擬南芥轉(zhuǎn)錄因子TCP2與CRY1相互作用的遺傳學(xué)與生化分析
發(fā)布時(shí)間：2018-07-22 21:19
【摘要】：光通過多種光受體調(diào)節(jié)植物的生長發(fā)育,這些光受體包含光敏色素與隱花色素。擬南芥隱花色素CRY1(CRYPTOCHROME 1)作為藍(lán)光信號受體蛋白,已有許多的研究報(bào)道了其在植物藍(lán)光信號轉(zhuǎn)導(dǎo)途徑方面的功能,如抑制下胚軸的伸長,控制幼苗的去黃化、生物鐘節(jié)律,促進(jìn)植物花青素的積累,影響葉片形態(tài)和開花時(shí)間等。CRY1蛋白通過與其下游的信號蛋白相互作用,啟動(dòng)藍(lán)光信號的傳遞,從而促進(jìn)植物光形態(tài)的建成。目前光受體調(diào)節(jié)光形態(tài)建成的機(jī)制已得到廣泛的研究,但是擬南芥隱花色素CRY1調(diào)節(jié)光形態(tài)建成的信號傳導(dǎo)機(jī)制目前仍不清楚。研究發(fā)現(xiàn)了許多參與CRY1藍(lán)光信號轉(zhuǎn)導(dǎo)并相互作用的蛋白,但轉(zhuǎn)錄因子蛋白參與CRY1藍(lán)光信號通路的研究報(bào)道目前幾乎沒有。為了更好地了解和完善CRY1藍(lán)光信號轉(zhuǎn)導(dǎo)機(jī)制,本研究通過酵母雙雜交的方法,用CRY1作為誘餌篩選轉(zhuǎn)錄因子酵母庫,獲得與CRY1相互作用并可能介導(dǎo)CRY1參與擬南芥光形態(tài)建成的轉(zhuǎn)錄因子蛋白。具體研究結(jié)果如下:通過多次的酵母雙雜交(yeast two hybrid assay)試驗(yàn)反復(fù)驗(yàn)證以及雙分子熒光互補(bǔ)Bi FC(Bimolecular Fluorescence Complementation assay)和免疫共沉淀co-IP(co-immunoprecipitation)試驗(yàn)的進(jìn)一步驗(yàn)證,證明CRY1與初步篩選到的轉(zhuǎn)錄因子蛋白TCP2(TEOSINTE-LIKE1,CYCLO IDEA,and PROLIFERATING CELL FACTOR 2)相互作用。通過煙草和擬南芥中亞細(xì)胞定位試驗(yàn),證明TCP2是一個(gè)核蛋白,其可能是與細(xì)胞核中的CRY1蛋白相互作用。本研究首次發(fā)現(xiàn)并證明了與CRY1直接相互作用的轉(zhuǎn)錄因子蛋白-TCP2,其相互作用在酵母細(xì)胞以及煙草細(xì)胞中表現(xiàn)出明顯的藍(lán)光特異性,而在擬南芥細(xì)胞中其藍(lán)光特異性依賴特征消失,推測可能是由于酵母及煙草系統(tǒng)中無擬南芥蛋白,而實(shí)際相互作用中可能存在其他擬南芥蛋白參與CRY1與TCP2的相互作用。通過生物信息學(xué)分析CRY1與TCP2的蛋白結(jié)構(gòu)域,分段克隆CRY1和TCP2的多個(gè)結(jié)構(gòu)域片段并通過酵母雙雜交以及Bi FC的方法,證明CRY1 N端結(jié)構(gòu)域(該結(jié)構(gòu)域包含1-515的氨基酸序列,涵蓋了光裂解酶結(jié)構(gòu)域PHR)能與TCP2的特征結(jié)構(gòu)域TCP(該區(qū)域包含TCP2 1-174氨基酸序列)相互作用。TCP結(jié)構(gòu)域中的R結(jié)構(gòu)域影響其與CRY1在酵母細(xì)胞中相互作用的藍(lán)光依賴性。遺傳學(xué)研究結(jié)果表明,TCP2藍(lán)光依賴性地抑制擬南芥下胚軸的伸長,這種藍(lán)光特異性不僅表現(xiàn)在光波長的特異性上還表現(xiàn)為光強(qiáng)度的特異性依賴;其在紅光和藍(lán)光下能調(diào)控?cái)M南芥幼苗的子葉形態(tài),而在遠(yuǎn)紅光下無此功能;其功能缺失突變體具有晚花的表型;我們的研究結(jié)果也進(jìn)一步驗(yàn)證了TCP2蛋白調(diào)控葉片形態(tài)和大小方面的功能。鑒于光作為環(huán)境影響因子在影響植物細(xì)胞內(nèi)蛋白的表達(dá)以及CRY1在藍(lán)光信號轉(zhuǎn)導(dǎo)途徑上的重要性,我們研究了TCP2蛋白在缺失CRY1與否的情況下對于不同光響應(yīng)而做出的表達(dá)水平變化。通過免疫印跡試驗(yàn)檢測Myc-TCP2/WT和Myc-TCP2/cry1轉(zhuǎn)基因材料中TCP2的蛋白變化,結(jié)果證明TCP2是一個(gè)光響應(yīng)的蛋白,其會在藍(lán)光下大幅積累而在黑暗和遠(yuǎn)紅光下降解,紅光下也會有積累但積累的量和速率遠(yuǎn)不及藍(lán)光下;同時(shí),CRY1的缺失會影響到TCP2蛋白的穩(wěn)定以及積累速度。蛋白酶抑制劑MG132的處理結(jié)果表明,TCP2蛋白是通過26S蛋白酶體泛素化途徑降解。通過Luciferase assay測定翻譯抑制劑CHX處理下不同背景(包括WT、CRY1突變體cry1、cry1cry2雙突變體和ZTL3突變體ztl3-1)中LUC-TCP2對藍(lán)光的響應(yīng),結(jié)果證明TCP2在藍(lán)光下的積累是受到多個(gè)藍(lán)光受體的影響,且m RNA的翻譯量會影響到蛋白的積累,這也側(cè)面地證明不同藍(lán)光受體都會通過影響TCP2的m RNA的表達(dá)量影響TCP2蛋白的積累。對于TCP2 m RNA光影響因子的研究結(jié)果表明,TCP2的m RNA表達(dá)量通過光特異性依賴的方式受到許多光受體的影響。TCP2作為一個(gè)核定位的轉(zhuǎn)錄因子蛋白正調(diào)控HY5、HYH、CAB和CHS的m RNA的表達(dá)。RBSS(Radom binding sites selection)結(jié)果表明TCP2的DNA結(jié)合位點(diǎn)為GGGGNCC。另外Ch IP-q PCR結(jié)果表明TCP2蛋白能藍(lán)光特異性結(jié)合HY5和HYH的啟動(dòng)子區(qū)域中的TCP2結(jié)合位點(diǎn)或相似性位點(diǎn)。以上結(jié)果表明TCP2作為轉(zhuǎn)錄因子作用于許多光受體的下游,其中就包括CRY1。為了更好地研究CRY1與TCP2蛋白在植物內(nèi)的相互作用,本研究設(shè)計(jì)了一套雙基因載體p DTs(p DT1、p DT7和p DT7G),以期通過一次植物轉(zhuǎn)化實(shí)現(xiàn)兩個(gè)蛋白的同時(shí)表達(dá)。本研究構(gòu)建多對基因至雙基因載體并轉(zhuǎn)入煙草或擬南芥中,通過定量、免疫印跡、熒光顯微鏡、免疫共沉淀、轉(zhuǎn)基因表型分析以及共表達(dá)效率分析試驗(yàn)分析雙基因載體的實(shí)用性,得到如下結(jié)果:(1)多對基因通過雙基因表達(dá)載體在植物細(xì)胞內(nèi)成功共表達(dá);(2)p DT7G載體上c GR實(shí)現(xiàn)了其核質(zhì)轉(zhuǎn)移功能;(3)共表達(dá)雙基因的生理生化功能未受影響;(4)雙基因載體能實(shí)現(xiàn)雙基因在植物細(xì)胞內(nèi)的高效共表達(dá)。
[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.
【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q943.2
本文編號：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.
【學(xué)位授予單位】：湖南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q943.2
【相似文獻(xiàn)】
相關(guān)博士學(xué)位論文 前1條
1 何志敏;擬南芥轉(zhuǎn)錄因子TCP2與CRY1相互作用的遺傳學(xué)與生化分析[D];湖南大學(xué);2016年
，本文編號：2138607
本文鏈接：http://sikaile.net/shoufeilunwen/jckxbs/2138607.html
最近更新
教材專著
