拟南芥转录因子ARF2响应低钾胁迫的分子机制研究

发布时间：2018-07-12 11:42

本文选题：拟南芥 + 低钾胁迫　；参考：《中国农业大学》2016年博士论文

【摘要】：钾是植物生长发育所必需的矿质营养元素之一。土壤缺钾可以导致植物生长发育受阻,并严重影响农作物的产量和品质。为了应对低钾胁迫,植物进化出一系列复杂的信号调控途径。研究发现,植物响应低钾胁迫主要通过转录水平及翻译后水平两个方面来调控下游功能基因或蛋白的表达量或活性,从而调节植物对钾的吸收、转运和再分配过程,最终使植物耐受低钾胁迫。HAK5是拟南芥中重要的高亲和钾转运体,它主要负责低钾条件下根部的钾吸收。在高钾条件下,HAK5的表达量极低,但是低钾胁迫可以显著诱导HAK5的转录升高。这是拟南芥响应低钾胁迫的重要调控机制之一,然而其分子调控机理目前仍不清楚。本论文工作主要研究了转录因子ARF2参与拟南芥钾吸收调控的生理功能,并深入解析了ARF2响应低钾胁迫并调控控HAK5转录的分子机制。本论文通过反向遗传学的方法,筛选并发现拟南芥转录因子ARF2的突变体具有低钾耐受表型。ARF2是生长素响应因子(Auxin Responsive Factor)家族的成员,它在拟南芥幼苗的根部和冠部都有较强的表达。在低钾(0.01 mM)条件下,arf2突变体表现出耐受表型,主要表现为：主根长于野生型,且植株钾含量显著高于野生型。而ARF2过表达植株则表现出主根生长受抑、钾含量降低的低钾敏感表型。回补材料可以恢复突变体的低钾表型及钾含量至野生型水平,表明arf2突变体的耐低钾表型是由ARF2基因的功能缺失导致。86Rb-同位素吸收实验证明arf2突变体的钾吸收能力显著高于野生型。作为转录因子,ARF2能够结合在含有生长素响应元件的基因启动子区,并抑制该基因的转录。本论文检测了钾吸收相关基因在arf2突变体及野生型中的表达差异,发现HAK5的表达量在arf2突变体中显著升高。烟草GUS/LUC实验结果也表明,ARF2能够抑制HAK5启动子驱动的GUS基因表达。凝胶阻滞实验与染色质免疫共沉淀实验证明,ARF2能够在体外和体内直接结合到HAK5启动子区的生长素响应元件上。遗传证据显示,ARF2过表达植株与hak5突变体表现出一致的低钾敏感表型。arf2突变体与HAK5过表达植株具有一致的耐低钾表型,但是arf2 hak5双突变体则表现出与hak5突变体一致的低钾敏感表型,由此认为HAK5是ARF2调控的下游基因。表型检测还发现,低钾条件下,arf2突变体与HAK5过表达的根毛都较野生型长,而hak5突变体与arf2 hak5双突变体的根毛都较野生型短。ChIP-qPCR的结果显示,在低钾条件下,ARF2对于HAK5启动子区的结合减弱,而这一过程受磷酸化作用调控。低钾处理可以增强ARF2的磷酸化修饰,从而解除ARF2对HAK5的转录抑制作用。低钾胁迫可以导致ARF2上第689位丝氨酸被磷酸化,该位点对于ARF2响应低钾胁迫至关重要。将689位丝氨酸突变为天冬氨酸后,ARF2对HAK5启动子的结合作用显著减弱。进一步研究还发现,在低钾条件下,ARF2蛋白发生降解,并且该降解依赖于26S蛋白酶体途径。本论文工作的研究表明,拟南芥转录因子ARF2通过磷酸化修饰来响应低钾胁迫。低钾引起的ARF2磷酸化可以解除其对HAK5的转录抑制,从而使HK5的表达升高,HAK5介导的高亲和钾吸收增强,利于植物耐受低钾胁迫。本论文的研究结果对于阐明植物响应低钾胁迫的转录调控机制提供了非常重要的实验证据。
[Abstract]:Potassium is one of the mineral nutrients necessary for plant growth and development. Potassium deficiency can cause plant growth and development to be hindered, and seriously affect crop yield and quality. In order to cope with low potassium stress, plants have evolved a series of complex signaling pathways. The study found that plants respond to low potassium stress mainly through transcription and turnover. The two aspects of the post translation level regulate the expression or activity of the downstream functional genes or proteins, thus regulating the plant's absorption, transport and redistribution of potassium, and ultimately the tolerance of plants to low potassium stress.HAK5 is an important Gao Qinhe potassium transporter in Arabidopsis, which is mainly responsible for the absorption of potassium in the roots under the condition of low potassium. Under the condition of high potassium, HAK5 The expression of HAK5 is very low, but low potassium stress can significantly induce the increase of transcription. This is one of the important regulatory mechanisms of Arabidopsis thaliana response to low potassium stress. However, the mechanism of its molecular regulation is still unclear. This paper mainly studies the physiological function of transcription factor ARF2 involved in the regulation of potassium uptake in Arabidopsis, and the ARF2 response is deeply analyzed. In this paper, we screened and found that the mutant of Arabidopsis thaliana transcriptional factor ARF2 with low potassium tolerance phenotype.ARF2 is a member of the auxin response factor (Auxin Responsive Factor) family, which is strongly expressed in the Wacom root of the root of Arabidopsis thaliana seedlings. Under the condition of low potassium (0.01 mM), the arf2 mutant showed the tolerance phenotype mainly: the main root was longer than the wild type, and the potassium content of the plant was significantly higher than that of the wild type. The ARF2 overexpressed plant showed the low potassium sensitive phenotype of the main root growth and the decrease of potassium content. The remedial material could restore the mutant's low potassium phenotypes and potassium content to the mutant. The wild type level showed that the low potassium tolerance of the arf2 mutant was caused by the function deletion of the ARF2 gene and the.86Rb- isotope absorption experiment proved that the potassium absorption ability of the arf2 mutant was significantly higher than that of the wild type. As a transcription factor, ARF2 could bind to the gene promoter region containing the auxin response element and inhibit the transcription of the gene. The expression of K absorption related genes in the arf2 mutant and the wild type was detected. The expression of HAK5 was significantly increased in the arf2 mutant. The results of GUS/LUC experiment also showed that ARF2 could inhibit the GUS gene expression driven by HAK5 promoter. The gel block experiment and chromatin immunoprecipitation experiment showed that ARF2 could be in body. The genetic evidence showed that the low potassium sensitive phenotypic.Arf2 mutant of the ARF2 overexpressed plant was consistent with the HAK5 overexpressed plant and had the same low potassium phenotype as the HAK5 overexpressed plant, but the arf2 hak5 double mutants showed the low potassium consistency with the hak5 mutants, according to the genetic evidence. It was found that HAK5 was a downstream gene of ARF2 regulation. The phenotypic detection also found that the arf2 mutant and HAK5 overexpressed root hairs were longer than the wild type under the low potassium condition, and the root hairs of the hak5 mutant and arf2 hak5 double mutant were both compared with the wild type short.ChIP-qPCR. This process is weakened, and this process is regulated by phosphorylation. Low potassium treatment can enhance the phosphorylation of ARF2 and release the transcriptional inhibition of ARF2 to HAK5. Low potassium stress can lead to phosphorylation of 689th serine on ARF2, which is crucial to the response of ARF2 to low potassium stress. After mutation of 689 serine into aspartic acid, AR The binding effect of F2 on the HAK5 promoter was significantly weakened. Further studies found that the ARF2 protein was degraded under the low potassium condition and that the degradation depended on the 26S proteasome pathway. The study in this paper showed that the Arabidopsis transcription factor ARF2 was phosphorylated to respond to the low potassium stress. The phosphorylation of ARF2 caused by low potassium could be relieved. The transcriptional inhibition of HAK5, thus increasing the expression of HK5, enhanced the absorption of high affinity potassium mediated by HAK5, is beneficial to plant tolerance to low potassium stress. The results of this paper provide very important experimental evidence for elucidating the mechanism of transcription regulation of plant response to low potassium stress.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

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