【作者】 张增林；

【导师】 林辰涛； 刘斌；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2014， 博士

【摘要】 蛋白降解作为一类重要的调控方式影响植物发育及响应外界环境变化；其中26S蛋白酶体途径是蛋白降解中最主要的一种方式；荧光素蛋白具有发光灵敏度高、稳定性好，易捕捉及量化等特性，目前已成为了研究蛋白动态变化的有力工具；本研究在合作的模式下，获得了近5000个拟南芥基因的融合荧光素酶植物表达载体，其中获得了1000个基因拟南芥转化材料；依此为基础，通过规模系统化筛选获得了一批在蛋白水平上响应光、生长素、细胞分裂素、独脚金内酯的候选蛋白；为下一步探索环境与植物互作提供了有重要价值的材料。在筛选获得响应环境变化的候选蛋白基础上，深入研究了光响应蛋白COL7的功能。研究表明，该蛋白在高比值红光/远红光条件下影响侧枝发育；进一步分析发现该基因与生长素代谢及光敏色素phyB的功能密t刀相关；遗传和分子证据揭示了COL7介导光信号影响侧枝发育的分子机理,为CO-Z7KE基因家族功能多样性提供了新证据；主要实验结果如下：(1)通过转化拟南芥获得了近1000个基因的融合荧光素酶蛋白转基因株系，为系统性筛选响应外界环境变化的候选蛋白提供了材料；以上述材料为基础，筛选获得了一批响应光、生长素、细胞分裂素、独脚金内酯四种环境信号的候选蛋白，为研究相关环境与植物互作提供了候选基因；(3)利用CHX可以阻断植物蛋白翻译的特点，系统研究了植物蛋白半衰期的规律；发现总体上植物蛋白半衰期呈现泊松分布，曲线的顶点出现在90分钟；并获得了一批半衰期异常的蛋白，推测这些蛋白异常的降解过程，可能作为一种调节手段而影响植物功能；(4)COL7作为CO-LIKE家族成员，蛋白水平有明显的光响应；选取2小时条件，黑暗中降解；远红光和蓝光条件下蛋白积累，在红光条件下出现先积累后降解，并且该蛋白在长日照和短日照下表现出不同的调控模式；(5)过量表达CQL7呈现多分枝的表型，并且随着种植密度的増加，该表型减弱；突变体co/7表型不明显，但掐顶实验表明CO/7与野生型相比，可以形成更少的侧枝；进一步研究发现，COL7仅在高比值红光/远红光条件下促进分枝，并且依赖于有功能的光敏色素phyB;(6)通过t刀根实验、嫁接实验及DR5rev?.GFP为材料的遗传实验等发现COU影响了生长素代谢，推测COU影响侧枝发育可能是通过影响生长素代谢途径实现的；(7)体内Dual-LUC实验表明COL7可以激活具有CCAAT-box顺式元件的启动子，并且酵母中COL7蛋白具有转录激活活性；(8)Q-PCR表明COL7抑制TAA1,7C/C2等一系列与生长素代谢相关基因的表达,但是上调了SUR2基因表达，进一步分析发现，CQL7上调5T/K2的表达是依赖于光敏色素phyB;综上所述，对COL7功能研究发现：光敏色素phyB在一定的光条件下稳定COL7蛋白，而COL7蛋白促进SUR2基因表达并抑制生长素合成，因此COL7可能作为一个关键的蛋白因子介导了光信号对拟南芥分枝发育的调控。更多还原

【Abstract】 Protein degradation is one of the most conspicuous processes involved in the regulations ofdevelopment in response to environmental variations.26s proteasome pathway plays a vital role in theprotein degradation. Luciferase is a powerful reporter for the detection of protein dynamic changesbecause of its high sensitivity, stability and authenticity. In this study, we obtained nearly5000constructs containing different genes fused with luciferase, and obtain a large population of transgenicplants representing1000independent constructs. Based on this transgenic populations, we screenedcandidate proteins, the levels of which is changed in response to light, auxin, cytokinin andstrigolactones. We totally identified262candidate proteins and chose one of them, named COL7for thefunctional study. We found that COL7is unstable in dark but stabilized by phyB in white light.Over-expression of COL7promotes branching in white light with a high red/far red light ratio (R/FR)but not in shade with a low F/FR. The major results of this study were described as following:1.We obtained a large transgenic populations representing1000independent genes fused withluciferase;2.We screen the transgenic population and obtained262candidate genes, the protein level ofwhich response to the light, auxin, cytokinin and strigolactones;3.We study the half-life of plant proteins, the results indicate that the overall half-life distributenormally with the peak at the90min;4.COL7is unstable in dark but accumulates continually in far-red and blue light condition;Interestingly, COL7protein accumulates first but then degrade in red light condition;5.Over-expression of COL7results a more branch phenotype, which could be suppressed whenthe plants were grown with a higher density; Moreover, the function of COL7in branchpromotion is dependent on the phyB;6.Root-excited assay, grafting assay and DR5rec::GFP genetic assay showed that COL7possiblyregulates branch development through the auxin synthesis pathway;7.In vivo dual-luc assay indicated that COL7could activate the recombinant promoter containingthe CCAAT-box, and COL7has the transcriptional activity in yeast. But in vitro DNA-bindingassay indicated that the COL7could not bind the CCAAT box directly, implying that COL7may form protein complex with unknown partners to target the promoter containing theCCAAT-box.8.Real-time PCR results indicated that the COL7could suppress the expression of auxin relatedgenes such as TAA, YUC2. Moreover, it could increase the expression of SUR2, a suppressor ofauxin biosynthesis.In vivo dual-LUC assay indicated that the COL7can activate the expressionof LUC reporter genes under the driven of SUR2promoter.Taking together that the protein stability of COL7is regulated by phyB in response to the lightradiations and COL7enhances branching via activating the expression of SUR2and suppressing the synthesis of auxin, we argue that C0L7isa key factor linking rfom the light perception to branchdevelopment.更多还原