蓝光诱导拟南芥隐花色素CRY2二聚体的形成及调控机制

发布时间：2018-02-11 07:13

本文关键词： HEK293T CRY2 二聚体二硫键 BICs　出处：《吉林大学》2016年博士论文　论文类型：学位论文

【摘要】：隐花色素Cryptochrome(CRY)是一类光裂合酶类似的黄素结合蛋白,包含两个结构域,N端(PHR)与光裂合酶高度同源,结合生色团FAD,C端结构域被认为是隐花素特有的一段延伸区(CCE),该区域呈现出高度的可变性、非结构化,但对于隐花素的功能至关重要。拟南芥CRY1和CRY2主要介导了蓝光对下胚轴生长的抑制和光周期控制的开花。已有证据表明,拟南芥CRY的二聚化对其起始蓝光信号转导至关重要,但各原初反应之间的相互关系尚不清楚。围绕这一课题的研究,首先建立了利用HEK293T表达植物隐花色素CRY2的蛋白表达系统,并对CRY2生物学功能进行了鉴定。其次利用该系统首次直接观察到CRY2蛋白受蓝光特异诱导的二聚及多聚化现象。在此基础上,利用多种氧化还原剂,进一步研究证明蓝光下形成的蛋白二聚体、多聚体的形成是依靠半胱氨酸介导的二硫键完成的。进一步证明了拟南芥CRY2的十一个半胱氨酸中,哪些半胱氨酸在这一过程中发挥作用。拟南芥中的功能未知蛋白BIC1/BIC2能够负调控隐花色素。过表达BIC1或BIC2均可获得类似cry1/cry2双突变体的表型。在此基础上,首先研究了CRY2与BICs的关系,通过Co-IP实验证明了CRY2与BICs能够互作,并且表现出较强的光响应。进而研究BIC如何影响CRY2,利用多种手段都证实了BICs能够直接影响CRY2蓝光下二聚体、多聚体的形成。进一步通过Co-IP,Split-luc实验证明了BIC存在的情况下,蓝光下能够影响CRY2-CRY2互作。通过上述研究获得以下结论:1、利用优化的HEK293T表达系统表达的隐花色素CRY2能够吸收蓝光、并且能够发挥出CRY2的正常生物学功能。2、在拟南芥隐花色素CRY2中,半胱氨酸介导的二硫键在蛋白蓝光特异的二聚体多聚体形成过程中发挥着重要的作用。并且蛋白二聚体、多聚体的形成依赖于细胞的氧化态微环境,强还原剂存在条件下不能形成蛋白二聚体、多聚体。CRY2十一个半胱氨酸中CRY2C89S,在这个点突变体中CRY2二聚体多聚体几乎全部消失,CRY2C39S、CRY2C158S、以及CRY2C583S蛋白二聚体多聚体也存在明显减弱,说明这些半胱氨酸是形成二聚体、多聚体的关键半胱氨酸。3、BICs能够抑制隐花色素调控的光形态建成,并且能够抑制CRY2蓝光下的磷酸化以及降解。这种抑制功能的发挥是依靠与CRY2的直接互作完成的。BIC蓝光下能够抑制CRY2蛋白二聚体以及多聚体的形成进而调控植物生长发育。
[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.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q945

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