發(fā)布時間：2018-05-10 22:41

  本文選題：雞 + SLIT2�。� 參考：《吉林農(nóng)業(yè)大學》2016年博士論文

【摘要】：雞的產(chǎn)蛋性狀是一類非常重要的經(jīng)濟性狀,在高產(chǎn)和低產(chǎn)品種(或品系)之間有著顯著差異,該差異主要取決于雞卵巢前等級卵泡周期性募集和等級化建立等發(fā)育階段。同時卵泡的生長發(fā)育是一個非常復(fù)雜而又高度協(xié)調(diào)的生理過程,此過程不僅受到下丘腦-垂體-性腺軸(HPG axis)內(nèi)分泌激素的控制,而且受到卵泡內(nèi)多種旁分泌和自分泌因子等的共同調(diào)節(jié)。SLIT/ROBO信號通路是神經(jīng)內(nèi)分泌信號轉(zhuǎn)導(dǎo)途徑之一,近年研究表明,該通路在哺乳動物卵巢發(fā)育過程中發(fā)揮著重要作用。然而,目前國內(nèi)外對于SLIT/ROBO信號通路調(diào)控雞前等級卵泡生長發(fā)育的作用則鮮有報道。本研究以海蘭褐蛋雞(Hy-Line Brown layers)為供試材料,分別采用組織原位雜交、免疫組化和免疫共沉淀等方法,對SLIT/ROBO通路中配體(SLITs)和受體(ROBOs)基因mRNA和蛋白質(zhì)在前等級卵泡中的時空表達模式、配體與受體分子在前等級卵泡中的互作關(guān)系進行測定分析的基礎(chǔ)上,采用熒光定量PCR技術(shù)和EDU試劑盒檢測SLIT/ROBO通路核心配體SLIT2和SLIT3基因mRNA超表達對顆粒細胞中FSHR、STAR、GDF9和CYP11A1基因(細胞分裂增殖生物標記基因)mRNA表達水平和顆粒細胞增殖的影響,探明SLIT2和SLIT3基因超表達對前等級卵泡生長發(fā)育的調(diào)控作用。并進一步利用siRNA干擾技術(shù)通過下調(diào)SLIT2和SLIT3基因表達對顆粒細胞中FSHR、STAR、GDF9和CYP11A1基因mRNA表達水平和顆粒細胞增殖所產(chǎn)生的生物學效應(yīng),證實配體SLIT2和SLIT3經(jīng)由其受體ROBO1和ROBO2分子介導(dǎo)調(diào)控前等級卵泡生長發(fā)育的作用,從而深入解析SLIT/ROBO信號通路主要配體和受體調(diào)節(jié)前等級卵泡生長發(fā)育的內(nèi)在聯(lián)系,揭示SLIT2和SLIT3分子在雞前等級卵泡生長發(fā)育中的調(diào)控作用。所開展的主要研究工作和結(jié)果如下:1.SLIT/ROBO通路配體和受體基因mRNA在卵巢中的時空表達模式利用組織原位雜交方法對雞SLIT/ROBO信號通路中配體SLITs和受體ROBOs基因在雞卵巢卵泡中mRNA的時空表達進行測定分析。結(jié)果顯示,雞SLITs(SLIT1、SLIT2、SLIT3)和ROBOs(ROBO1、ROBO2、ROBO3、ROBO4)基因在卵巢不同發(fā)育階段的前等級卵泡中的卵母細胞和顆粒細胞中均有表達分布,而未檢測到其在膜層細胞中的表達情況。表明SLIT/ROBO信號通路的配體和受體在mRNA轉(zhuǎn)錄水平上參與了調(diào)控卵巢前等級卵泡的生長發(fā)育。2.SLIT/ROBO通路配體和受體蛋白質(zhì)分子在卵巢中的時空表達模式在組織原位雜交的基礎(chǔ)上,利用免疫組化方法對雞slit/robo信號通路中配體(slits)和受體(robos)蛋白質(zhì)分子在雞卵巢卵泡中的時空表達、定位進行檢測分析。結(jié)果顯示,slits和robos蛋白質(zhì)分子在不同發(fā)育階段的前等級卵泡中的卵母細胞和顆粒細胞中均有表達,而在膜層細胞中均未發(fā)現(xiàn)其蛋白質(zhì)水平上的表達分布。該時空表達模式與其在mrna水平上的時空表達模式相吻合,此證實,slit/robo信號通路中配體slits分子可能以自分泌或旁分泌方式經(jīng)由其受體robos所介導(dǎo)共同參與了前等級卵泡不同生長發(fā)育時期的調(diào)節(jié)控制,對卵泡生長發(fā)育發(fā)揮著關(guān)鍵生物學功能。3.slit2和slit3分子與受體robo1和robo2之間互作關(guān)系的檢測用pas(pcr-basedaccuratesynthesis)方法合成帶有ha標簽的slit2和slit3基因的完整讀碼框cdna序列,再將其亞克隆至表達載體pyr-adshuttle-4質(zhì)粒上,分別將已新重組的表達載體pyr-adshuttle-4-slit2和pyr-adshuttle-4-slit3及其空載質(zhì)粒(陰性對照)經(jīng)由lipofectamine2000試劑轉(zhuǎn)染至顆粒細胞(源于前等級卵泡),利用westernblotting方法對配體slit2、slit3分別與受體robo1、robo2的互作關(guān)系進行檢測、鑒定。結(jié)果顯示,配體slit2、slit3分子分別與受體robo1、robo2相結(jié)合,此表明,受體robo1、robo2分別在介導(dǎo)配體slit2和slit3調(diào)控前等級卵泡的生長發(fā)育中發(fā)揮著重要橋梁作用。4.slit2和slit3分子超表達對前等級卵泡生長發(fā)育的抑制作用采用基因超表達和熒光定量pcr等技術(shù)對顆粒細胞中fshr、star、gdf9和cyp11a1基因mrna表達水平和顆粒細胞增殖情況進行檢測,結(jié)果顯示,fshr、star、gdf9和cyp11a1基因mrna表達水平均顯著下降(p0.05),顆粒細胞增殖顯著減少(p0.05),說明slit2和slit3基因mrna超表達會抑制前等級卵泡的生長發(fā)育;在此過程中,受體robo1和robo2基因mrna和蛋白質(zhì)表達水平顯著上調(diào)(p0.05),此表明slit2、slit3分子對前等級卵泡的生長發(fā)育具有抑制作用,且該抑制效應(yīng)是在其共用受體robo1和robo2分子的介導(dǎo)下協(xié)同實現(xiàn)的。5.slit2和slit3分子抑制前等級卵泡生長發(fā)育作用的鑒定為進一步驗證slit2和slit3分子抑制前等級卵泡生長發(fā)育的作用,本實驗采用sirna干擾的方法通過下調(diào)配體slit2和slit3分子的表達水平,用qpcr技術(shù)、edu試劑盒檢測其對顆粒細胞中fshr、star、gdf9和cyp11a1基因mrna表達水平和顆粒細胞增殖的影響,結(jié)果顯示,顆粒細胞中fshr、star、gdf9和cyp11a1基因mrna表達水平均顯著上調(diào)(p0.05),顆粒細胞增殖顯著(p0.05),表明下調(diào)slit2和slit3基因mrna表達會顯著刺激顆粒細胞的分裂增殖,進而促進前等級卵泡的生長發(fā)育;在此過程中,受體ROBO1和ROBO2基因mRNA和蛋白質(zhì)表達水平顯著下調(diào)(P0.05)。此進一步證明,配體SLIT2和SLIT3與其受體ROBO1和ROBO2以協(xié)同一致的時空調(diào)節(jié)模式,共同發(fā)揮著抑制前等級卵泡生長發(fā)育的作用。本研究首次明確了雞SLIT/ROBO通路中配體(SLITs)和受體(ROBOs)基因mRNA和蛋白質(zhì)在前等級卵泡中的時空表達定位模式、配體和受體分子在前等級卵泡中的互作關(guān)系。在此基礎(chǔ)上,分別通過超表達和抑制內(nèi)源核心配體SLIT2和SLIT3基因mRNA表達等實驗發(fā)現(xiàn),SLIT2和SLIT3基因分別對顆粒細胞中FSHR、STAR、GDF9和CYP11A1基因mRNA表達和顆粒細胞的分裂增殖具有抑制效應(yīng),證實了SLIT2和SLIT3分子由其受體ROBO1和ROBO2所介導(dǎo)調(diào)控前等級卵泡生長發(fā)育的作用,揭示了SLIT2和SLIT3分子在雞前等級卵泡生長發(fā)育中的調(diào)控作用,從而進一步完善了雞卵泡發(fā)育的調(diào)控網(wǎng)絡(luò)。此結(jié)果的闡明可為深入研究維持卵泡募集、優(yōu)勢選擇和等級化建立的遺傳機理提供理論依據(jù);也為深入發(fā)掘控制雞產(chǎn)蛋性狀的關(guān)鍵基因,開展以提高產(chǎn)蛋性狀為育種目標的地方雞種資源遺傳改良和雜種優(yōu)勢利用奠定分子基礎(chǔ),具有十分重要的科學意義和應(yīng)用前景。
[Abstract]:The egg laying traits of chickens are a very important economic character. There is a significant difference between high and low yield varieties (or strains). This difference is mainly determined by the developmental stages such as the periodic recruitment and hierarchical establishment of the follicle in the pre ovaries of the chicken, and the growth and development of the follicles is a very complex and highly coordinated physiological process. The process is not only controlled by the endocrine hormones of the hypothalamus pituitary - gonadal axis (HPG axis), but also the common regulation of.SLIT/ROBO signaling pathway in the follicular and autocrine factors, which is one of the neuroendocrine signal transduction pathways. In recent years, the research shows that this pathway plays an important role in the development of mammalian ovary. However, there are few reports on the role of SLIT/ROBO signaling pathway in regulating the growth and development of pre chicken follicles at home and abroad. In this study, Hy-Line Brown layers (Hy-Line Brown) was used as a test material, and the methods of tissue in situ hybridization, immunohistochemistry and immunoprecipitation were used to determine the ligand (SLITs) and receptor (ROBOs) in the SLIT/ROBO pathway. ) the spatio-temporal expression pattern of gene mRNA and protein in the pre grade follicles, and on the basis of the interaction between ligand and receptor molecules in the pre grade follicles, based on the fluorescence quantitative PCR and EDU kits for detecting FSHR, STAR, GDF9 and CYP in granular cells by mRNA overexpression of the SLIT/ROBO pathway core ligand SLIT2 and SLIT3 gene. The expression level of 11A1 gene (cell division and proliferation biomarker gene) mRNA expression level and the effect of granulosa cell proliferation, the regulation of SLIT2 and SLIT3 overexpression on the growth and development of pre grade follicles was explored. The siRNA interference technique was further used to express FSHR, STAR, GDF9 and CYP11A1 genes in granular cells by down regulation of SLIT2 and SLIT3 genes. The biological effects of expression level and granulosa cell proliferation confirm that the ligand SLIT2 and SLIT3 mediate the growth and development of the pre grade follicle through its receptor ROBO1 and ROBO2 molecules, thus deeply analyzing the internal relationship between the main ligand of the SLIT/ROBO signaling pathway and the growth and development of the pre regulated follicle of the receptor and revealing SLIT2 and SLIT3. The regulatory role of molecules in the growth and development of pre - chicken follicle growth. The main research work and results are as follows: the spatio-temporal expression pattern of the 1.SLIT/ROBO pathway ligand and receptor gene mRNA in the ovary by tissue in situ hybridization method for the mRNA in the chicken ovarian follicles by the ligand SLITs and the receptor ROBOs gene in the chicken SLIT/ROBO signaling pathway The results showed that the SLITs (SLIT1, SLIT2, SLIT3) and ROBOs (ROBO1, ROBO2, ROBO3, ROBO4) genes were expressed in the oocytes and granulosa cells of the pre grade follicles of different developmental stages of the ovary, but the expression of the genes in the membrane cells was not detected. The ligand of the SLIT/ROBO signaling pathway was shown. On the mRNA transcriptional level, it participates in the.2.SLIT/ROBO pathway ligand regulating the ovarian follicle and the spatio-temporal expression pattern of the receptor protein molecules in the ovary. On the basis of the tissue in situ hybridization, the ligand (slits) and the receptor (Robos) protein molecules in the chicken slit/robo signaling pathway are used by the immunohistochemical method. The spatial and temporal expression and location of the ovarian follicles were detected and analyzed. The results showed that slits and Robos protein molecules were expressed in the oocytes and granulosa cells in the pre grade follicles of different developmental stages, but the protein level was not found in the membrane cells. The spatio-temporal expression pattern and the level of the protein were at the level of mRNA. The spatio-temporal expression pattern coincides with that, which confirms that the ligand slits molecule in the slit/robo signaling pathway may be mediated by the autocrine or paracrine way via its receptor Robos and participates in the regulatory control of the different growth stages of the anterior follicles. The key biological functions of the follicle growth and development are.3.slit2 and slit3 molecules. The interaction between body Robo1 and Robo2 is detected by PAS (pcr-basedaccuratesynthesis) method to synthesize the complete reading frame cDNA sequence of Slit2 and slit3 genes with HA tags, and then subcloned to the expression vector pyr-adshuttle-4 plasmids, and the newly recombinant expression carrier pyr-adshuttle-4-slit2 and pyr-adshuttle-4-slit3, and The unloaded plasmid (negative control) was transfected into granular cells (derived from the anterior follicles) via the lipofectamine2000 reagent. The interaction between the ligand Slit2, slit3 and the receptor Robo1, Robo2, respectively, was detected by the westernblotting method. The results showed that the ligand Slit2, slit3 molecules were combined with the receptor Robo1 and Robo2 respectively. This indicates that the receptor rob is rob. O1 and Robo2 play an important bridge role in the growth and development of pre grade follicles, which are mediated by the mediating ligand Slit2 and slit3, respectively. The inhibition of.4.slit2 and slit3 molecule overexpression on the growth and development of the pre grade follicles by gene overexpression and fluorescence quantitative PCR are used to express the level of FSHR, star, GDF9 and CYP11A1 genes in granular cells and the expression level of FSHR, star, GDF9 and CYP11A1. The proliferation of granulosa cells was detected. The results showed that the mRNA expression level of FSHR, star, GDF9 and CYP11A1 genes decreased significantly (P0.05), and the proliferation of granulosa cells decreased significantly (P0.05), indicating that Slit2 and slit3 gene mRNA overexpression could inhibit the growth and development of the pre grade follicles. In this process, the receptor Robo1 and Robo2 genes were expressed and protein expressions were expressed. The level of Slit2 and slit3 molecules can inhibit the growth and development of the pre grade follicles, and the inhibitory effect is identified by the synergistic effect of.5.slit2 and slit3 molecular inhibition on the growth and development of the follicles in the pre grade follicles under the guidance of the common receptor Robo1 and Robo2 molecules, which further verify the inhibition of Slit2 and slit3 molecules. In this experiment, the effect of the growth and development of the follicle of the pre made follicles was studied by using the siRNA interference method. The expression level of the ligand Slit2 and slit3 molecules was downregulated, and the expression level of FSHR, star, GDF9 and CYP11A1 gene mRNA expression level and granulosa cell proliferation in granular cells were detected by qPCR technology and edu kit. The results showed that FSHR, star, and star were in granular cells. The expression level of 9 and CYP11A1 gene mRNA was significantly up-regulated (P0.05), and the proliferation of granular cells was significant (P0.05), indicating that down regulation of Slit2 and slit3 gene mRNA expression could significantly stimulate the proliferation of granular cells and promote the growth and development of the pre grade follicles. In this process, the expression level of mRNA and protein of the receptor ROBO1 and ROBO2 genes was significantly down (P0) .05). It is further demonstrated that ligands, SLIT2 and SLIT3, and their receptor ROBO1 and ROBO2, together play a role in inhibiting the growth and development of pre grade follicles. This study was the first to clarify the temporal and spatial expression of the ligand (SLITs) and receptor (ROBOs) based mRNA and protein in the pre grade follicles of the chicken SLIT/ROBO pathway. The interaction between the ligand and the receptor molecule in the anterior follicles. On this basis, we found that SLIT2 and SLIT3 genes have inhibitory effects on the expression of FSHR, STAR, GDF9 and CYP11A1 genes in granular cells and the proliferation of granular cells in granular cells, respectively, by overexpression and inhibition of the expression of the endogenous SLIT2 and the SLIT3 gene mRNA expression, respectively. The effect of SLIT2 and SLIT3 molecules was confirmed by its receptor ROBO1 and ROBO2 to regulate the growth and development of the follicle of the pre grade follicles, which revealed the regulatory role of SLIT2 and SLIT3 molecules in the growth and development of the follicle of the pre chicken, which further improved the regulation network of the development of chicken follicles. It provides a theoretical basis for the genetic mechanism established by recruitment, advantage selection and hierarchical, and is of great scientific significance and application prospects for exploring the key genes controlling the egg laying traits and developing the molecular basis of genetic improvement and Heterosis Utilization of local chicken seed resources to improve the laying traits as the breeding targets.

【學位授予單位】：吉林農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S831

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