本文選題：擬南芥 + 低鉀脅迫　； 參考：《中國(guó)農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：鉀是植物生長(zhǎng)發(fā)育所必需的礦質(zhì)營(yíng)養(yǎng)元素之一。土壤缺鉀可以導(dǎo)致植物生長(zhǎng)發(fā)育受阻,并嚴(yán)重影響農(nóng)作物的產(chǎn)量和品質(zhì)。為了應(yīng)對(duì)低鉀脅迫,植物進(jìn)化出一系列復(fù)雜的信號(hào)調(diào)控途徑。研究發(fā)現(xiàn),植物響應(yīng)低鉀脅迫主要通過(guò)轉(zhuǎn)錄水平及翻譯后水平兩個(gè)方面來(lái)調(diào)控下游功能基因或蛋白的表達(dá)量或活性,從而調(diào)節(jié)植物對(duì)鉀的吸收、轉(zhuǎn)運(yùn)和再分配過(guò)程,最終使植物耐受低鉀脅迫。HAK5是擬南芥中重要的高親和鉀轉(zhuǎn)運(yùn)體,它主要負(fù)責(zé)低鉀條件下根部的鉀吸收。在高鉀條件下,HAK5的表達(dá)量極低,但是低鉀脅迫可以顯著誘導(dǎo)HAK5的轉(zhuǎn)錄升高。這是擬南芥響應(yīng)低鉀脅迫的重要調(diào)控機(jī)制之一,然而其分子調(diào)控機(jī)理目前仍不清楚。本論文工作主要研究了轉(zhuǎn)錄因子ARF2參與擬南芥鉀吸收調(diào)控的生理功能,并深入解析了ARF2響應(yīng)低鉀脅迫并調(diào)控控HAK5轉(zhuǎn)錄的分子機(jī)制。本論文通過(guò)反向遺傳學(xué)的方法,篩選并發(fā)現(xiàn)擬南芥轉(zhuǎn)錄因子ARF2的突變體具有低鉀耐受表型。ARF2是生長(zhǎng)素響應(yīng)因子(Auxin Responsive Factor)家族的成員,它在擬南芥幼苗的根部和冠部都有較強(qiáng)的表達(dá)。在低鉀(0.01 mM)條件下,arf2突變體表現(xiàn)出耐受表型,主要表現(xiàn)為：主根長(zhǎng)于野生型,且植株鉀含量顯著高于野生型。而ARF2過(guò)表達(dá)植株則表現(xiàn)出主根生長(zhǎng)受抑、鉀含量降低的低鉀敏感表型�；匮a(bǔ)材料可以恢復(fù)突變體的低鉀表型及鉀含量至野生型水平,表明arf2突變體的耐低鉀表型是由ARF2基因的功能缺失導(dǎo)致。86Rb-同位素吸收實(shí)驗(yàn)證明arf2突變體的鉀吸收能力顯著高于野生型。作為轉(zhuǎn)錄因子,ARF2能夠結(jié)合在含有生長(zhǎng)素響應(yīng)元件的基因啟動(dòng)子區(qū),并抑制該基因的轉(zhuǎn)錄。本論文檢測(cè)了鉀吸收相關(guān)基因在arf2突變體及野生型中的表達(dá)差異,發(fā)現(xiàn)HAK5的表達(dá)量在arf2突變體中顯著升高。煙草GUS/LUC實(shí)驗(yàn)結(jié)果也表明,ARF2能夠抑制HAK5啟動(dòng)子驅(qū)動(dòng)的GUS基因表達(dá)。凝膠阻滯實(shí)驗(yàn)與染色質(zhì)免疫共沉淀實(shí)驗(yàn)證明,ARF2能夠在體外和體內(nèi)直接結(jié)合到HAK5啟動(dòng)子區(qū)的生長(zhǎng)素響應(yīng)元件上。遺傳證據(jù)顯示,ARF2過(guò)表達(dá)植株與hak5突變體表現(xiàn)出一致的低鉀敏感表型。arf2突變體與HAK5過(guò)表達(dá)植株具有一致的耐低鉀表型,但是arf2 hak5雙突變體則表現(xiàn)出與hak5突變體一致的低鉀敏感表型,由此認(rèn)為HAK5是ARF2調(diào)控的下游基因。表型檢測(cè)還發(fā)現(xiàn),低鉀條件下,arf2突變體與HAK5過(guò)表達(dá)的根毛都較野生型長(zhǎng),而hak5突變體與arf2 hak5雙突變體的根毛都較野生型短。ChIP-qPCR的結(jié)果顯示,在低鉀條件下,ARF2對(duì)于HAK5啟動(dòng)子區(qū)的結(jié)合減弱,而這一過(guò)程受磷酸化作用調(diào)控。低鉀處理可以增強(qiáng)ARF2的磷酸化修飾,從而解除ARF2對(duì)HAK5的轉(zhuǎn)錄抑制作用。低鉀脅迫可以導(dǎo)致ARF2上第689位絲氨酸被磷酸化,該位點(diǎn)對(duì)于ARF2響應(yīng)低鉀脅迫至關(guān)重要。將689位絲氨酸突變?yōu)樘於彼岷?ARF2對(duì)HAK5啟動(dòng)子的結(jié)合作用顯著減弱。進(jìn)一步研究還發(fā)現(xiàn),在低鉀條件下,ARF2蛋白發(fā)生降解,并且該降解依賴于26S蛋白酶體途徑。本論文工作的研究表明,擬南芥轉(zhuǎn)錄因子ARF2通過(guò)磷酸化修飾來(lái)響應(yīng)低鉀脅迫。低鉀引起的ARF2磷酸化可以解除其對(duì)HAK5的轉(zhuǎn)錄抑制,從而使HK5的表達(dá)升高,HAK5介導(dǎo)的高親和鉀吸收增強(qiáng),利于植物耐受低鉀脅迫。本論文的研究結(jié)果對(duì)于闡明植物響應(yīng)低鉀脅迫的轉(zhuǎn)錄調(diào)控機(jī)制提供了非常重要的實(shí)驗(yàn)證據(jù)。
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2

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1 趙帥;擬南芥轉(zhuǎn)錄因子ARF2響應(yīng)低鉀脅迫的分子機(jī)制研究[D];中國(guó)農(nóng)業(yè)大學(xué);2016年

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本文編號(hào)：2117037