【作者】 王琴；

【导师】 刘选明； 林辰涛；

【作者基本信息】 湖南大学 ， 分析化学， 2013， 博士

【摘要】 植物的生长发育都依赖光，光不仅是植物光合作用所必需的，还是植物感受外部环境信息所必需的。这种光依赖的植物生长发育过程为植物的光形态建成过程。在模式植物拟南芥中，光形态建成的分子机制已被广泛研究。目前已知有几种光受体介导光形态建成的信号传递，包括红光和远红光光受体光敏色素、蓝光和紫外光光受体隐花色素、向光素及含LOV结构域的F-box家族蛋白ZTL/FKF/LKP2。这些蛋白通过与它们各自的信号蛋白发生光依赖的相互作用而启动信号的传递过程，从而调节植物的光形态建成。然而目前对于这些信号传递的具体过程的研究尚不清楚。本论文主要探讨与隐花色素CRY2互作的γCAL2和CIU1的遗传学功能与生物化学特性，以及蓝光依赖的CRY2降解的生化特性及参与CRY2降解的相关遗传因子，获得了如下研究结果。亚细胞定位研究发现，γCAL2仅定位在线粒体中，由于CRY2是核蛋白，因此推测γCAL2可能不是CRY2的信号传递蛋白。对γCAL2的遗传学功能及生物化学特性进行研究，发现拟南芥中的γCAL1和γCAL2都突变后导致胚胎发育异常及种子不萌发；表达了γCAL2RNAi载体的γcal1突变体(c1c2i)幼苗具有类似cop的持续光形态建成表型，而且c1c2i成年植物对光周期的敏感性降低。这些结果表明γCAL2及与之同源的γCAL1在植物的光形态建成过程中起重要作用，首次证明线粒体复合体I中的γCA亚复合体对植物发育的重要性。对CRY2与CIU1的相互作用进行深入研究发现，CIU1在蓝光下能够与CRY2相互作用，它们的互作不仅仅是蓝光依赖性的也是蓝光强度依赖性的；CIU1蛋白与CRY2的N端PHR结构域相互作用，CIU1通过其N端的前135个氨基酸（CIU1N135）与CRY2互作。免疫共沉淀实验进一步证明CRY2与CIU1相互作用，表明这两个蛋白在体内通过形成复合体而对蓝光作出响应。将CIU1过量表达到拟南芥中，发现该转基因植物表现出晚花表型，由于CRY2的主要功能之一是调节光周期开花，因此CIU1很可能参与调节CRY2的功能。鉴于蓝光依赖的CRY2蛋白的泛素化及降解在整个CRY2信号转导与调节过程中的重要性，本论文的第二部分着重于对蓝光依赖的CRY2蛋白泛素化及降解的生物化学及遗传方面的机理研究。第一，通过检测CRY2色氨酸三联体突变体在蓝光下的降解，发现CRY2色氨酸三联体突变体蛋白的降解减慢了，且CRY2色氨酸三联体突变体蛋白降解后会出现提前累积的现象，后经翻译抑制剂CHX处理植物后，发现CRY2色氨酸三联体突变体蛋白不再有累积现象，说明这种累积现象是由翻译的加强引起的。第二，通过检测CRY2在co，fd，ft，ft tsf，flc，toc1，cca1lhy，prr1579，ztl，ztllkp2，ztllkp2fkf1和hmr-1不同光信号传递分子突变体内的降解情况，发现CRY2在ft，ft tsf，flc和cca1lhy突变体内能够正常降解，且CRY2在ztl，ztllkp2和ztllkp2fkf1突变体中也降解正常，说明ZTL/LKP2/FKF1并不是蓝光下介导CRY2降解的光受体，CRY2在co，fd，toc1和prr1579突变体内的降解变慢，而在hmr-1突变体内的降解加快。第三，通过免疫印迹检测CRY2在ubR48过表达植株体内的降解情况，发现诱导表达ubR48能够抑制CRY2蛋白的降解，从而说明CRY2蛋白泛素链的形成依赖于赖氨酸48（Lys48）。第四，通过检测CRY2在E3泛素连接酶复合体突变体cul1和cop1突变体内的降解发现，CRY2在这两类突变体内的降解被抑制，证明光激活态的CRY2蛋白的泛素化至少需要基于Cul1的SCF类E3泛素连接酶及基于Cul4的COP1E3泛素连接酶的参与。第五，通过定量分析蓝光下野生型植物体内CRY2mRNA的变化，首次发现蓝光下调CRY2的mRNA表达，这说明蓝光不仅调节CRY2蛋白泛素化与降解，且调节CRY2mRNA的表达。为了找到参与CRY2泛素化降解的基因，利用LUC-CRY2转基因拟南芥，通过EMS诱变，构建了突变体库。目前已从中筛选到多个影响CRY2降解的突变体，这为今后深入研究CRY2在植物光形态建成中的作用及其信号转导与调节机制奠定了基础。更多还原

【Abstract】 Plant growth and development are dependent on light for not onlyphotosynthetic energy supply but also for environmental information acquisition.This light-dependent plant developmental process is referred to asphotomorphogenesis. The molecular mechanism underlying photomorphogenesishas been extensively studied in the model plant Arabidopsis. It is currently knownthat several photoreceptors, including the red/far-red light receptor phytochromes,and blue/UV-A light receptors cryptochromes, phototropins and LOV-domain F-box receptors ZTL/FKF/LKP2mediate light signals to regulatephotomorphogenesis. It has also been discovered that those photoreceptorsundergo light-dependent interaction with respective signaling partners to triggersignal transduction process controlling photomorphogenesis. However, manydetails of the process remain unclear. The dissertation research mainly concernson the genetic function and biochemical property of two putative CRY-interactingproteins, γCAL2and CIU1, and also concerns on the biochemical property of bluelight-dependent CRY2degradation and the genetic components associated withCRY2degradation.γCAL2is located exclusively in the mitochondria, according to thesubcellular localization experiment, whereas CRY2is an exclusively nuclearphotoreceptor, thus γCAL2is unlikely a bona fide CRY2-signaling protein. Anumber of genetics and photomorphogenic analyses were performed to furtherinvestigate the function of γCAL2. It was found that mutations of the γCAL1andγCAL2in Arabidopsis result in defective embryogenesis and non-germinatingseeds, and the γcal1mutant plant expressing the RNAi (referred as c1c2i)construct of the γCAL2gene showed a partial cop (constitutive photomorphogenic)phenotype in young seedlings, and a reduced photoperiodi c sensitivity in adultplants. All these results demonstrate that γ-CAL2and its close homolog γCAL1play important roles in photomorphogenesis and for the first time demonstrate thatthe functional significance of the γCA subcomplex of mitochondrial complex I inplant development.A detailed investigation of the physical interaction between CRY2and CIU1demonstrates that CIU1undergoes blue light-dependent interaction with CRY2, which is not only wavelength-specific but also fluence rate-dependent. Moreover,the interacting domains for the two proteins have been narrowed down to theCRY2-PHR domain and the CIU1N terminal135amino acid region (CIU1N135).The blue light-stimulated CRY2-CIU1interaction has also been shown by the co-IP assay, suggesting that the two proteins indeed form complex in response toblue light. CIU1-overexpressing transgenic plants showed delayed floweringphenotype. Because regulation of photoperiodic flowering is a major function ofCRY2, this result indicates that CIU1is likely to involve in CRY2functionregulation.Given the importance of the blue light-dependent ubiquitination anddegradation of CRY2in the overall CRY2signal transduction and regulation, thesecond part of this dissertation is focused on the biochemistry and geneticsmechanism studies underlying the blue light-dependent CRY2ubiquitination anddegradation. And the following results are obtained:(1) CRY2trp-triad mutantproteins degrade slower than wild type CRY2protein, and the mutant proteinsaccumulate right after degradation. The accumulation of the mutant proteins afterexposing to blue light can be eliminated by treating with CHX, suggestion that theaccumulation of CRY2trp-triad mutant proteins is caused by acceleratingtranslation.(2) After checking the degradation of CRY2in co, fd, ft, ft tsf, flc,toc1, cca1lhy, prr1579, ztl, ztllkp2, ztllkp2fkf1and hmr-1mutants, we found thatCRY2degraded normally in ft, ft tsf, flc,cca1lhy, ztl, ztllkp2and ztllkp2fkf1mutants and degraded slower in co, fd, toc1and prr1579, but accelerated itsdegradation in hmr-1mutant. The normal degradations of CRY2in ztl, ztllkp2andztllkp2fkf1indicate that ZTL/LKP2/FKF1are not the photoreceptor mediatingCRY2ubiquitination and degradation in response to blue light.(3) Thedegradation of CRY2is impaired in ubR48overexpressing plants, indicating thatubiquitination of CRY2is involved with Lysine48-dependent polyubiquitinationpathway.(4) The slow degradation of CRY2in cul1and cop1mutants suggest thatthe photoexcited CRY2is ubiquitinated by at least two types of E3ubiquitinligases, the Cul1-dependend SCF E3ubiquitin ligase and Cul4-dependent COP1E3ubiquitin ligase.(5) For the first time we found that the blue light-dependentregulation of CRY2expression may involve not only ubiquitination anddegradation of the CRY2protein, but also altered homeostasis of the CRY2mRNA.A LUC-CRY2EMS based genetics study was initiated to screen for genes involving in the blue light-dependent CRY2ubiquitination and degradation. Manymutations were isolated and studied. The results of this experiment will pave theway for future studies of CRY2signal transduction and regulation as well as therole of CRY photoreceptor signal transduction and regulation in plantphotomorphogenesis.更多还原