時(shí)鐘基因在母雞生殖系統(tǒng)中的節(jié)律性表達(dá)及顆粒細(xì)胞中時(shí)控基因的篩選
發(fā)布時(shí)間：2018-08-18 13:53
【摘要】：晝夜節(jié)律幾乎影響了生物體生命活動(dòng)的方方面面,幫助其趨利避害,實(shí)現(xiàn)和外界環(huán)境的同步和適應(yīng),是生命的基本特征之一。雌禽的排卵-產(chǎn)蛋循環(huán)便具有明顯的節(jié)律性和環(huán)境適應(yīng)性,具體表現(xiàn)在排卵(產(chǎn)蛋)時(shí)間偏好、連產(chǎn)間歇等方面。時(shí)間生物學(xué)和生殖內(nèi)分泌學(xué)的發(fā)展為解釋上述現(xiàn)象提供了理論基礎(chǔ),但性腺軸上的調(diào)控復(fù)雜交錯(cuò),卵泡的成熟,排卵和蛋的形成是多組織、多過程、多層次參與的生理事件,其間不同生理進(jìn)程在時(shí)間上的吻合顯示了機(jī)體自身的協(xié)調(diào)統(tǒng)一。單獨(dú)研究神經(jīng)內(nèi)分泌或卵泡的發(fā)育無法全面的解釋排卵-產(chǎn)蛋這一復(fù)雜又特殊的生理過程,本研究從晝夜生物鐘系統(tǒng)切入,整合各過程的時(shí)間控制因素,解析排卵-產(chǎn)蛋中的節(jié)律現(xiàn)象。1.產(chǎn)蛋監(jiān)測與產(chǎn)蛋規(guī)律分析本研究通過記錄母雞在特定時(shí)間段里的具體產(chǎn)蛋時(shí)間,得到了商品蛋雞羅曼粉殼蛋雞在常規(guī)飼養(yǎng)條件下的產(chǎn)蛋性狀,包括：產(chǎn)蛋時(shí)間分布、連產(chǎn)間歇、間歇前一天產(chǎn)蛋時(shí)間分布和產(chǎn)蛋間隔時(shí)間。結(jié)果發(fā)現(xiàn)：母雞產(chǎn)蛋有極強(qiáng)的時(shí)間偏好性,主要集中在8：00一12：00這個(gè)時(shí)段：產(chǎn)蛋性能好的母雞產(chǎn)蛋時(shí)間間隔以24小時(shí)為軸心左右波動(dòng)。連產(chǎn)間歇前一天的產(chǎn)蛋時(shí)間在早、晚兩個(gè)時(shí)段密集分布,說明接近開放期的前后邊緣都會(huì)造成產(chǎn)蛋間歇。證實(shí)了產(chǎn)蛋開放期模型。通過產(chǎn)蛋規(guī)律的監(jiān)測,挑選出了生理狀態(tài)一致(產(chǎn)蛋時(shí)間和產(chǎn)蛋周期一致)的雞群作為后續(xù)的試驗(yàn)材料。2.時(shí)鐘基因的節(jié)律性表達(dá)與發(fā)條機(jī)制的定位本研究通過檢測時(shí)鐘基因(Bmall,Bmal2,Clock, Per2, Per3,Cryl,Cry2)在等級(jí)卵泡的顆粒細(xì)胞和膜細(xì)胞以及輸卵管的四個(gè)部位中的表達(dá)情況,運(yùn)用余弦分析法檢測其表達(dá)是否呈現(xiàn)顯著性節(jié)律震蕩,結(jié)合生物鐘運(yùn)行的分子機(jī)制,將時(shí)鐘基因在一個(gè)晝夜周期內(nèi)的表達(dá)規(guī)律加以組合,根據(jù)峰值時(shí)間關(guān)系確定生物鐘發(fā)條機(jī)制在該組織中是否存在。結(jié)果表明：生物鐘發(fā)條機(jī)制在F1-F3卵泡的顆粒細(xì)胞以及輸卵管的傘部和子宮部中存在,并且確定了在其他檢測組織中(如膜細(xì)胞)不存在運(yùn)行的生物鐘發(fā)條機(jī)制。比較顆粒細(xì)胞中生物鐘節(jié)律的強(qiáng)度和時(shí)相狀態(tài)(標(biāo)志性時(shí)鐘基因的峰值時(shí)間)發(fā)現(xiàn),F1中節(jié)律震蕩最強(qiáng),F2次之,F3最小,但它們之間的時(shí)相狀態(tài)沒有明顯區(qū)別。輸卵管的子宮部時(shí)相狀態(tài)要顯著早于傘部。3.促黃體素對時(shí)鐘基因表達(dá)的調(diào)控及機(jī)理研究本研究通過添加特異性的信號(hào)通路阻斷劑,從時(shí)鐘基因的表達(dá)變化來探索促黃體素(Luteinizing hormone, LH)對時(shí)鐘基因調(diào)控的分子機(jī)理。體外培養(yǎng)的F1顆粒細(xì)胞施加地塞米松同步化處理作為對照組,試驗(yàn)組添加LH處理。結(jié)果發(fā)現(xiàn),24小時(shí)周期內(nèi),對照組和試驗(yàn)組中時(shí)鐘基因表達(dá)趨勢都較平緩,余弦分析節(jié)律性時(shí)均未達(dá)到顯著水平。施加LH處理4個(gè)小時(shí)后(Zeitgeber Time 4, ZT4),Bmall的表達(dá)量顯著增高；Per2在ZT4和ZT8兩個(gè)時(shí)間點(diǎn)試驗(yàn)組均顯著的高于對照組；Clock在LH處理后表達(dá)不再有任何波動(dòng)變化；Cryl在ZT12,ZT20,ZT24三個(gè)時(shí)間點(diǎn)表達(dá)量都是試驗(yàn)組顯著的高于對照組。添加cAMP抑制劑(H89)可以阻斷LH對時(shí)鐘基因Bmal1和Per2的促進(jìn)作用,證明了LH對時(shí)鐘基因的調(diào)控依賴于cAMP/PKA信號(hào)通路。單獨(dú)抑制Erkl/2和p38MAPK信號(hào)通路對Bmal1的阻斷作用不顯著,但都顯著的阻斷了對Per2的促進(jìn)作用,當(dāng)同時(shí)抑制Erkl/2和p38MAPK時(shí),Bmal1和Per2的表達(dá)均接近對照組,顯著的低于LH處理組,證明了Erkl/2和p38MAPK兩條通路協(xié)同作用傳導(dǎo)LH帶來的胞外信號(hào)。4.全面篩選F1卵泡顆粒細(xì)胞中的時(shí)控基因本研究采用高通量測序技術(shù)檢測了F1卵泡中顆粒細(xì)胞在一個(gè)完整晝夜周期內(nèi)的表達(dá)譜,通過"JTK CYCLE"算法篩選時(shí)控基因(受生物鐘發(fā)條機(jī)制調(diào)控的基因),全景式的描述卵泡顆粒細(xì)胞生物鐘參與的生理過程和調(diào)控通路。結(jié)果表明：365個(gè)基因具有晝夜節(jié)律性表達(dá),調(diào)控和參與了眾多的生理功能和生物學(xué)過程,主要富集在核糖體蛋白和剪切蛋白通路。核糖體蛋白和腫瘤發(fā)生有密切的關(guān)系,因此母雞卵泡可作為研究卵巢癌的模型；剪切蛋白負(fù)責(zé)的剪切作用是一種重要的轉(zhuǎn)錄后修飾,表明發(fā)條機(jī)制不僅通過轉(zhuǎn)錄因子來調(diào)控下游基因的表達(dá),還可以通過剪切作用影響下游基因的最終豐度。綜上,本研究以晝夜生物節(jié)律為主線,通過檢測時(shí)鐘基因的節(jié)律性表達(dá)在母雞繁殖系統(tǒng)中對生物鐘發(fā)條機(jī)制進(jìn)行定位,探索了顆粒細(xì)胞中影響時(shí)鐘基因表達(dá)的同步化信號(hào)通路,并采用轉(zhuǎn)錄組測序篩選得到了顆粒細(xì)胞中的時(shí)控基因,整體上解析了排卵-產(chǎn)蛋過程中的節(jié)律現(xiàn)象,為后續(xù)繁殖性能的深入研究提供了借鑒與參考。
[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.
【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S831
本文編號(hào)：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.
【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S831
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 耿照玉;邱祥聘;曾繁同;周鐵茅;;蛋雞快慢羽純系及其雜交雞連產(chǎn)對產(chǎn)蛋性能影響的研究[J];四川農(nóng)業(yè)大學(xué)學(xué)報(bào);1988年03期
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