拟南芥转录因子TCP2与CRY1相互作用的遗传学与生化分析
发布时间：2018-07-22 21:19
【摘要】：光通过多种光受体调节植物的生长发育,这些光受体包含光敏色素与隐花色素。拟南芥隐花色素CRY1(CRYPTOCHROME 1)作为蓝光信号受体蛋白,已有许多的研究报道了其在植物蓝光信号转导途径方面的功能,如抑制下胚轴的伸长,控制幼苗的去黄化、生物钟节律,促进植物花青素的积累,影响叶片形态和开花时间等。CRY1蛋白通过与其下游的信号蛋白相互作用,启动蓝光信号的传递,从而促进植物光形态的建成。目前光受体调节光形态建成的机制已得到广泛的研究,但是拟南芥隐花色素CRY1调节光形态建成的信号传导机制目前仍不清楚。研究发现了许多参与CRY1蓝光信号转导并相互作用的蛋白,但转录因子蛋白参与CRY1蓝光信号通路的研究报道目前几乎没有。为了更好地了解和完善CRY1蓝光信号转导机制,本研究通过酵母双杂交的方法,用CRY1作为诱饵筛选转录因子酵母库,获得与CRY1相互作用并可能介导CRY1参与拟南芥光形态建成的转录因子蛋白。具体研究结果如下:通过多次的酵母双杂交(yeast two hybrid assay)试验反复验证以及双分子荧光互补Bi FC(Bimolecular Fluorescence Complementation assay)和免疫共沉淀co-IP(co-immunoprecipitation)试验的进一步验证,证明CRY1与初步筛选到的转录因子蛋白TCP2(TEOSINTE-LIKE1,CYCLO IDEA,and PROLIFERATING CELL FACTOR 2)相互作用。通过烟草和拟南芥中亚细胞定位试验,证明TCP2是一个核蛋白,其可能是与细胞核中的CRY1蛋白相互作用。本研究首次发现并证明了与CRY1直接相互作用的转录因子蛋白-TCP2,其相互作用在酵母细胞以及烟草细胞中表现出明显的蓝光特异性,而在拟南芥细胞中其蓝光特异性依赖特征消失,推测可能是由于酵母及烟草系统中无拟南芥蛋白,而实际相互作用中可能存在其他拟南芥蛋白参与CRY1与TCP2的相互作用。通过生物信息学分析CRY1与TCP2的蛋白结构域,分段克隆CRY1和TCP2的多个结构域片段并通过酵母双杂交以及Bi FC的方法,证明CRY1 N端结构域(该结构域包含1-515的氨基酸序列,涵盖了光裂解酶结构域PHR)能与TCP2的特征结构域TCP(该区域包含TCP2 1-174氨基酸序列)相互作用。TCP结构域中的R结构域影响其与CRY1在酵母细胞中相互作用的蓝光依赖性。遗传学研究结果表明,TCP2蓝光依赖性地抑制拟南芥下胚轴的伸长,这种蓝光特异性不仅表现在光波长的特异性上还表现为光强度的特异性依赖;其在红光和蓝光下能调控拟南芥幼苗的子叶形态,而在远红光下无此功能;其功能缺失突变体具有晚花的表型;我们的研究结果也进一步验证了TCP2蛋白调控叶片形态和大小方面的功能。鉴于光作为环境影响因子在影响植物细胞内蛋白的表达以及CRY1在蓝光信号转导途径上的重要性,我们研究了TCP2蛋白在缺失CRY1与否的情况下对于不同光响应而做出的表达水平变化。通过免疫印迹试验检测Myc-TCP2/WT和Myc-TCP2/cry1转基因材料中TCP2的蛋白变化,结果证明TCP2是一个光响应的蛋白,其会在蓝光下大幅积累而在黑暗和远红光下降解,红光下也会有积累但积累的量和速率远不及蓝光下;同时,CRY1的缺失会影响到TCP2蛋白的稳定以及积累速度。蛋白酶抑制剂MG132的处理结果表明,TCP2蛋白是通过26S蛋白酶体泛素化途径降解。通过Luciferase assay测定翻译抑制剂CHX处理下不同背景(包括WT、CRY1突变体cry1、cry1cry2双突变体和ZTL3突变体ztl3-1)中LUC-TCP2对蓝光的响应,结果证明TCP2在蓝光下的积累是受到多个蓝光受体的影响,且m RNA的翻译量会影响到蛋白的积累,这也侧面地证明不同蓝光受体都会通过影响TCP2的m RNA的表达量影响TCP2蛋白的积累。对于TCP2 m RNA光影响因子的研究结果表明,TCP2的m RNA表达量通过光特异性依赖的方式受到许多光受体的影响。TCP2作为一个核定位的转录因子蛋白正调控HY5、HYH、CAB和CHS的m RNA的表达。RBSS(Radom binding sites selection)结果表明TCP2的DNA结合位点为GGGGNCC。另外Ch IP-q PCR结果表明TCP2蛋白能蓝光特异性结合HY5和HYH的启动子区域中的TCP2结合位点或相似性位点。以上结果表明TCP2作为转录因子作用于许多光受体的下游,其中就包括CRY1。为了更好地研究CRY1与TCP2蛋白在植物内的相互作用,本研究设计了一套双基因载体p DTs(p DT1、p DT7和p DT7G),以期通过一次植物转化实现两个蛋白的同时表达。本研究构建多对基因至双基因载体并转入烟草或拟南芥中,通过定量、免疫印迹、荧光显微镜、免疫共沉淀、转基因表型分析以及共表达效率分析试验分析双基因载体的实用性,得到如下结果:(1)多对基因通过双基因表达载体在植物细胞内成功共表达;(2)p DT7G载体上c GR实现了其核质转移功能;(3)共表达双基因的生理生化功能未受影响;(4)双基因载体能实现双基因在植物细胞内的高效共表达。
[Abstract]:Light receptors regulate the growth and development of plants through a variety of photoreceptors. These light receptors contain photosensitive pigments and coloring pigments. Arabidopsis pigment CRY1 (CRYPTOCHROME 1) is a blue light signal receptor protein. Many studies have reported its function in plant blue light signal transduction pathway, such as inhibiting the elongation of hypocotyl, controlling the seedling Chlorosis, circadian rhythm, accelerating the accumulation of anthocyanins, affecting leaf morphology and flowering time, and other.CRY1 proteins interact with the downstream signal proteins to initiate the transmission of blue light signals, thus promoting the formation of plant light forms. The signal transduction mechanism for the modulation of the light morphogenesis of mustard pigment CRY1 is still unclear. Many proteins involved in CRY1 blue light signal transduction and interaction have been found, but the study of the transcription factor protein involved in the CRY1 blue light signaling pathway is currently rarely reported. In order to better understand and improve the CRY1 blue light signal transduction mechanism, The yeast library of transcriptional factors was screened by CRY1 as a bait by yeast two hybrid methods. The interaction between CRY1 and CRY1 could mediate the transcription factor protein of Arabidopsis thaliana. The results were as follows: repeated verification by yeast two hybrid (yeast two hybrid assay) and double molecular fluorescence Further validation of the complementary Bi FC (Bimolecular Fluorescence Complementation assay) and immunoprecipitation co-IP (co-immunoprecipitation) test demonstrated the interaction between CRY1 and the preliminarily screened transcription factor protein TCP2 (TEOSINTE-LIKE1, CYCLO), and the localization of Central Asian cells in tobacco and Arabidopsis. The experiment shows that TCP2 is a nucleoprotein, which may interact with the CRY1 protein in the nucleus. This study has first discovered and demonstrated the transcription factor protein -TCP2, which is directly interacting with CRY1, and its interaction in yeast cells and tobacco cells shows a clear blue light specificity, and the blue light specific in Arabidopsis cells. The characteristics of sexual dependence disappear, presumably due to the absence of Arabidopsis in yeast and tobacco systems, and the interaction of other Arabidopsis proteins in the actual interaction may be involved in the interaction between CRY1 and TCP2. By bioinformatics analysis of the protein domain of CRY1 and TCP2, multiple domains of CRY1 and TCP2 are cloned and by yeast double Hybridization and Bi FC methods prove that the CRY1 N terminal domain (the domain containing 1-515 amino acid sequences, covering the photolyase domain PHR) can interact with the TCP2 characteristic domain TCP, which contains the TCP2 1-174 amino acid sequence, and the R domain in the.TCP domain affects its blue light interacting with CRY1 in yeast cells. The genetic study showed that TCP2 blue light depended on the inhibition of the elongation of the Hypocotyl in Arabidopsis, which not only showed the specificity of the light wavelength, but also the specific dependence of light intensity; it could regulate the subleaf morphology of Arabidopsis seedlings under red and blue light, but had no function under the far red light. The deletion mutants have phenotypes of late flowers; our results also further verify the function of TCP2 protein in regulating leaf morphology and size. In view of the importance of light as an environmental impact factor in the expression of protein in plant cells and the importance of CRY1 in the blue light signal transduction pathway, we have studied the TCP2 protein in the absence of CRY1 and The changes in the expression level of different photoresponse were made. The changes in the protein of TCP2 in the Myc-TCP2/WT and Myc-TCP2/cry1 transgenic materials were detected by Western blot test. The results showed that TCP2 was a light responsive protein, which would accumulate in the blue light and degraded in the dark and far red light, and the red light would accumulate in the red light. The amount and rate of accumulation were far below the blue light; at the same time, the loss of CRY1 could affect the stability and accumulation of TCP2 protein. The results of the protease inhibitor MG132 treatment showed that the TCP2 protein was degraded through the ubiquitination pathway of the 26S proteasome. The Luciferase assay was used to determine the different background (including WT, CRY1 mutation) under the translational inhibitor CHX treatment. The response of LUC-TCP2 to blue light in body cry1, cry1cry2 double mutants and ZTL3 mutant ztl3-1) shows that the accumulation of TCP2 under blue light is influenced by several blue light receptors, and the translation of M RNA affects the accumulation of protein, which also proves that the different blue light receptors will affect the TCP2 eggs through the expression of M RNA that affects TCP2. The results of the study of the TCP2 m RNA light influence factors show that the m RNA expression of TCP2 is affected by the light specific dependence and the effect of many optical receptors on the.TCP2 as a nuclear transcription factor protein that regulates HY5, HYH, CAB and CHS M The binding site is GGGGNCC. and the Ch IP-q PCR results indicate that the TCP2 protein can bind to the TCP2 binding site or similarity loci in the promoter region of HY5 and HYH. The above results indicate that TCP2 acts as a transcription factor downstream of many optical receptors, including CRY1. for the better study of CRY1 and TCP2 protein in plants. In this study, a set of double gene carriers, P DTs (P DT1, P DT7 and P DT7G), was designed to achieve simultaneous expression of two proteins through one plant transformation. Analysis and co expression efficiency analysis test to analyze the practicability of double gene vector, and get the following results: (1) multiple pairs of genes were successfully co expressed in plant cells through double gene expression vector; (2) C GR on P DT7G vector realized its nuclear transfer function; (3) the physiological and biochemical functions of co expression of double genes were not affected; (4) double gene carrier can be real High efficient co expression of the present double genes in plant cells.
【学位授予单位】：湖南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2
本文编号：2138607
[Abstract]:Light receptors regulate the growth and development of plants through a variety of photoreceptors. These light receptors contain photosensitive pigments and coloring pigments. Arabidopsis pigment CRY1 (CRYPTOCHROME 1) is a blue light signal receptor protein. Many studies have reported its function in plant blue light signal transduction pathway, such as inhibiting the elongation of hypocotyl, controlling the seedling Chlorosis, circadian rhythm, accelerating the accumulation of anthocyanins, affecting leaf morphology and flowering time, and other.CRY1 proteins interact with the downstream signal proteins to initiate the transmission of blue light signals, thus promoting the formation of plant light forms. The signal transduction mechanism for the modulation of the light morphogenesis of mustard pigment CRY1 is still unclear. Many proteins involved in CRY1 blue light signal transduction and interaction have been found, but the study of the transcription factor protein involved in the CRY1 blue light signaling pathway is currently rarely reported. In order to better understand and improve the CRY1 blue light signal transduction mechanism, The yeast library of transcriptional factors was screened by CRY1 as a bait by yeast two hybrid methods. The interaction between CRY1 and CRY1 could mediate the transcription factor protein of Arabidopsis thaliana. The results were as follows: repeated verification by yeast two hybrid (yeast two hybrid assay) and double molecular fluorescence Further validation of the complementary Bi FC (Bimolecular Fluorescence Complementation assay) and immunoprecipitation co-IP (co-immunoprecipitation) test demonstrated the interaction between CRY1 and the preliminarily screened transcription factor protein TCP2 (TEOSINTE-LIKE1, CYCLO), and the localization of Central Asian cells in tobacco and Arabidopsis. The experiment shows that TCP2 is a nucleoprotein, which may interact with the CRY1 protein in the nucleus. This study has first discovered and demonstrated the transcription factor protein -TCP2, which is directly interacting with CRY1, and its interaction in yeast cells and tobacco cells shows a clear blue light specificity, and the blue light specific in Arabidopsis cells. The characteristics of sexual dependence disappear, presumably due to the absence of Arabidopsis in yeast and tobacco systems, and the interaction of other Arabidopsis proteins in the actual interaction may be involved in the interaction between CRY1 and TCP2. By bioinformatics analysis of the protein domain of CRY1 and TCP2, multiple domains of CRY1 and TCP2 are cloned and by yeast double Hybridization and Bi FC methods prove that the CRY1 N terminal domain (the domain containing 1-515 amino acid sequences, covering the photolyase domain PHR) can interact with the TCP2 characteristic domain TCP, which contains the TCP2 1-174 amino acid sequence, and the R domain in the.TCP domain affects its blue light interacting with CRY1 in yeast cells. The genetic study showed that TCP2 blue light depended on the inhibition of the elongation of the Hypocotyl in Arabidopsis, which not only showed the specificity of the light wavelength, but also the specific dependence of light intensity; it could regulate the subleaf morphology of Arabidopsis seedlings under red and blue light, but had no function under the far red light. The deletion mutants have phenotypes of late flowers; our results also further verify the function of TCP2 protein in regulating leaf morphology and size. In view of the importance of light as an environmental impact factor in the expression of protein in plant cells and the importance of CRY1 in the blue light signal transduction pathway, we have studied the TCP2 protein in the absence of CRY1 and The changes in the expression level of different photoresponse were made. The changes in the protein of TCP2 in the Myc-TCP2/WT and Myc-TCP2/cry1 transgenic materials were detected by Western blot test. The results showed that TCP2 was a light responsive protein, which would accumulate in the blue light and degraded in the dark and far red light, and the red light would accumulate in the red light. The amount and rate of accumulation were far below the blue light; at the same time, the loss of CRY1 could affect the stability and accumulation of TCP2 protein. The results of the protease inhibitor MG132 treatment showed that the TCP2 protein was degraded through the ubiquitination pathway of the 26S proteasome. The Luciferase assay was used to determine the different background (including WT, CRY1 mutation) under the translational inhibitor CHX treatment. The response of LUC-TCP2 to blue light in body cry1, cry1cry2 double mutants and ZTL3 mutant ztl3-1) shows that the accumulation of TCP2 under blue light is influenced by several blue light receptors, and the translation of M RNA affects the accumulation of protein, which also proves that the different blue light receptors will affect the TCP2 eggs through the expression of M RNA that affects TCP2. The results of the study of the TCP2 m RNA light influence factors show that the m RNA expression of TCP2 is affected by the light specific dependence and the effect of many optical receptors on the.TCP2 as a nuclear transcription factor protein that regulates HY5, HYH, CAB and CHS M The binding site is GGGGNCC. and the Ch IP-q PCR results indicate that the TCP2 protein can bind to the TCP2 binding site or similarity loci in the promoter region of HY5 and HYH. The above results indicate that TCP2 acts as a transcription factor downstream of many optical receptors, including CRY1. for the better study of CRY1 and TCP2 protein in plants. In this study, a set of double gene carriers, P DTs (P DT1, P DT7 and P DT7G), was designed to achieve simultaneous expression of two proteins through one plant transformation. Analysis and co expression efficiency analysis test to analyze the practicability of double gene vector, and get the following results: (1) multiple pairs of genes were successfully co expressed in plant cells through double gene expression vector; (2) C GR on P DT7G vector realized its nuclear transfer function; (3) the physiological and biochemical functions of co expression of double genes were not affected; (4) double gene carrier can be real High efficient co expression of the present double genes in plant cells.
【学位授予单位】：湖南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2
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1 何志敏;拟南芥转录因子TCP2与CRY1相互作用的遗传学与生化分析[D];湖南大学;2016年
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