本文關鍵詞： HEK293T CRY2 二聚體 二硫鍵 BICs　出處：《吉林大學》2016年博士論文　論文類型：學位論文

【摘要】：隱花色素Cryptochrome(CRY)是一類光裂合酶類似的黃素結合蛋白,包含兩個結構域,N端(PHR)與光裂合酶高度同源,結合生色團FAD,C端結構域被認為是隱花素特有的一段延伸區(qū)(CCE),該區(qū)域呈現(xiàn)出高度的可變性、非結構化,但對于隱花素的功能至關重要。擬南芥CRY1和CRY2主要介導了藍光對下胚軸生長的抑制和光周期控制的開花。已有證據(jù)表明,擬南芥CRY的二聚化對其起始藍光信號轉導至關重要,但各原初反應之間的相互關系尚不清楚。圍繞這一課題的研究,首先建立了利用HEK293T表達植物隱花色素CRY2的蛋白表達系統(tǒng),并對CRY2生物學功能進行了鑒定。其次利用該系統(tǒng)首次直接觀察到CRY2蛋白受藍光特異誘導的二聚及多聚化現(xiàn)象。在此基礎上,利用多種氧化還原劑,進一步研究證明藍光下形成的蛋白二聚體、多聚體的形成是依靠半胱氨酸介導的二硫鍵完成的。進一步證明了擬南芥CRY2的十一個半胱氨酸中,哪些半胱氨酸在這一過程中發(fā)揮作用。擬南芥中的功能未知蛋白BIC1/BIC2能夠負調控隱花色素。過表達BIC1或BIC2均可獲得類似cry1/cry2雙突變體的表型。在此基礎上,首先研究了CRY2與BICs的關系,通過Co-IP實驗證明了CRY2與BICs能夠互作,并且表現(xiàn)出較強的光響應。進而研究BIC如何影響CRY2,利用多種手段都證實了BICs能夠直接影響CRY2藍光下二聚體、多聚體的形成。進一步通過Co-IP,Split-luc實驗證明了BIC存在的情況下,藍光下能夠影響CRY2-CRY2互作。通過上述研究獲得以下結論:1、利用優(yōu)化的HEK293T表達系統(tǒng)表達的隱花色素CRY2能夠吸收藍光、并且能夠發(fā)揮出CRY2的正常生物學功能。2、在擬南芥隱花色素CRY2中,半胱氨酸介導的二硫鍵在蛋白藍光特異的二聚體多聚體形成過程中發(fā)揮著重要的作用。并且蛋白二聚體、多聚體的形成依賴于細胞的氧化態(tài)微環(huán)境,強還原劑存在條件下不能形成蛋白二聚體、多聚體。CRY2十一個半胱氨酸中CRY2C89S,在這個點突變體中CRY2二聚體多聚體幾乎全部消失,CRY2C39S、CRY2C158S、以及CRY2C583S蛋白二聚體多聚體也存在明顯減弱,說明這些半胱氨酸是形成二聚體、多聚體的關鍵半胱氨酸。3、BICs能夠抑制隱花色素調控的光形態(tài)建成,并且能夠抑制CRY2藍光下的磷酸化以及降解。這種抑制功能的發(fā)揮是依靠與CRY2的直接互作完成的。BIC藍光下能夠抑制CRY2蛋白二聚體以及多聚體的形成進而調控植物生長發(fā)育。
[Abstract]:Cryptochromefon (CRYY) is a kind of flavin-binding protein similar to photolysin, which contains two domains (N-terminal PHRs) and has high homology with photolytic synthase. The C-terminal domain of the binding chromophore FADN is considered to be an extension of cryptosin, which is highly variable and unstructured. In Arabidopsis thaliana, CRY1 and CRY2 mainly mediate the inhibition of hypocotyls growth and the flowering of photoperiod controlled by blue light. It has been shown that dimerization of Arabidopsis CRY is very important for its initiation of blue light signal transduction. However, the relationship between the primary reactions is still unclear. A protein expression system for the expression of plant cryptoanthocyanin CRY2 by HEK293T was established in this paper. The biological function of CRY2 was identified. Secondly, the dimerization and poly-polymerization of CRY2 protein induced by blue light were observed for the first time by using this system. On the basis of this, a variety of oxidizing reductants were used. Further studies have demonstrated that the formation of protein dimers and polymers under blue light depends on cysteine-mediated disulfide bonds. It is further demonstrated that eleven cysteines in Arabidopsis CRY2, Which cysteine plays a role in this process. BIC1/BIC2, a functional unknown protein in Arabidopsis, can negatively regulate cryptoanthocyanin. Overexpression of BIC1 or BIC2 can produce phenotypes similar to cry1/cry2 double mutants. Firstly, the relationship between CRY2 and BICs is studied, and it is proved by Co-IP experiment that CRY2 and BICs can interact and exhibit strong light response. Then, how BIC affects CRY2 is studied. It is proved by various means that BICs can directly affect the dimer under CRY2 blue light. The formation of polymers. Further, the co-IPN Split-luc experiment proved that in the presence of BIC, the interaction of CRY2-CRY2 can be affected by blue light. From the above study, the following conclusions can be obtained: 1, using the optimized HEK293T expression system, cryptosin CRY2 can absorb blue light. In Arabidopsis thaliana cryptoanthocyanidin CRY2, cysteine-mediated disulfide bonds play an important role in the formation of protein blue-light-specific dimer. The formation of polymer depends on the oxidized microenvironment of the cell, and the protein dimer can not be formed in the presence of strong reductant. CRY2C89Sin 11 cysteines, CRY2C89Sin this point mutant, CRY2 dimer in CRY2C39SMA-CRY2C158S, and CRY2C158Sin CRY2C39Sm, and CRY2C583S protein dimer were also significantly weakened, indicating that these cysteines are dimers. The key cysteine. 3BICs of the polymer can inhibit the photomorphogenesis regulated by cryptoanthocyanin. It can inhibit the phosphorylation and degradation of CRY2 by direct interaction with CRY2, which can inhibit the formation of CRY2 protein dimer and polymer, and then regulate the growth and development of plants.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q945

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