本文選題：鵝　切入點(diǎn)：等級(jí)前卵泡　出處：《吉林農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：鵝的卵泡發(fā)育是復(fù)雜且相互協(xié)調(diào)的生物過(guò)程,其發(fā)育直接影響鵝的生產(chǎn)性能。因此,對(duì)鵝卵泡發(fā)育調(diào)控機(jī)制進(jìn)行研究將有助于我國(guó)鵝業(yè)發(fā)展。近年研究表明精氨酸-多胺(Arg-PA)代謝通路對(duì)鵝的卵泡發(fā)育具有調(diào)控作用。為了解其具體作用機(jī)制,生物學(xué)信息及功能。本試驗(yàn)以產(chǎn)蛋高峰期籽鵝等級(jí)前卵泡為研究對(duì)象,構(gòu)建基因共表達(dá)網(wǎng)絡(luò),初步分析相關(guān)基因的生物學(xué)功能。通過(guò)RNA-Seq技術(shù)對(duì)鵝等級(jí)前卵泡組織進(jìn)行測(cè)序分析,篩選確定Arg-PA代謝通路及通路中相關(guān)基因,得到相關(guān)基因表達(dá)情況,并通過(guò)qRT-PCR和Western Blot技術(shù)從RNA和蛋白水平進(jìn)行表達(dá)趨勢(shì)驗(yàn)證,同時(shí)結(jié)合卵泡發(fā)育結(jié)構(gòu)變化,初步探索Arg-PA代謝通路及通路上的相關(guān)基因,為鵝等級(jí)前卵泡發(fā)育的作用機(jī)制提供理論依據(jù)。研究結(jié)果如下:(1)通過(guò)HE染色對(duì)產(chǎn)蛋高峰期籽鵝等級(jí)前卵泡進(jìn)行組織結(jié)構(gòu)觀察,結(jié)果發(fā)現(xiàn)隨著卵泡的發(fā)育卵泡膜結(jié)構(gòu)發(fā)生變化。其中卵泡膜層和顆粒細(xì)胞層的厚度在各等級(jí)卵泡組織中存在顯著差異(P0.05),但厚度變化趨勢(shì)相同,先增加后降低,且卵泡膜在M等級(jí)卵泡中厚度最大。(2)本試驗(yàn)通過(guò)RNA-seq技術(shù)對(duì)處于產(chǎn)蛋高峰期的籽鵝等級(jí)前卵泡組織進(jìn)行轉(zhuǎn)錄組測(cè)序分析,確定Arg-PA代謝通路及通路上相關(guān)基因:ADC、ARG、OAZ2、ODC、NOS和SMS、SRM、SMOX。通過(guò)qRT-PCR和Western Blot技術(shù)分別從關(guān)鍵基因的mRNA水平和蛋白質(zhì)水平進(jìn)行表達(dá)趨勢(shì)驗(yàn)證,研究結(jié)果顯示ODC、ARG、OAZ2、ODC、NOS和SMS、SRM、SMOX的RNA在鵝等級(jí)前卵泡組織中均有表達(dá),表達(dá)趨勢(shì)相同。(3)通過(guò)qRT-PCR試驗(yàn)方法驗(yàn)證發(fā)現(xiàn)Arg-PA代謝通路上相關(guān)基因在鵝等級(jí)前卵泡組織中表達(dá)存在顯著差異。其中ARG,ODC,NOS,OAZ2,SRM和SMS基因在PE等級(jí)卵泡組織中的表達(dá)量最高,ADC和SMOX基因mRNA在PE等級(jí)卵泡組織中的表達(dá)量最低;ADC基因mRNA在S等級(jí)卵泡中表達(dá)量最高,SMOX基因mRNA在M等級(jí)卵泡中表達(dá)量最高,而ARG,ODC,NOS,SRM和SMS基因mRNA在M等級(jí)卵泡組織中表達(dá)量最低,OAZ2基因mRNA在L等級(jí)卵泡中表達(dá)量最低。各基因mRNA的最高與最低表達(dá)量之間差異顯著(P0.05)。(4)通過(guò)Western Blot技術(shù)對(duì)Arg-PA代謝中關(guān)鍵基因ODC,ARG和NOS進(jìn)行蛋白表達(dá)水平分析,結(jié)果發(fā)現(xiàn):ARG和NOS在PE等級(jí)卵泡中的蛋白表達(dá)最高,在M等級(jí)卵泡中的蛋白表達(dá)量最低;ODC基因在鵝L等級(jí)卵泡中的蛋白表達(dá)量最高,在SY等級(jí)卵泡中的蛋白表達(dá)最低,各基因蛋白最高表達(dá)量與最低表達(dá)量之間存在顯著差異(P0.05)。(5)將Arg-PA代謝通路上相關(guān)基因表達(dá)情況與卵泡發(fā)育結(jié)合分析,結(jié)果推測(cè)Arg-PA代謝可能參與原始卵泡生長(zhǎng)啟動(dòng)調(diào)控,同時(shí)ARG、ODC、NOS、SMS和SRM可通過(guò)調(diào)控顆粒細(xì)胞增殖發(fā)育來(lái)促進(jìn)卵泡發(fā)育,而ADC,OAZ,SMOX抑制卵泡發(fā)育。
[Abstract]:Follicular development of geese is a complex and coordinated biological process, which directly affects the performance of geese.Therefore, studies on the mechanism of follicle development in geese will contribute to the development of goose industry in China.Recent studies have shown that arginine-polyamines (Arg-PA) metabolic pathway regulates follicular development in geese.In order to understand its specific mechanism, biological information and function.In this experiment, the pre-grade follicles of the egg-laying peak period of the seed goose were studied, and the gene coexpression network was constructed, and the biological function of the related genes was preliminarily analyzed.The prefollicular tissues of goose were sequenced by RNA-Seq technique, and the related genes in the metabolic pathway and pathway of Arg-PA were screened and identified, and the expression trend of the related genes was verified by qRT-PCR and Western Blot from RNA and protein levels.At the same time, combined with the changes of follicular development structure, the related genes in Arg-PA metabolic pathway and pathway were preliminarily explored in order to provide theoretical basis for the mechanism of follicle development in goose pre-grade.The results were as follows: (1) the tissue structure of follicles before egg-laying peak stage was observed by HE staining. The results showed that the membrane structure of follicles changed with the development of follicles.The thickness of follicular membrane layer and granulosa cell layer was significantly different in the follicular tissues of different grades, but the change trend of the thickness was the same, which increased at first and then decreased.The follicular membrane is the thickest in M grade follicles. In this experiment, RNA-seq technique was used to analyze the transcription sequence of follicular tissue in the pre-grade follicles of the goose at the peak stage of laying eggs, and to identify the related genes of Arg-PA metabolic pathway and its related genes: ADCA, ARG, OAZ2ODCN, NOS and SMSSSRMMOX.QRT-PCR and Western Blot techniques were used to verify the expression trend of mRNA and protein levels of the key genes, respectively. The results showed that the RNA of ODCA ARGN OAZ2ODCP and SMSS SRM SMOX were expressed in the pregrade follicular tissues of geese.The results of qRT-PCR test showed that there were significant differences in the expression of genes related to Arg-PA metabolic pathway in the pregrade follicle tissues of goose.The expression of ADCs and SMOX genes in PE grade follicles was the lowest. MRNA gene expression was the highest in S grade follicles and SMOX gene mRNA was the highest in M grade follicles.The expression of SMS gene mRNA and NOSN SRM gene were the lowest in M-grade follicles. The expression of mRNA was the lowest in L-grade follicles.There was significant difference between the highest and lowest expression levels of mRNA. (4) the protein expression levels of ODCARG and NOS, the key genes of Arg-PA metabolism, were analyzed by Western Blot. The results showed that the protein expression of Arg-PA and NOS was the highest in PE grade follicles.The protein expression of ODC gene was the lowest in M grade follicles and the lowest in goose L grade follicles and sy grade follicles.There was a significant difference between the highest and lowest expression levels of each gene protein. (P0.05) the expression of genes related to the metabolic pathway of Arg-PA was combined with follicular development. The results suggested that Arg-PA metabolism might be involved in the regulation of primordial follicle growth and initiation.At the same time, SRM and SRM can promote follicular development by regulating the proliferation and development of granulosa cells, while ADCOZ SMOX inhibits follicular development.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S835

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