Arg-PA代谢通路关键基因在鹅等级前卵泡中的表达规律研究

发布时间：2018-04-03 17:57

本文选题：鹅　切入点：等级前卵泡　出处：《吉林农业大学》2017年硕士论文

【摘要】：鹅的卵泡发育是复杂且相互协调的生物过程,其发育直接影响鹅的生产性能。因此,对鹅卵泡发育调控机制进行研究将有助于我国鹅业发展。近年研究表明精氨酸-多胺(Arg-PA)代谢通路对鹅的卵泡发育具有调控作用。为了解其具体作用机制,生物学信息及功能。本试验以产蛋高峰期籽鹅等级前卵泡为研究对象,构建基因共表达网络,初步分析相关基因的生物学功能。通过RNA-Seq技术对鹅等级前卵泡组织进行测序分析,筛选确定Arg-PA代谢通路及通路中相关基因,得到相关基因表达情况,并通过qRT-PCR和Western Blot技术从RNA和蛋白水平进行表达趋势验证,同时结合卵泡发育结构变化,初步探索Arg-PA代谢通路及通路上的相关基因,为鹅等级前卵泡发育的作用机制提供理论依据。研究结果如下:(1)通过HE染色对产蛋高峰期籽鹅等级前卵泡进行组织结构观察,结果发现随着卵泡的发育卵泡膜结构发生变化。其中卵泡膜层和颗粒细胞层的厚度在各等级卵泡组织中存在显著差异(P0.05),但厚度变化趋势相同,先增加后降低,且卵泡膜在M等级卵泡中厚度最大。(2)本试验通过RNA-seq技术对处于产蛋高峰期的籽鹅等级前卵泡组织进行转录组测序分析,确定Arg-PA代谢通路及通路上相关基因:ADC、ARG、OAZ2、ODC、NOS和SMS、SRM、SMOX。通过qRT-PCR和Western Blot技术分别从关键基因的mRNA水平和蛋白质水平进行表达趋势验证,研究结果显示ODC、ARG、OAZ2、ODC、NOS和SMS、SRM、SMOX的RNA在鹅等级前卵泡组织中均有表达,表达趋势相同。(3)通过qRT-PCR试验方法验证发现Arg-PA代谢通路上相关基因在鹅等级前卵泡组织中表达存在显著差异。其中ARG,ODC,NOS,OAZ2,SRM和SMS基因在PE等级卵泡组织中的表达量最高,ADC和SMOX基因mRNA在PE等级卵泡组织中的表达量最低;ADC基因mRNA在S等级卵泡中表达量最高,SMOX基因mRNA在M等级卵泡中表达量最高,而ARG,ODC,NOS,SRM和SMS基因mRNA在M等级卵泡组织中表达量最低,OAZ2基因mRNA在L等级卵泡中表达量最低。各基因mRNA的最高与最低表达量之间差异显著(P0.05)。(4)通过Western Blot技术对Arg-PA代谢中关键基因ODC,ARG和NOS进行蛋白表达水平分析,结果发现:ARG和NOS在PE等级卵泡中的蛋白表达最高,在M等级卵泡中的蛋白表达量最低;ODC基因在鹅L等级卵泡中的蛋白表达量最高,在SY等级卵泡中的蛋白表达最低,各基因蛋白最高表达量与最低表达量之间存在显著差异(P0.05)。(5)将Arg-PA代谢通路上相关基因表达情况与卵泡发育结合分析,结果推测Arg-PA代谢可能参与原始卵泡生长启动调控,同时ARG、ODC、NOS、SMS和SRM可通过调控颗粒细胞增殖发育来促进卵泡发育,而ADC,OAZ,SMOX抑制卵泡发育。
[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.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S835

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