时钟基因在母鸡生殖系统中的节律性表达及颗粒细胞中时控基因的筛选
发布时间：2018-08-18 13:53
【摘要】：昼夜节律几乎影响了生物体生命活动的方方面面,帮助其趋利避害,实现和外界环境的同步和适应,是生命的基本特征之一。雌禽的排卵-产蛋循环便具有明显的节律性和环境适应性,具体表现在排卵(产蛋)时间偏好、连产间歇等方面。时间生物学和生殖内分泌学的发展为解释上述现象提供了理论基础,但性腺轴上的调控复杂交错,卵泡的成熟,排卵和蛋的形成是多组织、多过程、多层次参与的生理事件,其间不同生理进程在时间上的吻合显示了机体自身的协调统一。单独研究神经内分泌或卵泡的发育无法全面的解释排卵-产蛋这一复杂又特殊的生理过程,本研究从昼夜生物钟系统切入,整合各过程的时间控制因素,解析排卵-产蛋中的节律现象。1.产蛋监测与产蛋规律分析本研究通过记录母鸡在特定时间段里的具体产蛋时间,得到了商品蛋鸡罗曼粉壳蛋鸡在常规饲养条件下的产蛋性状,包括：产蛋时间分布、连产间歇、间歇前一天产蛋时间分布和产蛋间隔时间。结果发现：母鸡产蛋有极强的时间偏好性,主要集中在8：00一12：00这个时段：产蛋性能好的母鸡产蛋时间间隔以24小时为轴心左右波动。连产间歇前一天的产蛋时间在早、晚两个时段密集分布,说明接近开放期的前后边缘都会造成产蛋间歇。证实了产蛋开放期模型。通过产蛋规律的监测,挑选出了生理状态一致(产蛋时间和产蛋周期一致)的鸡群作为后续的试验材料。2.时钟基因的节律性表达与发条机制的定位本研究通过检测时钟基因(Bmall,Bmal2,Clock, Per2, Per3,Cryl,Cry2)在等级卵泡的颗粒细胞和膜细胞以及输卵管的四个部位中的表达情况,运用余弦分析法检测其表达是否呈现显著性节律震荡,结合生物钟运行的分子机制,将时钟基因在一个昼夜周期内的表达规律加以组合,根据峰值时间关系确定生物钟发条机制在该组织中是否存在。结果表明：生物钟发条机制在F1-F3卵泡的颗粒细胞以及输卵管的伞部和子宫部中存在,并且确定了在其他检测组织中(如膜细胞)不存在运行的生物钟发条机制。比较颗粒细胞中生物钟节律的强度和时相状态(标志性时钟基因的峰值时间)发现,F1中节律震荡最强,F2次之,F3最小,但它们之间的时相状态没有明显区别。输卵管的子宫部时相状态要显著早于伞部。3.促黄体素对时钟基因表达的调控及机理研究本研究通过添加特异性的信号通路阻断剂,从时钟基因的表达变化来探索促黄体素(Luteinizing hormone, LH)对时钟基因调控的分子机理。体外培养的F1颗粒细胞施加地塞米松同步化处理作为对照组,试验组添加LH处理。结果发现,24小时周期内,对照组和试验组中时钟基因表达趋势都较平缓,余弦分析节律性时均未达到显著水平。施加LH处理4个小时后(Zeitgeber Time 4, ZT4),Bmall的表达量显著增高；Per2在ZT4和ZT8两个时间点试验组均显著的高于对照组；Clock在LH处理后表达不再有任何波动变化；Cryl在ZT12,ZT20,ZT24三个时间点表达量都是试验组显著的高于对照组。添加cAMP抑制剂(H89)可以阻断LH对时钟基因Bmal1和Per2的促进作用,证明了LH对时钟基因的调控依赖于cAMP/PKA信号通路。单独抑制Erkl/2和p38MAPK信号通路对Bmal1的阻断作用不显著,但都显著的阻断了对Per2的促进作用,当同时抑制Erkl/2和p38MAPK时,Bmal1和Per2的表达均接近对照组,显著的低于LH处理组,证明了Erkl/2和p38MAPK两条通路协同作用传导LH带来的胞外信号。4.全面筛选F1卵泡颗粒细胞中的时控基因本研究采用高通量测序技术检测了F1卵泡中颗粒细胞在一个完整昼夜周期内的表达谱,通过"JTK CYCLE"算法筛选时控基因(受生物钟发条机制调控的基因),全景式的描述卵泡颗粒细胞生物钟参与的生理过程和调控通路。结果表明：365个基因具有昼夜节律性表达,调控和参与了众多的生理功能和生物学过程,主要富集在核糖体蛋白和剪切蛋白通路。核糖体蛋白和肿瘤发生有密切的关系,因此母鸡卵泡可作为研究卵巢癌的模型；剪切蛋白负责的剪切作用是一种重要的转录后修饰,表明发条机制不仅通过转录因子来调控下游基因的表达,还可以通过剪切作用影响下游基因的最终丰度。综上,本研究以昼夜生物节律为主线,通过检测时钟基因的节律性表达在母鸡繁殖系统中对生物钟发条机制进行定位,探索了颗粒细胞中影响时钟基因表达的同步化信号通路,并采用转录组测序筛选得到了颗粒细胞中的时控基因,整体上解析了排卵-产蛋过程中的节律现象,为后续繁殖性能的深入研究提供了借鉴与参考。
[Abstract]:The circadian rhythm affects almost every aspect of organism's life activities. It is one of the basic characteristics of life to help them seek advantages and avoid disadvantages, realize synchronization and adaptation with the external environment. The development of interbiology and reproductive endocrinology provides a theoretical basis for explaining the above phenomena. However, the regulation of the gonadal axis is complicated and interlaced. The maturation of follicles, ovulation and egg formation are multi-tissue, multi-process and multi-level physiological events. Neuroendocrine or follicular development alone can not fully explain the complex and special physiological process of ovulation-laying. This study, starting from the circadian biological clock system, integrates the time control factors of each process, and analyzes the rhythmic phenomena of ovulation-laying. 1. Egg production monitoring and analysis of laying laws. This study recorded the hens in a specific physiological process. The laying traits of commercial laying hens under conventional feeding conditions were obtained, including laying time distribution, continuous laying interval, laying time distribution one day before the interval and laying interval. The laying time of the hens with good egg-laying performance fluctuated about 24 hours. The laying time of the day before the laying interval was densely distributed in the early and late periods, which indicated that the laying interval would be caused by the front and back edges of the laying interval near the opening period. Chickens with the same state (laying time and laying cycle) were used as follow-up materials. 2. The rhythmic expression of clock genes and the localization of clockwise mechanism This study examined the expression of clock genes (Bmall, Bmal2, Clock, Per2, Per3, Cryl, Cry2) in granulosa cells and membranous cells of graded follicles and four parts of fallopian tubes. Cosine analysis was used to detect whether the clocks exhibited significant rhythmic oscillations. Combined with the molecular mechanism of biological clock operation, the clocks were combined in a circadian cycle to determine whether there was a clockwork mechanism in the tissue according to the peak time relationship. The granulosa cells of F3 follicles, the umbrella and uterus of the fallopian tube were present, and the clockwork mechanism of the biological clock was determined to be absent in other tissues (e.g. membranous cells). Comparing the intensity and phase state of the biological clock rhythm in granulosa cells (the peak time of the marker clock gene), the rhythmic oscillation was strongest in F1, followed by F2. Luteinizing hormone (Luteinizing hormone) was studied by adding specific signal pathway blockers to explore the regulation and mechanism of clock gene expression. Dexamethasone synchronization was applied to F1 granulosa cells cultured in vitro as the control group and LH was added to the experimental group. The results showed that the clock gene expression trend in the control group and the experimental group was more gentle within 24 hours, and the rhythm of cosine analysis did not reach a significant level. Four hours later (Zeitgeber Time 4, ZT4), the expression of Bmall was significantly increased; Per2 was significantly higher in the experimental group than in the control group at ZT4 and ZT8 time points; Clock was no longer fluctuated after LH treatment; Cryl was significantly higher in the experimental group at ZT12, ZT20, ZT24 time points than in the control group. H89) blocked the promotion of LH on clock genes Bmal1 and Per2, and proved that the regulation of LH on clock genes depended on the cAMP/PKA signaling pathway. Inhibition of Erkl/2 and p38MAPK signaling pathways alone did not significantly block the promotion of Bmal1, but both significantly blocked the promotion of Per2. When Erkl/2 and p38MAPK were inhibited simultaneously, the expression of Bmal1 and Per2 was inhibited. The expression profiles of granulosa cells in F1 follicles during a full day-night cycle were detected by high-throughput sequencing. The expression profiles of granulosa cells in F1 follicles during a full day-night cycle were determined by "J The TK CYCLE algorithm screens time-controlled genes (genes regulated by the clockwork mechanism of biological clock) and describes the physiological processes and regulatory pathways involved in the biological clock of follicular granulosa cells panoramically. The ribosomal protein is closely related to tumorigenesis, so the hen follicles can be used as a model for studying ovarian cancer; the shearing of shearing proteins is an important post-transcriptional modification, indicating that the cloning mechanism not only regulates the expression of downstream genes by transcription factors, but also by shearing. To sum up, this study focused on circadian biological rhythm, by detecting the circadian expression of clock genes in hen reproductive system to locate the clockwise mechanism of biological clock, explored the synchronization signal pathway affecting clock gene expression in granulosa cells, and screened the granules by transcriptome sequencing. The time-regulated genes in the cells, as a whole, analyze the rhythm of ovulation-laying process, and provide a reference for further study of reproductive performance.
【学位授予单位】：四川农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S831
本文编号：2189668
[Abstract]:The circadian rhythm affects almost every aspect of organism's life activities. It is one of the basic characteristics of life to help them seek advantages and avoid disadvantages, realize synchronization and adaptation with the external environment. The development of interbiology and reproductive endocrinology provides a theoretical basis for explaining the above phenomena. However, the regulation of the gonadal axis is complicated and interlaced. The maturation of follicles, ovulation and egg formation are multi-tissue, multi-process and multi-level physiological events. Neuroendocrine or follicular development alone can not fully explain the complex and special physiological process of ovulation-laying. This study, starting from the circadian biological clock system, integrates the time control factors of each process, and analyzes the rhythmic phenomena of ovulation-laying. 1. Egg production monitoring and analysis of laying laws. This study recorded the hens in a specific physiological process. The laying traits of commercial laying hens under conventional feeding conditions were obtained, including laying time distribution, continuous laying interval, laying time distribution one day before the interval and laying interval. The laying time of the hens with good egg-laying performance fluctuated about 24 hours. The laying time of the day before the laying interval was densely distributed in the early and late periods, which indicated that the laying interval would be caused by the front and back edges of the laying interval near the opening period. Chickens with the same state (laying time and laying cycle) were used as follow-up materials. 2. The rhythmic expression of clock genes and the localization of clockwise mechanism This study examined the expression of clock genes (Bmall, Bmal2, Clock, Per2, Per3, Cryl, Cry2) in granulosa cells and membranous cells of graded follicles and four parts of fallopian tubes. Cosine analysis was used to detect whether the clocks exhibited significant rhythmic oscillations. Combined with the molecular mechanism of biological clock operation, the clocks were combined in a circadian cycle to determine whether there was a clockwork mechanism in the tissue according to the peak time relationship. The granulosa cells of F3 follicles, the umbrella and uterus of the fallopian tube were present, and the clockwork mechanism of the biological clock was determined to be absent in other tissues (e.g. membranous cells). Comparing the intensity and phase state of the biological clock rhythm in granulosa cells (the peak time of the marker clock gene), the rhythmic oscillation was strongest in F1, followed by F2. Luteinizing hormone (Luteinizing hormone) was studied by adding specific signal pathway blockers to explore the regulation and mechanism of clock gene expression. Dexamethasone synchronization was applied to F1 granulosa cells cultured in vitro as the control group and LH was added to the experimental group. The results showed that the clock gene expression trend in the control group and the experimental group was more gentle within 24 hours, and the rhythm of cosine analysis did not reach a significant level. Four hours later (Zeitgeber Time 4, ZT4), the expression of Bmall was significantly increased; Per2 was significantly higher in the experimental group than in the control group at ZT4 and ZT8 time points; Clock was no longer fluctuated after LH treatment; Cryl was significantly higher in the experimental group at ZT12, ZT20, ZT24 time points than in the control group. H89) blocked the promotion of LH on clock genes Bmal1 and Per2, and proved that the regulation of LH on clock genes depended on the cAMP/PKA signaling pathway. Inhibition of Erkl/2 and p38MAPK signaling pathways alone did not significantly block the promotion of Bmal1, but both significantly blocked the promotion of Per2. When Erkl/2 and p38MAPK were inhibited simultaneously, the expression of Bmal1 and Per2 was inhibited. The expression profiles of granulosa cells in F1 follicles during a full day-night cycle were detected by high-throughput sequencing. The expression profiles of granulosa cells in F1 follicles during a full day-night cycle were determined by "J The TK CYCLE algorithm screens time-controlled genes (genes regulated by the clockwork mechanism of biological clock) and describes the physiological processes and regulatory pathways involved in the biological clock of follicular granulosa cells panoramically. The ribosomal protein is closely related to tumorigenesis, so the hen follicles can be used as a model for studying ovarian cancer; the shearing of shearing proteins is an important post-transcriptional modification, indicating that the cloning mechanism not only regulates the expression of downstream genes by transcription factors, but also by shearing. To sum up, this study focused on circadian biological rhythm, by detecting the circadian expression of clock genes in hen reproductive system to locate the clockwise mechanism of biological clock, explored the synchronization signal pathway affecting clock gene expression in granulosa cells, and screened the granules by transcriptome sequencing. The time-regulated genes in the cells, as a whole, analyze the rhythm of ovulation-laying process, and provide a reference for further study of reproductive performance.
【学位授予单位】：四川农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S831
【参考文献】
相关期刊论文 前1条
1 耿照玉;邱祥聘;曾繁同;周铁茅;;蛋鸡快慢羽纯系及其杂交鸡连产对产蛋性能影响的研究[J];四川农业大学学报;1988年03期
，本文编号：2189668
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