體中(myosin regulatory light chain MRCL3、glyceraldehyde-3-phosphate dehydrogenase和nitric oxide-inducible gene protein),其他14種蛋白質(zhì)定位于其他部位;GO分析表明,基于GO分析中的細(xì)胞組件結(jié)果得到21個(gè)注釋,在細(xì)胞質(zhì)(cytoplasm)、細(xì)胞質(zhì)基質(zhì)(cytosol)、細(xì)胞核(nucleus)和鈣離子復(fù)合物(calcium complex)呈富集狀態(tài),基于GO分析中的分子功能得到31個(gè)注釋,在在ATP結(jié)合(ATP binding)、信號(hào)轉(zhuǎn)導(dǎo)(signal transducer activity)、鈣離子結(jié)合(calcium ion binding)、氧的轉(zhuǎn)運(yùn)活性(oxygen transporter activity)、生長因子活性(growth factor activity)、DNA結(jié)合(DNA binding)和碳水化合物結(jié)合(carbohydrate binding)呈現(xiàn)富集狀態(tài),基于GO分析中的生物過程得到35個(gè)注釋,在氧化還原過程(oxidation-reduction process)、卵泡生長發(fā)育(ovarian follicle development)、信號(hào)轉(zhuǎn)導(dǎo)(signal transduction)、BMP信號(hào)通路(BMP signaling pathway)、生長因子受體信號(hào)通路(growth factor receptor signaling pathway)、C21甾體激素的代謝過程(C21-steroid hormone metabolic process)和代謝過程(metabolic process)呈現(xiàn)富集狀態(tài);KEGG分析共得到23個(gè)注釋,其中在氧化磷酸化通路(Oxidative phosphorylation pathway)、卵母細(xì)胞減數(shù)分裂(Oocyte meiosis)、TGF-β信號(hào)通路(TGF-beta signaling pathway)和GnRH信號(hào)通路(GnRH signaling pathway)呈現(xiàn)富集狀態(tài)。⑶篩選與綿羊產(chǎn)羔性狀相關(guān)的關(guān)鍵蛋白有:Calmodulin,GDF9,BMPR-1B,MTHFR和SOD-[Cu-Zn]-like。
[Abstract]:Lambing character is one of the important economic traits of sheep, which directly affects the efficiency and cost of sheep production, and plays a key role in the development of sheep industry. The lambing traits of sheep are divided into three traits, two lambs and multiple lambs, and the lambing rate of different sheep is different. The ideal lambing character of sheep is selected. The most effective method is to master the molecular genetic mechanism and regulation mechanism of lambing traits in sheep. From the angle of DNA, the main genes and molecular markers of lambing traits are found, and molecular breeding is carried out. The number of lambs in sheep is controlled by the number of ovulation. In the process of follicle development and ovulation, there are a series of cell regulation mechanisms involved in the process of ovarian follicle development and ovulation. Therefore, in order to have a comprehensive and deep understanding of the regulation mechanism of lambing traits in sheep, it is necessary to study the protein of lambing traits on the whole, dynamic and regulatory ways, to determine the main signal subdivisions and transmission pathways of different lambing traits in sheep, and to reveal the different lambing traits of sheep. Molecular genetic mechanism and regulatory mechanism provide a theoretical basis for molecular breeding and molecular breeding of lambing traits in sheep. This study carried out a comparison, screening and optimization of TCA/ acetone precipitation and direct lysis in sheep ovarian tissue, using two dimensional gel electrophoresis and mass spectrometry, for the Anoestrus (Mongolia sheep). The Mongolia sheep breeding ewes (including single lambs and double lambs, abbreviated as single lambs, Mongolia sheep, double lambs, Mongolia sheep), and non horns Dorset sheep breeding ewes (including single lambs and double Lambs), were found in the absence of estrus, with first days of estrus and eleventh days of estrous cycle. The dynamic changes in the protein composition and content of the ovaries of the two lambs and Small Tailed Han sheep were analyzed. The proteins expressed differently in the ovaries of the sheep were screened with different lambing traits. The subcellular localization of the differential proteins was carried out by bioinformatics method, and the GO analysis (fine) Cell components, molecular functions and biological processes) and KEGG pathway analysis to construct differential protein interaction networks in order to explore the molecular mechanism and regulation mechanism of different lambing traits in sheep. (1) analysis of the total protein 2-DE Atlas of ovine ovaries obtained under the same electrophoresis process and operation steps The extraction effect of total ovarian protein is better than that of TCA/ acetone precipitation, in which lysate formula is 7M urea, 2M thiourea, 4%CHAPS, 0.5% amphoteric electrolyte pH 3-10100mmol/L DTT, 60 mmol/L Tris, and 20 mu L/mL protease inhibitor mixture is suitable for the extraction of all protein in ovaries. 2. Bi-gel electrophoresis, biological mass spectrometry and eggs White matter bioinformatics was screened and analyzed for single lamb Mongolia sheep, double lamb Mongolia sheep, single lamb without horseback dovette sheep, double lambs without horseback Dorset sheep and multi lambs Small Tail Han sheep in the hypoestrus, first days of estrous cycle and eleventh days of estrous cycle, the results showed that 19 kinds of differential proteins were screened, among them, 15 species of hypoestrus were found. The first day of the estrous cycle is 10 species, and the estrous cycle is eleventh days 15. The difference protein subcellular location prediction results show that 19 kinds of protein 2 proteins are located in the secretory pathway (GDF9 and glutathione S-transferase Mu 1-like isoform3), and the 3 proteins are in the mitochondria (myosin regulatory light chain MRCL3, glyceraldehyde-3-phosphat). E dehydrogenase and nitric oxide-inducible gene protein), the other 14 proteins are located at other sites; GO analysis shows that the cell component results in the GO analysis are 21 annotations, in the cytoplasm (cytoplasm), the cytoplasmic matrix (cytosol), the nucleus (nucleus) and the calcium ion complex (calcium), based on the enrichment state. The molecular functions in the analysis are 31 annotations, in ATP binding (ATP binding), signal transduction (signal transducer activity), calcium ion binding (calcium ion binding), oxygen transport activity (oxygen transporter activity), growth factor activity and carbohydrate binding Binding) presents a state of enrichment, and 35 annotations are obtained in the biological process based on GO analysis, in the redox process (oxidation-reduction process), follicle growth and development (ovarian follicle development), signal transduction (signal transduction), BMP signaling pathway (BMP signaling), and growth factor receptor signaling pathway Eptor signaling pathway), the metabolic process (C21-steroid hormone metabolic process) and metabolic process (metabolic process) of C21 steroid were enriched, and 23 notes were obtained in the KEGG analysis, in which the oxidative phosphorylation pathway (Oxidative), oocyte meiosis, and beta signal TGF-beta signaling pathway and GnRH signaling pathway (GnRH signaling pathway) present enrichment status. (3) key proteins related to lamb traits of sheep are selected: Calmodulin, GDF9, BMPR-1B, MTHFR and SOD-[Cu-Zn]-like..
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：S826

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