拟南芥转录因子MYB30通过胞内钙信号调控胁迫响应的机制研究

发布时间：2018-07-05 15:38

本文选题：拟南芥 + 钙信号　；参考：《中国农业大学》2017年博士论文

【摘要】：钙离子(Ca2+)是植物细胞内重要的第二信使,它对植物响应外界环境胁迫有着至关重要的作用。许多非生物胁迫或生物胁迫,如氧化胁迫、热胁迫、渗透胁迫、盐胁迫和病原菌胁迫等,都会导致胞内游离Ca2+(Cytosolicfree Ca2+,[Ca2]浓度的升高。不同胁迫下,[Ca2+]cyt浓度升高的持续时间、强度、频率、空间分布等都具有一定的特异性,因而被称作"钙信号"(Calcium signature)。钙信号被细胞内钙响应元件解码,并转化为下游的磷酸化活动和转录活动。目前对钙信号的研究主要集中在Ca2+通道和Ca2+转运蛋白的生物学作用,以及钙响应元件调控下游信号转导的过程,但是对特异的钙信号如何产生,又如何调控植物的胁迫响应,还了解得比较少。为了寻找钙信号形成过程中的重要基因,本研究通过正向遗传学的方法,在稳定表达钙监测蛋白AEQUORIN的拟南芥转基因株系背景下,筛选钙信号异常的突变体。经过筛选发现,在H202刺激下,MYB30基因的突变体[Ca2+]cyt浓度的升高明显高于野生型。拟南芥MYB30转录因子是植物R2R3-MYB家族的重要成员。在H202刺激下,回补材料可以将突变体中[Ca2+]cyt浓度恢复至野生型水平,MYB30过表达植株[Ca2+]cyt浓度的升高明显低于野生型。这些结果表明,H202刺激下MYB30影响[Ca2+]cyt浓度的升高过程。Real-time RT-PCR结果表明,MYB30抑制植物ANNEXINs(ANNs)基因的表达。ANNs蛋白是一种Ca2+依赖的膜结合蛋白,它影响多种胁迫刺激下植物胞内钙信号的形成。荧光素酶实验,染色质免疫共沉淀实验(ChIP),凝胶阻滞实验(EMSA)和酵母单杂交实验结果表明,MYB30在体内和体外都可以结合ANN1和ANN4基因的启动子,并抑制ANN1和ANN4基因的表达。表型分析结果显示,myb30对甲基紫精(MV)敏感,对热胁迫耐受。氧化胁迫和热胁迫下,myb30的钙信号异常表型、氧化胁迫敏感表型和热胁迫耐受表型,都被ANNs的缺失或Ca2+通道抑制剂]LaCl3所部分恢复。这说明,ANN1和ANN4介导的钙信号通路参与了 MYB30对氧化胁迫和热胁迫响应的调节过程。多种胁迫都导致myb30产生异常的钙信号,但只有部分胁迫下胞内钙信号的产生依赖于ANNs。myb30的某些胁迫敏感表型,如ABA敏感表型,不被ANNs的缺失或LaCl3所恢复。这说明不同胁迫下钙信号的产生具有特异性。综上所述,本论文的研究发现了拟南芥MYB30转录因子通过影响ANNs介导的特异的胞内钙信号的产生,调控了植物的氧化胁迫响应和热胁迫响应过程。这些发现使我们能更深入地了解胁迫刺激下植物胞内钙信号的特异性产生,与其在植物胁迫响应过程中的重要作用。
[Abstract]:Calcium ion (Ca2+) is an important second messenger in plant cells. It plays an important role in plant response to environmental stress. Many abiotic stress or biological stress, such as oxidative stress, heat stress, osmotic stress, salt stress and pathogenic bacteria stress, will lead to intracellular free Ca2+ (Cytosolicfree Ca2+, [Ca2] concentration increase. Under the same stress, the duration, intensity, frequency and spatial distribution of [Ca2+]cyt concentration have certain specificity, so it is called "calcium signal" (Calcium signature). Calcium signals are decoded by intracellular calcium response elements and converted into phosphorylation and transcription activities downstream. The current research on calcium signals is mainly focused on the Ca2+ pass. The biological role of the pathway and Ca2+ transporters, as well as the process of regulating downstream signal transduction by calcium responsive elements, is not known for how specific calcium signals produce and how to regulate plant stress responses. In order to find important genes in the process of calcium signal formation, this study is based on a positive genetic method in the stable table. Under the background of transgenic plant of Arabidopsis thaliana with calcium monitoring protein AEQUORIN, the mutant of abnormal calcium signal was screened. After screening, it was found that the increase of [Ca2+]cyt concentration of MYB30 gene was obviously higher than that of wild type under the stimulation of H202. The MYB30 transcription factor of Arabidopsis thaliana was an important member of the plant R2R3-MYB family. Under the stimulation of H202, the remedial material was available. In order to restore the [Ca2+]cyt concentration in the mutant to the wild type level, the increase of [Ca2+]cyt concentration in the MYB30 overexpressed plant was significantly lower than that in the wild type. These results showed that the MYB30 effect of MYB30 on the increase of [Ca2+]cyt concentration under the stimulation of H202 indicated that the MYB30 inhibition plant ANNEXINs (ANNs) gene expressed the.ANNs protein as a kind. The dependent membrane binding protein, which affects the formation of intracellular calcium signals of plants under a variety of stress stimuli. Luciferase experiment, chromatin immunoprecipitation test (ChIP), gel block test (EMSA) and yeast single hybridization experiment results show that MYB30 can bind the promoter of ANN1 and ANN4 genes in both in vivo and in vitro, and inhibit the ANN1 and ANN4 genes. The results of phenotypic analysis showed that myb30 was sensitive to methyl violet (MV) and was tolerant to heat stress. Under oxidative stress and heat stress, the abnormal calcium signal phenotype of myb30, the sensitive phenotypes of oxidative stress and the tolerance phenotype of heat stress were partially restored by the deletion of ANNs or by the Ca2+ channel inhibitor]LaCl3. This shows that ANN1 and ANN4 mediated calcium signaling. The pathway involved in the regulation of MYB30's response to oxidative stress and heat stress. A variety of stresses cause myb30 to produce abnormal calcium signals, but the production of intracellular calcium signals only under some stress depends on some of the stress sensitive phenotypes of ANNs.myb30, such as ABA sensitive phenotype, not by the loss of ANNs or by LaCl3. This indicates that the calcium signal under different stresses is under different stress. In summary, this study found that the MYB30 transcription factor of Arabidopsis thaliana regulates plant oxidative stress response and heat stress response through the production of specific intracellular calcium signaling mediated by ANNs, which enables us to understand more deeply about the specific intracellular calcium signals under stress stimulation. Heterosexual production plays an important role in plant stress response.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q943.2

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