發(fā)布時(shí)間：2018-04-17 00:21

  本文選題：蛋白質(zhì)結(jié)構(gòu) + 功能域　； 參考：《中國(guó)農(nóng)業(yè)科學(xué)院》2016年博士論文

【摘要】：在長(zhǎng)期的進(jìn)化中,植物形成了一套有效的免疫系統(tǒng)應(yīng)對(duì)逆境環(huán)境。植物免疫是基于對(duì)環(huán)境中誘導(dǎo)子的識(shí)別,蛋白質(zhì)激發(fā)子是最主要的誘導(dǎo)子之一,已成為植物免疫領(lǐng)域的一個(gè)研究熱點(diǎn)。蛋白質(zhì)激發(fā)子MoHrip2是從稻瘟菌中分離獲得的一種外分泌蛋白,能夠誘導(dǎo)煙草免疫系統(tǒng)的激活,提高水稻對(duì)白葉枯病和稻瘟病的抗性。然而,MoHrip2作為激發(fā)子的作用機(jī)理尚不清楚。本研究從蛋白質(zhì)結(jié)構(gòu)、結(jié)構(gòu)與功能的關(guān)系、互作蛋白的篩選與驗(yàn)證以及生物學(xué)功能等方面對(duì)MoHrip2進(jìn)行研究,獲得了以下研究成果:1、解析獲得MoHrip2的三維結(jié)構(gòu)通過原核表達(dá)系統(tǒng)獲得了MoHrip2蛋白以及摻入硒原子的蛋白衍生物,對(duì)蛋白質(zhì)結(jié)晶條件進(jìn)行篩選并優(yōu)化,最終獲得MoHrip2蛋白母體及衍生物的高質(zhì)量單晶;利用X-射線衍射技術(shù)獲得了MoHrip2蛋白母體及衍生物的衍射數(shù)據(jù),分辨率分別可達(dá)2?和1.8?;通過硒原子產(chǎn)生的反常散射數(shù)據(jù),確定了MoHrip2蛋白晶體的相位,每個(gè)不對(duì)稱單元含有兩個(gè)單體;利用COOT軟件進(jìn)行模型搭建和精細(xì)修改,解析獲得了MoHrip2的三維結(jié)構(gòu),每個(gè)單體形成一個(gè)β-桶狀結(jié)構(gòu),包括11個(gè)β-折疊片和一個(gè)很短的α-螺旋,分子內(nèi)形成3對(duì)二硫鍵;通過DALI軟件進(jìn)行結(jié)構(gòu)比對(duì),發(fā)現(xiàn)MoHrip2與TL-XI蛋白在結(jié)構(gòu)上具有很高的相似度。2、確定了MoHrip2的功能域基于二級(jí)結(jié)構(gòu),對(duì)MoHrip2蛋白進(jìn)行截短并利用原核表達(dá)系統(tǒng)表達(dá)和純化,獲得了8個(gè)截短突變體;對(duì)各截短突變體進(jìn)行誘導(dǎo)煙草HR和抗病性分析,確定位于中間部位的第76-89位共14個(gè)氨基酸殘基組成的短肽段,是MoHrip2發(fā)揮激發(fā)子活性的功能域。3、獲得MoHrip2在擬南芥中的互作蛋白Mo2BP1以MoHrip2為誘餌蛋白,通過酵母雙雜交技術(shù)篩選擬南芥cDNA文庫(kù),獲得了4個(gè)候選互作蛋白;通過雙分子熒光互補(bǔ)實(shí)驗(yàn)進(jìn)行體內(nèi)驗(yàn)證,其中一個(gè)蛋白能夠在煙草細(xì)胞中與MoHrip2互作,命名為Mo2BP1;Mo2BP1為一個(gè)類奇異果甜-病程相關(guān)蛋白,在植物體內(nèi)參與響應(yīng)外源刺激;對(duì)Mo2BP1進(jìn)行適應(yīng)大腸桿菌表達(dá)的密碼子優(yōu)化,并重組表達(dá)、純化獲得GST-Mo2BP1融合蛋白;通過GST pull-down實(shí)驗(yàn)驗(yàn)證兩個(gè)蛋白在體外能夠相互作用。4、Mo2BP1缺失影響MoHrip2的功能在煙草葉片細(xì)胞中瞬時(shí)表達(dá)Mo2BP1-GFP融合蛋白,使用共聚焦激光掃描顯微鏡觀察熒光,確定Mo2BP1在煙草細(xì)胞中定位于細(xì)胞膜上;購(gòu)買獲得Mo2BP1缺失突變雜合擬南芥,通過培育、篩選獲得其純合突變體;通過分析MoHrip2在野生型和突變體擬南芥中誘導(dǎo)HR和抗病性的不同效果,確定Mo2BP1在MoHrip2誘導(dǎo)擬南芥產(chǎn)生HR和提高抗病性中發(fā)揮重要作用。5、MoHrip2在擬南芥中過表達(dá)提高抗病性通過農(nóng)桿菌介導(dǎo)法轉(zhuǎn)化擬南芥,獲得MoHrip2穩(wěn)定遺傳表達(dá)的轉(zhuǎn)基因植株;分析轉(zhuǎn)基因擬南芥對(duì)灰葡萄孢菌的抗性,表明MoHrip2過表達(dá)能夠提高擬南芥的抗病性。
[Abstract]:Over the course of long-term evolution, plants have developed an effective immune system for coping with adverse environments.Plant immunity is based on the recognition of elicitors in the environment. Protein elicitor is one of the most important elicitors and has become a hotspot in the field of plant immunity.Protein elicitor MoHrip2 is an exocrine protein isolated from Magnaporthe grisea. It can induce the activation of tobacco immune system and increase the resistance of rice to bacterial blight and blast.However, the mechanism of MoHrip2 as excitator is unclear.In this study, MoHrip2 was studied from the aspects of protein structure, relationship between structure and function, screening and verification of interacting proteins and biological functions.The following research results were obtained: 1: 1. The three-dimensional structure of MoHrip2 was analyzed and obtained by prokaryotic expression system. MoHrip2 protein and protein derivatives doped with selenium atom were obtained by using prokaryotic expression system. The crystallization conditions of proteins were screened and optimized.The high quality single crystal of MoHrip2 protein parent and derivative was obtained, and the diffraction data of MoHrip2 protein parent and derivative were obtained by using X-ray diffraction technique, the resolution of MoHrip2 protein parent and derivative were up to 2?Using anomalous scattering data from selenium atoms, the phase of MoHrip2 protein crystal was determined, each asymmetric unit containing two monomers. The three-dimensional structure of MoHrip2 was obtained by modeling and fine modification by COOT software.Each monomer forms a 尾 -barrel structure, consisting of 11 尾 -folds and a very short 偽 -helix, in which three pairs of disulfide bonds are formed within the molecule.The structural similarity between MoHrip2 and TL-XI protein was found to be very high. The functional domain of MoHrip2 was determined based on secondary structure. The MoHrip2 protein was truncated and expressed and purified by prokaryotic expression system. Eight truncated mutants were obtained.HR and disease resistance of the truncated mutants were analyzed, and the short peptides composed of 14 amino acid residues at position 76-89 were identified.The interaction protein of MoHrip2 in Arabidopsis thaliana (Mo2BP1) was obtained by using MoHrip2 as bait protein. CDNA library of Arabidopsis thaliana was screened by yeast two-hybrid technique, and four candidate interaction proteins were obtained.In vivo, a protein was identified to interact with MoHrip2 in tobacco cells by bimolecular fluorescence complementary assay. It was named Mo2BP1Mo2BP1 as a kiwifruit like sweet disease-course related protein, which was involved in response to exogenous stimuli in plants.The codon of Mo2BP1 adapted to E. coli expression was optimized and recombined to obtain GST-Mo2BP1 fusion protein.GST pull-down assay showed that the function of MoHrip2 could be affected by the deletion of MoHrip2 in vitro. The Mo2BP1-GFP fusion protein was transiently expressed in tobacco leaf cells. The fluorescence was observed by confocal laser scanning microscope.The homozygous mutant Arabidopsis thaliana (Arabidopsis thaliana) with Mo2BP1 deletion mutation was obtained and its homozygous mutants were obtained by breeding.By analyzing the different effects of MoHrip2 on inducing HR and disease resistance in wild type and mutant Arabidopsis thaliana,It was confirmed that Mo2BP1 plays an important role in the production of HR and the improvement of disease resistance in Arabidopsis thaliana induced by MoHrip2. The overexpression of MoHrip2 in Arabidopsis thaliana and the improvement of resistance to disease were transformed into Arabidopsis thaliana by Agrobacterium tumefaciens. Transgenic plants with stable genetic expression of MoHrip2 were obtained.The analysis of resistance of transgenic Arabidopsis thaliana to Grapevine showed that overexpression of MoHrip2 could improve the resistance of Arabidopsis thaliana.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S435.11
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本文編號(hào)：1761237