本文關(guān)鍵詞：微絲骨架Actin介導(dǎo)的擬南芥免疫反應(yīng)及其抗小麥條銹病機(jī)制研究　出處：《西北農(nóng)林科技大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 小麥 擬南芥 Actin RIN4 CAP1

【摘要】：微絲骨架作為真核細(xì)胞中動(dòng)態(tài)變化的三維網(wǎng)狀結(jié)構(gòu),在植物感知和抵抗病原菌侵染中發(fā)揮重要作用。微絲骨架參與植物的免疫反應(yīng),但其如何參與免疫信號(hào)傳導(dǎo)過(guò)程,目前所知甚少。小麥條銹病是由條形柄銹菌(Puccinia striiformis f.sp.tritici)引起的小麥生產(chǎn)上危害嚴(yán)重的一種真菌病害,但是小麥抗條銹病機(jī)制研究尚不完全清楚。因此,從模式植物擬南芥以及小麥重要病害條銹病入手,研究微絲骨架actin(肌動(dòng)蛋白)介導(dǎo)的植物免疫機(jī)制,對(duì)全面了解微絲骨架參與擬南芥免疫機(jī)制,以及進(jìn)一步闡明小麥與條銹菌互作的分子機(jī)理,具有重要意義。本研究以擬南芥和小麥作為研究對(duì)象,主要通過(guò)微絲骨架熒光標(biāo)記、酵母雙雜交、免疫共沉淀、蛋白磷酸化、病毒誘導(dǎo)基因沉默、LC/MS(liquid chromatography/mass spectrometry,液相色譜/質(zhì)譜)技術(shù)和MAPK(mitogen-activated protein kinase,絲裂原活化蛋白激酶)等實(shí)驗(yàn)技術(shù)和方法,篩選與微絲骨架肌動(dòng)蛋白ACT7互作蛋白,對(duì)擬南芥微絲骨架肌動(dòng)蛋白ACT7、肌動(dòng)蛋白結(jié)合蛋白CAP1和小麥肌動(dòng)蛋白解聚因子ADF4的功能進(jìn)行了深入研究。主要研究結(jié)果如下:1.對(duì)擬南芥cDNA文庫(kù)進(jìn)行篩選,選出24個(gè)與ACT7互作的蛋白,其中包括actin結(jié)合蛋白ADF1、ADF4和ARPC3等,與植物免疫相關(guān)的RIN4、ROC4、LOS2蛋白,以及CAP1等多個(gè)目前在植物免疫中功能尚不清楚的蛋白。利用酵母雙雜交和免疫共沉淀技術(shù),進(jìn)一步驗(yàn)證了ACT7與RIN4、CAP1的蛋白相互作用。2.利用擬南芥-丁香假單胞菌模式互作系統(tǒng),發(fā)現(xiàn)ACT7和CAP1參與DC3000和DC3000AvrRpm1引起的擬南芥免疫反應(yīng),ACT7負(fù)調(diào)控?cái)M南芥抗病性,CAP1正調(diào)控?cái)M南芥抗病性。與野生型Col-0相比,act7-2突變體抑制DC3000繁殖,相反cap1突變體促進(jìn)DC3000大量繁殖。cap1突變體產(chǎn)生嚴(yán)重壞死斑,act7-2突變體幾乎無(wú)壞死斑產(chǎn)生。與野生型Col-0相比,act7-2突變體抑制DC3000AvrRpm1生長(zhǎng),壞死面積降低,表現(xiàn)更為抗病;cap1突變體促進(jìn)DC3000AvrRpm1生長(zhǎng),抑制HR反應(yīng),壞死面積增加,表現(xiàn)更為感病。3.利用flg22誘導(dǎo)的MAPK實(shí)驗(yàn),證明突變CAP1顯著抑制flg22誘導(dǎo)的MAPK3/MAPK6的積累,而突變ACT7促進(jìn)flg22誘導(dǎo)的MAPK3/MAPK6的積累。與對(duì)照相比,flg22處理后10 min,cap1突變體中MAPK3/MAPK6積累量顯著降低;act7-2中MAPK3/MAPK6積累量升高。qRT-PCR檢測(cè)發(fā)現(xiàn),PTI信號(hào)途徑marker基因FRK1在act7-2突變體中顯著上調(diào)表達(dá);在cap1突變體中顯著抑制表達(dá)。表明ACT7和CAP1參與flg22誘導(dǎo)的MAPK信號(hào)級(jí)聯(lián)反應(yīng)PTI途徑。4.采用Phos-tag技術(shù)對(duì)DC3000AvrRpm1誘導(dǎo)RIN4在act7-2和cap1突變體中磷酸化情況進(jìn)行分析,發(fā)現(xiàn)與野生型相比,ACT7突變后,RIN4磷酸化被促進(jìn)并增強(qiáng),而RIN4磷酸化在CAP1突變后受到抑制。接種病原菌DC3000AvrRpm1后,act7-2中RIN4積累量顯著降低,而cap1中RIN4的積累卻未降低。qRT-PCR分析顯示,ETI免疫相關(guān)基因RPM1、PR1以及WRKY70在ACT7突變后表達(dá)量升高,在CAP1突變后表達(dá)量顯著降低。表明ACT7和CAP1參與RIN4介導(dǎo)的擬南芥ETI免疫信號(hào)途徑。5.從小麥cDNA文庫(kù)中,克隆了6個(gè)小麥ADF基因,分別命名為TaADF3、TaADF4、TaADF5、TaADF6、TaADF8和TaADF11。酵母雙雜交及免疫共沉淀結(jié)果,證明TaADF4與TaACT1、AtADF4與AtACT7直接互作,同時(shí)TaADF4與AtACT7、AtADF4與TaACT1交叉互作。蛋白三維結(jié)構(gòu)建模,TaADF4與AtADF4蛋白模型幾乎完全重合,表明TaADF4和AtADF4結(jié)構(gòu)功能相似。小麥ADFs與TaACT1特異性互作分析顯示,TaADF8不與TaACT1互作,且TaADF8蛋白結(jié)構(gòu)模型獨(dú)立于另外5個(gè)小麥ADF蛋白結(jié)構(gòu)模型,表明不同的小麥ADFs可能存在功能特異性。激光共聚焦顯微觀察,發(fā)現(xiàn)TaADF4-RFP與TaACT1-GFP熒光標(biāo)記蛋白信號(hào)共定位于微絲骨架。蛋白磷酸化實(shí)驗(yàn),證明AtADF4和TaADF4活性被CPK3磷酸化調(diào)節(jié)。6.qRT-PCR分析顯示,TaADF4參與小麥生物與非生物應(yīng)答,TaADF4被高溫、茉莉酸甲酯和脫落酸誘導(dǎo)上調(diào)表達(dá),鹽和低溫顯著抑制其表達(dá)。TaADF4在小麥與條銹菌CYR23非親和互作中表達(dá)量顯著高于小麥與條銹菌CYR31親和互作。利用VIGS技術(shù)沉默TaADF4后,小麥對(duì)條銹菌CYR23的抗性降低。LatB處理微絲骨架誘導(dǎo)TaADF4上調(diào)表達(dá),抑制條銹菌菌絲生長(zhǎng),促進(jìn)活性氧的積累和過(guò)敏性壞死。利用LC/MS技術(shù),對(duì)植物激素JA(jasmonic acid茉莉酸)和SA(salicylic acid水楊酸)的積累進(jìn)行檢測(cè),結(jié)果顯示TaADF4沉默后,JA的積累量較對(duì)照組明顯降低。以上結(jié)果證明,TaADF4通過(guò)參與小麥JA信號(hào)途徑轉(zhuǎn)導(dǎo),積極調(diào)節(jié)小麥對(duì)條銹菌小種CYR23的抗性作用。綜上所述,本研究進(jìn)一步解析了微絲骨架在植物免疫過(guò)程中的重要作用,首次發(fā)現(xiàn)并證明了微絲骨架肌動(dòng)蛋白ACT7和環(huán)化酶相關(guān)蛋白CAP1,參與flg22誘導(dǎo)的MAPK擬南PTI信號(hào)途徑和RIN4介導(dǎo)的ETI信號(hào)傳導(dǎo)途徑,最后探索了小麥肌動(dòng)蛋白解聚因子TaADF4在小麥抵抗條銹病侵染過(guò)程中的作用,為全面了解植物的病害分子機(jī)制提供了理論依據(jù)。
[Abstract]:As a three-dimensional network structure of actin cytoskeleton dynamics in eukaryotic cells, play an important role in plant resistance to pathogen infection and perception. The immune response of actin cytoskeleton in plant, but how to participate in the process of immune signaling. Little is known about the wheat stripe rust by Puccinia striiformis (Puccinia striiformis f.sp.tritici) caused by the wheat production of a fungal disease of serious harm, but the study of wheat stripe rust resistance mechanism is still not clear. Therefore, starting from the model plant Arabidopsis and wheat stripe rust disease, actin of microfilaments (actin) plant immune mediated mechanism, to fully understand the actin cytoskeleton in Arabidopsis thaliana and further molecular immune mechanism. To clarify the interaction between wheat and stripe rust mechanism, which is of great significance. In this study, Arabidopsis and wheat as the research object, mainly through micro Wire skeleton fluorescent labeling, yeast two hybrid, CO immunoprecipitation, protein phosphorylation, virus induced gene silencing, LC/MS (liquid chromatography/mass spectrometry, liquid chromatography / mass spectrometry (mitogen-activated) technology and MAPK protein kinase, mitogen activated protein kinase) and other experimental techniques and methods of screening and actin cytoskeleton actin ACT7 interacting protein, the Arabidopsis ACT7 actin microfilaments, actin binding protein CAP1 and wheat actin depolymerizing factor ADF4 function are studied. The main results are as follows: 1. of the Arabidopsis cDNA library was screened and selected 24 proteins interacting with ACT7, including the actin binding protein ADF1, ADF4 and ARPC3, related to plant immune RIN4 ROC4, CAP1, LOS2 protein, and other functions in plant immunity is unclear. The protein using yeast two hybrid and co immunoprecipitation Technology, further validation of the ACT7 and RIN4, CAP1 protein.2. interaction using Arabidopsis Pseudomonas syringae interaction system model, found that Arabidopsis immune response of ACT7 and CAP1 in DC3000 and DC3000AvrRpm1 by ACT7, the negative regulation of disease resistance in Arabidopsis, CAP1 positively regulates Arabidopsis resistance. Compared with the wild type Col-0, act7-2 inhibition of DC3000 mutant breeding, instead of cap1 mutant promote DC3000 multiply.Cap1 mutants have serious necrosis, act7-2 mutant almost no necrosis spot. Compared with wild-type Col-0, mutant act7-2 inhibit the growth of DC3000AvrRpm1, the necrotic area decreased, especially for disease resistance; cap1 mutant promote DC3000AvrRpm1 growth inhibition, HR reaction, necrosis area increased more for the experiment of MAPK susceptible.3. induced by flg22, demonstrated that the mutant CAP1 significantly inhibited flg22 induced MAPK3/MAPK6 accumulation and mutation of ACT7 promoted flg2 2 induced the accumulation of MAPK3/MAPK6. Compared with the control group, after treatment with flg22 10 min MAPK3/MAPK6 cap1 mutant accumulation was significantly decreased; MAPK3/MAPK6 accumulation increased.QRT-PCR detected in act7-2, up regulate the expression of PTI signaling pathway of marker gene FRK1 in act7-2 mutant; in mutant cap1 inhibit the expression of ACT7 and CAP1 in the.4. show. The MAPK signaling cascade PTI pathway induced by flg22 using Phos-tag technology on DC3000AvrRpm1 induced phosphorylation of RIN4 in act7-2 and cap1 mutants in the analysis of the situation, found that compared with the wild type, ACT7 mutant, RIN4 phosphorylation was promoted and enhanced, and the inhibition of RIN4 phosphorylation in the CAP1 mutant after inoculation. DC3000AvrRpm1, act7-2 in the accumulation of RIN4 was obviously decreased, while cap1 did not reduce the accumulation of RIN4 in.QRT-PCR analysis showed that ETI gene related to immune RPM1, PR1 and WRKY70 in ACT7 mutant After the expression increased, significantly reduced the expression of CAP1 in mutant. The results indicated that ACT7 and CAP1 participate in the.5. of Arabidopsis ETI immune signaling pathway mediated by RIN4 from wheat cDNA library, cloned 6 ADF genes of wheat, which were named TaADF3, TaADF4, TaADF5, TaADF6, TaADF8 and TaADF11. yeast two hybrid and co the results showed that TaADF4 and TaACT1 precipitation, AtADF4, direct interaction with AtACT7, and TaADF4 and AtACT7, AtADF4 and TaACT1 cross interaction. Protein three-dimensional structure modeling, TaADF4 model and AtADF4 protein almost completely overlap, indicating that TaADF4 and AtADF4 structure. ADFs and TaACT1 function similar to wheat specific interaction analysis showed that the TaADF8 interaction with TaACT1, TaADF8 and protein structure model is independent of the other 5 wheat ADF protein structure model shows that different wheat ADFs may have specific function. Laser confocal microscopic observation, TaADF4-RFP and TaACT1-GFP. Label protein signals were localized in the actin cytoskeleton. Protein phosphorylation experiments show that AtADF4 and TaADF4 activity by CPK3 phosphorylation of.6.qRT-PCR analysis showed that TaADF4 is involved in wheat biological and non biological response, TaADF4 temperature, methyl jasmonate and abscisic acid induced expression, salt and low temperature significantly inhibited the expression of.TaADF4 in wheat and stripe rust CYR23 incompatible expression was significantly higher than that of wheat stripe rust and CYR31 affinity interaction. Using VIGS technology to silence TaADF4, resistance to stripe rust of wheat CYR23.LatB can reduce the expression of actin cytoskeleton induced by TaADF4, the growth inhibition of stripe rust hypha, promote the accumulation of active oxygen and hypersensitive. Using LC/MS technology. JA (jasmonic acid on plant hormone jasmonic acid) and SA (salicylic acid salicylic acid) accumulation were detected, the results showed that TaADF4 gene silencing, JA accumulation was lower than the control group Low. The above results proved that the TaADF4 of wheat JA by participating in signal transduction, actively regulate the resistance of wheat to stripe rust race CYR23. In conclusion, this study further analyzes the important role of actin cytoskeleton in plant immune process, first discovered and proved the microfilaments and actin ACT7 cyclase associated protein CAP1, participate in MAPK flg22 induced quasi ETI signal transduction pathway South PTI pathway and RIN4 mediated, and finally explore the actin depolymerizing factor TaADF4 of wheat resistance to stripe rust infection function in the process of wheat, and provides a theoretical basis for the comprehensive understanding of the molecular mechanism of plant disease.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S435.121.42

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本文編號(hào)：1438004