SLIT2和SLIT3分子在鸡前等级卵泡生长发育中的调控作用

发布时间：2018-05-10 22:41

  本文选题：鸡 + SLIT2　； 参考：《吉林农业大学》2016年博士论文

【摘要】：鸡的产蛋性状是一类非常重要的经济性状,在高产和低产品种(或品系)之间有着显著差异,该差异主要取决于鸡卵巢前等级卵泡周期性募集和等级化建立等发育阶段。同时卵泡的生长发育是一个非常复杂而又高度协调的生理过程,此过程不仅受到下丘脑-垂体-性腺轴(HPG axis)内分泌激素的控制,而且受到卵泡内多种旁分泌和自分泌因子等的共同调节。SLIT/ROBO信号通路是神经内分泌信号转导途径之一,近年研究表明,该通路在哺乳动物卵巢发育过程中发挥着重要作用。然而,目前国内外对于SLIT/ROBO信号通路调控鸡前等级卵泡生长发育的作用则鲜有报道。本研究以海兰褐蛋鸡(Hy-Line Brown layers)为供试材料,分别采用组织原位杂交、免疫组化和免疫共沉淀等方法,对SLIT/ROBO通路中配体(SLITs)和受体(ROBOs)基因mRNA和蛋白质在前等级卵泡中的时空表达模式、配体与受体分子在前等级卵泡中的互作关系进行测定分析的基础上,采用荧光定量PCR技术和EDU试剂盒检测SLIT/ROBO通路核心配体SLIT2和SLIT3基因mRNA超表达对颗粒细胞中FSHR、STAR、GDF9和CYP11A1基因(细胞分裂增殖生物标记基因)mRNA表达水平和颗粒细胞增殖的影响,探明SLIT2和SLIT3基因超表达对前等级卵泡生长发育的调控作用。并进一步利用siRNA干扰技术通过下调SLIT2和SLIT3基因表达对颗粒细胞中FSHR、STAR、GDF9和CYP11A1基因mRNA表达水平和颗粒细胞增殖所产生的生物学效应,证实配体SLIT2和SLIT3经由其受体ROBO1和ROBO2分子介导调控前等级卵泡生长发育的作用,从而深入解析SLIT/ROBO信号通路主要配体和受体调节前等级卵泡生长发育的内在联系,揭示SLIT2和SLIT3分子在鸡前等级卵泡生长发育中的调控作用。所开展的主要研究工作和结果如下:1.SLIT/ROBO通路配体和受体基因mRNA在卵巢中的时空表达模式利用组织原位杂交方法对鸡SLIT/ROBO信号通路中配体SLITs和受体ROBOs基因在鸡卵巢卵泡中mRNA的时空表达进行测定分析。结果显示,鸡SLITs(SLIT1、SLIT2、SLIT3)和ROBOs(ROBO1、ROBO2、ROBO3、ROBO4)基因在卵巢不同发育阶段的前等级卵泡中的卵母细胞和颗粒细胞中均有表达分布,而未检测到其在膜层细胞中的表达情况。表明SLIT/ROBO信号通路的配体和受体在mRNA转录水平上参与了调控卵巢前等级卵泡的生长发育。2.SLIT/ROBO通路配体和受体蛋白质分子在卵巢中的时空表达模式在组织原位杂交的基础上,利用免疫组化方法对鸡slit/robo信号通路中配体(slits)和受体(robos)蛋白质分子在鸡卵巢卵泡中的时空表达、定位进行检测分析。结果显示,slits和robos蛋白质分子在不同发育阶段的前等级卵泡中的卵母细胞和颗粒细胞中均有表达,而在膜层细胞中均未发现其蛋白质水平上的表达分布。该时空表达模式与其在mrna水平上的时空表达模式相吻合,此证实,slit/robo信号通路中配体slits分子可能以自分泌或旁分泌方式经由其受体robos所介导共同参与了前等级卵泡不同生长发育时期的调节控制,对卵泡生长发育发挥着关键生物学功能。3.slit2和slit3分子与受体robo1和robo2之间互作关系的检测用pas(pcr-basedaccuratesynthesis)方法合成带有ha标签的slit2和slit3基因的完整读码框cdna序列,再将其亚克隆至表达载体pyr-adshuttle-4质粒上,分别将已新重组的表达载体pyr-adshuttle-4-slit2和pyr-adshuttle-4-slit3及其空载质粒(阴性对照)经由lipofectamine2000试剂转染至颗粒细胞(源于前等级卵泡),利用westernblotting方法对配体slit2、slit3分别与受体robo1、robo2的互作关系进行检测、鉴定。结果显示,配体slit2、slit3分子分别与受体robo1、robo2相结合,此表明,受体robo1、robo2分别在介导配体slit2和slit3调控前等级卵泡的生长发育中发挥着重要桥梁作用。4.slit2和slit3分子超表达对前等级卵泡生长发育的抑制作用采用基因超表达和荧光定量pcr等技术对颗粒细胞中fshr、star、gdf9和cyp11a1基因mrna表达水平和颗粒细胞增殖情况进行检测,结果显示,fshr、star、gdf9和cyp11a1基因mrna表达水平均显著下降(p0.05),颗粒细胞增殖显著减少(p0.05),说明slit2和slit3基因mrna超表达会抑制前等级卵泡的生长发育;在此过程中,受体robo1和robo2基因mrna和蛋白质表达水平显著上调(p0.05),此表明slit2、slit3分子对前等级卵泡的生长发育具有抑制作用,且该抑制效应是在其共用受体robo1和robo2分子的介导下协同实现的。5.slit2和slit3分子抑制前等级卵泡生长发育作用的鉴定为进一步验证slit2和slit3分子抑制前等级卵泡生长发育的作用,本实验采用sirna干扰的方法通过下调配体slit2和slit3分子的表达水平,用qpcr技术、edu试剂盒检测其对颗粒细胞中fshr、star、gdf9和cyp11a1基因mrna表达水平和颗粒细胞增殖的影响,结果显示,颗粒细胞中fshr、star、gdf9和cyp11a1基因mrna表达水平均显著上调(p0.05),颗粒细胞增殖显著(p0.05),表明下调slit2和slit3基因mrna表达会显著刺激颗粒细胞的分裂增殖,进而促进前等级卵泡的生长发育;在此过程中,受体ROBO1和ROBO2基因mRNA和蛋白质表达水平显著下调(P0.05)。此进一步证明,配体SLIT2和SLIT3与其受体ROBO1和ROBO2以协同一致的时空调节模式,共同发挥着抑制前等级卵泡生长发育的作用。本研究首次明确了鸡SLIT/ROBO通路中配体(SLITs)和受体(ROBOs)基因mRNA和蛋白质在前等级卵泡中的时空表达定位模式、配体和受体分子在前等级卵泡中的互作关系。在此基础上,分别通过超表达和抑制内源核心配体SLIT2和SLIT3基因mRNA表达等实验发现,SLIT2和SLIT3基因分别对颗粒细胞中FSHR、STAR、GDF9和CYP11A1基因mRNA表达和颗粒细胞的分裂增殖具有抑制效应,证实了SLIT2和SLIT3分子由其受体ROBO1和ROBO2所介导调控前等级卵泡生长发育的作用,揭示了SLIT2和SLIT3分子在鸡前等级卵泡生长发育中的调控作用,从而进一步完善了鸡卵泡发育的调控网络。此结果的阐明可为深入研究维持卵泡募集、优势选择和等级化建立的遗传机理提供理论依据;也为深入发掘控制鸡产蛋性状的关键基因,开展以提高产蛋性状为育种目标的地方鸡种资源遗传改良和杂种优势利用奠定分子基础,具有十分重要的科学意义和应用前景。
[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.

【学位授予单位】：吉林农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S831

【参考文献】

相关博士学位论文 前3条

1 张学敬;前列腺素和孕酮对小鼠黄体退化中Slit/Robo表达的调节及相关机理[D];中国农业大学;2014年

2 陶灵;JAK-STATs通路对CTGF刺激人增生性瘢痕成纤维细胞增殖分化作用的研究[D];第三军医大学;2008年

3 金艳梅;鸡等级前卵泡颗粒细胞发育的激素和营养调控及机理研究[D];浙江大学;2007年

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