本文選題：擬南芥 + 鈣信號��； 參考：《中國農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：鈣離子(Ca2+)是植物細胞內(nèi)重要的第二信使,它對植物響應(yīng)外界環(huán)境脅迫有著至關(guān)重要的作用。許多非生物脅迫或生物脅迫,如氧化脅迫、熱脅迫、滲透脅迫、鹽脅迫和病原菌脅迫等,都會導(dǎo)致胞內(nèi)游離Ca2+(Cytosolicfree Ca2+,[Ca2]濃度的升高。不同脅迫下,[Ca2+]cyt濃度升高的持續(xù)時間、強度、頻率、空間分布等都具有一定的特異性,因而被稱作"鈣信號"(Calcium signature)。鈣信號被細胞內(nèi)鈣響應(yīng)元件解碼,并轉(zhuǎn)化為下游的磷酸化活動和轉(zhuǎn)錄活動。目前對鈣信號的研究主要集中在Ca2+通道和Ca2+轉(zhuǎn)運蛋白的生物學(xué)作用,以及鈣響應(yīng)元件調(diào)控下游信號轉(zhuǎn)導(dǎo)的過程,但是對特異的鈣信號如何產(chǎn)生,又如何調(diào)控植物的脅迫響應(yīng),還了解得比較少。為了尋找鈣信號形成過程中的重要基因,本研究通過正向遺傳學(xué)的方法,在穩(wěn)定表達鈣監(jiān)測蛋白AEQUORIN的擬南芥轉(zhuǎn)基因株系背景下,篩選鈣信號異常的突變體。經(jīng)過篩選發(fā)現(xiàn),在H202刺激下,MYB30基因的突變體[Ca2+]cyt濃度的升高明顯高于野生型。擬南芥MYB30轉(zhuǎn)錄因子是植物R2R3-MYB家族的重要成員。在H202刺激下,回補材料可以將突變體中[Ca2+]cyt濃度恢復(fù)至野生型水平,MYB30過表達植株[Ca2+]cyt濃度的升高明顯低于野生型。這些結(jié)果表明,H202刺激下MYB30影響[Ca2+]cyt濃度的升高過程。Real-time RT-PCR結(jié)果表明,MYB30抑制植物ANNEXINs(ANNs)基因的表達。ANNs蛋白是一種Ca2+依賴的膜結(jié)合蛋白,它影響多種脅迫刺激下植物胞內(nèi)鈣信號的形成。熒光素酶實驗,染色質(zhì)免疫共沉淀實驗(ChIP),凝膠阻滯實驗(EMSA)和酵母單雜交實驗結(jié)果表明,MYB30在體內(nèi)和體外都可以結(jié)合ANN1和ANN4基因的啟動子,并抑制ANN1和ANN4基因的表達。表型分析結(jié)果顯示,myb30對甲基紫精(MV)敏感,對熱脅迫耐受。氧化脅迫和熱脅迫下,myb30的鈣信號異常表型、氧化脅迫敏感表型和熱脅迫耐受表型,都被ANNs的缺失或Ca2+通道抑制劑]LaCl3所部分恢復(fù)。這說明,ANN1和ANN4介導(dǎo)的鈣信號通路參與了 MYB30對氧化脅迫和熱脅迫響應(yīng)的調(diào)節(jié)過程。多種脅迫都導(dǎo)致myb30產(chǎn)生異常的鈣信號,但只有部分脅迫下胞內(nèi)鈣信號的產(chǎn)生依賴于ANNs。myb30的某些脅迫敏感表型,如ABA敏感表型,不被ANNs的缺失或LaCl3所恢復(fù)。這說明不同脅迫下鈣信號的產(chǎn)生具有特異性。綜上所述,本論文的研究發(fā)現(xiàn)了擬南芥MYB30轉(zhuǎn)錄因子通過影響ANNs介導(dǎo)的特異的胞內(nèi)鈣信號的產(chǎn)生,調(diào)控了植物的氧化脅迫響應(yīng)和熱脅迫響應(yīng)過程。這些發(fā)現(xiàn)使我們能更深入地了解脅迫刺激下植物胞內(nèi)鈣信號的特異性產(chǎn)生,與其在植物脅迫響應(yīng)過程中的重要作用。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q943.2

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相關(guān)期刊論文 前2條

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2 ;Microfilament Dynamics is Required for Root Growth under Alkaline Stress in Arabidopsis[J];Journal of Integrative Plant Biology;2010年11期

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本文編號：2100740