發(fā)布時間：2018-04-26 05:07

  本文選題：生物鐘節(jié)律 + CRY基因��； 參考：《中國農(nóng)業(yè)大學》2016年博士論文

【摘要】：地球上幾乎所有的生物都具有生物鐘。在哺乳動物中,生物的主生物鐘定位于大腦內(nèi)被稱之為視交叉上核(Suprachiasmatic nucleus, SCN)的核團中,SCN核團能夠通過光信號同步化生物體外周生物鐘。實際上,在單個細胞的水平就存在生物鐘,生物鐘可以是細胞水平的獨立行為。最被廣泛接受的生物鐘分子機制是一個基于轉(zhuǎn)錄翻譯的負反饋環(huán)路模型,包括兩個轉(zhuǎn)錄激活因子(BMAL1蛋白和CLOCK蛋白)和兩類轉(zhuǎn)錄抑制因子(PERIOD蛋白,或稱作PER1/2/3蛋白；和CRYPTOCHROME蛋白,即CRY1/2蛋白)維持的近二十四小時的負反饋環(huán)路�；谶@個負反饋模型,生物鐘系統(tǒng)再通過一系列鐘控基因(Clock-Controlled Gene, CCG基因)調(diào)控著生物的行為及生理節(jié)律。在哺乳動物細胞內(nèi),至少有10%的轉(zhuǎn)錄組顯示出周期性的轉(zhuǎn)錄表達,而其中大部分的基因都是生物鐘控制的基因。正向轉(zhuǎn)錄因子BMAL1基因或(CLOCK基因的周期性表達對于生物鐘的產(chǎn)生不是必需的,但是周期性的負向調(diào)控因子(PER基因或CRY基因)的表達對于生物鐘的形成至關(guān)重要。在生物鐘的研究中PER蛋白與CRY蛋白被視為一個PER復合物來研究,PER蛋白的重要性被廣泛接受。雖然很早就發(fā)現(xiàn)CRY1基因和CRY2基因可以導致不同的生物鐘周期,但是關(guān)于CRY基因調(diào)控生物鐘周期的機制卻沒有完善的研究。盡管到目前為止CRY蛋白的穩(wěn)定性模型是解釋CRY蛋白調(diào)控生物鐘周期最為廣泛接受的模型。在我們的實驗中,我們首先發(fā)現(xiàn)了在內(nèi)源CRY1基因啟動子驅(qū)動下的CRY2基因單獨表達也可以恢復CRY基因缺失細胞系的生物鐘表型。在對CRY蛋白穩(wěn)定性進行分析時,我們發(fā)現(xiàn)雖然CRY2蛋白的穩(wěn)定性較強,但是相比穩(wěn)定性較弱的CRY1蛋白卻表現(xiàn)出短周期的生物鐘節(jié)律,這與之前關(guān)于CRY蛋白穩(wěn)定性調(diào)控生物鐘周期的模型相矛盾。我們還發(fā)現(xiàn)CRY2蛋白的N端序列決定了CRY2蛋白比CRY1蛋白更加穩(wěn)定,此外CRY2蛋白與CRYI蛋白中賴氨酸殘基的數(shù)量可能也是蛋白穩(wěn)定性的決定因素。由CRY2蛋白N端序列決定的蛋白穩(wěn)定性不能直接影響生物鐘的周期長度,反而是不能夠明顯影響CRY1蛋白與CRY2蛋白穩(wěn)定性的C端序列決定了生物鐘的周期長度。最后,我們還發(fā)現(xiàn)了CRY1蛋白與轉(zhuǎn)錄延伸因子P-TEFb(positive transcriptional elongation factor b)復合物以及7SK snRNP (small nuclear ribonucleoprotein complex)復合物之間的緊密聯(lián)系,從而揭示了CRY蛋白通過7SK RNA行使生物鐘轉(zhuǎn)錄抑制活性的可能機制。我們的研究證明了長久以來一直存在的通過CRY1蛋白與CRY2蛋白的比例調(diào)控生物鐘周期的假設(shè)。更精確的說,我們的實驗證明了不是通過單獨的PER蛋白或者CRY蛋白,而是由這兩個蛋白組成的PER復合物的入核速率決定了生物鐘的周期長度。同時我們的研究顯示哺乳動物CRY1蛋白可能是通過靶向失活P-TEFb復合物完成轉(zhuǎn)錄抑制功能。我們的研究解釋了生物鐘的周期調(diào)控機制和負反饋抑制機制,完整了生物鐘負反饋環(huán)路的模型。
[Abstract]:Almost all living things on earth have biological clocks. In mammals, the main biological clock of organisms is located in the nucleus Suprachiasmatic nucleus (SCN) of the brain called Suprachiasmatic nucleus (SCNs). In fact, there is a biological clock at the level of a single cell, which can be an independent behavior at the cellular level. The most widely accepted molecular mechanism of the biological clock is a negative feedback loop model based on transcriptional translation, consisting of two transcriptional activators, BMAL1 and CLOCK, and two types of transcription suppressor, PERIOD, or PER1/2/3 protein; and CRYPTOCHROME protein. CRY1/2 protein) maintains a negative feedback loop for nearly 24 hours. Based on the negative feedback model, the biological clock system regulates the biological behavior and physiological rhythm through a series of clock-controlled genes (CCG gene). In mammalian cells, at least 10% of the transcriptome shows cyclic transcriptional expression, and most of the genes are controlled by the biological clock. The periodic expression of forward transcription factor (BMAL1) gene or CRY gene is not necessary for the production of biological clock, but the expression of periodic negative regulatory factor (per gene or CRY gene) is very important for the formation of biological clock. In the study of biological clock, the importance of PER protein and CRY protein being regarded as a PER complex to study per protein is widely accepted. Although it has long been discovered that CRY1 gene and CRY2 gene can lead to different circadian cycle, the mechanism of CRY gene regulating clock cycle is not well studied. Although by far the stability model of CRY protein is the most widely accepted model to explain the circadian clock cycle regulated by CRY protein. In our experiment, we first found that the expression of CRY2 gene driven by endogenous CRY1 gene promoter could also restore the clock phenotype of CRY gene deletion cell line. In the analysis of the stability of CRY protein, we found that although the stability of CRY2 protein is stronger than that of CRY1 protein with less stability, it shows a short cycle circadian rhythm. This contradicts previous models for the stability of CRY proteins to regulate the circadian clock cycle. We also found that the N-terminal sequence of CRY2 protein determines that CRY2 protein is more stable than CRY1 protein, and the number of lysine residues in CRY2 protein and CRYI protein may also be the determinants of protein stability. The protein stability determined by the N-terminal sequence of CRY2 protein does not directly affect the cycle length of the clock, but the C-terminal sequence, which can not affect the stability of the CRY1 protein and the CRY2 protein, determines the cycle length of the clock. Finally, we also found the close relationship between CRY1 protein and P-TEFb(positive transcriptional elongation factor b) complex and 7SK snRNP small nuclear ribonucleoprotein complex) complex, which revealed the possible mechanism of CRY protein exerting transcriptional inhibitory activity through 7SK RNA. Our research supports the long-established hypothesis that the circadian cycle is regulated by the ratio of CRY1 protein to CRY2 protein. To be more precise, our experiments show that the cycle length of the biological clock is determined not by a single PER protein or CRY protein, but by the nucleation rate of the PER complex composed of these two proteins. At the same time, our study suggests that mammalian CRY1 protein may be transcriptional inhibitory by targeting inactivated P-TEFb complexes. Our study explains the periodic regulation mechanism and negative feedback inhibition mechanism of biological clock, and completes the model of biological clock negative feedback loop.
【學位授予單位】：中國農(nóng)業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q953

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