本文選題：短柄草 + 甲硫氨酸亞砜還原酶��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：鹽、旱等非生物脅迫會(huì)誘發(fā)細(xì)胞內(nèi)產(chǎn)生過(guò)量活性氧(Reactive Oxygen Species,ROS),致使蛋白質(zhì)等生物大分子發(fā)生氧化損傷。其中,蛋白質(zhì)表面含有甲硫氨酸(methionine,Met)和半胱氨酸等殘基的蛋白最易受到這種氧化損傷。機(jī)體內(nèi)進(jìn)化的甲硫氨酸亞砜還原酶(methionine sulfoxide reductase,MSR)可以將氧化的甲硫氨酸還原,保護(hù)細(xì)胞免受氧化損傷。盡管研究表明生物中MSR基因參與了多種氧化還原反應(yīng),但它們?cè)诙瘫?B.distachyon)中的作用未見(jiàn)報(bào)道。我們從短柄草測(cè)序公共數(shù)據(jù)庫(kù)中發(fā)掘并克隆出6個(gè)短柄草MSR基因,系統(tǒng)研究了其應(yīng)對(duì)非生物脅迫的功能與作用機(jī)制。主要研究?jī)?nèi)容包括:1短柄草MSR基因家族及轉(zhuǎn)基因酵母的抗逆分析短柄草MSRR基因家族包括6個(gè)基因,其中3個(gè)MSRR4(MSRA2,-A4,-A5)和 3 個(gè) MSRB(MSRB1,-B3,-B5);8 個(gè) cDNA,其中BdMSRB 和BdMSRB5分別有一對(duì)不同的剪接產(chǎn)物(BdMSRB1.1/-B1.2和BdMSRB5.1/-B5.2)。BdMS和家族成員的蛋白序列中都含有同源的保守結(jié)構(gòu)域,催化性的半胱氨酸分布于其中,具有典型的MSR結(jié)構(gòu)特點(diǎn)。這些基因在短柄草的根、莖、葉以及不同發(fā)育時(shí)期的小穗中都有不同豐度的表達(dá)。啟動(dòng)子序列分析結(jié)果顯示它們的啟動(dòng)子上有多種已知功能的脅迫響應(yīng)的順式作用元件,誘導(dǎo)實(shí)驗(yàn)也證明BdMSRR家族成員受到如鹽、旱、低溫、CdCl2、過(guò)氧化氫以及ABA的誘導(dǎo)。底物特異性分析結(jié)果顯示,短柄草MSRB1.1能夠特異還原R型的甲硫氨酸亞砜(Met-R-SO),屬于B亞家族。轉(zhuǎn)化酵母實(shí)驗(yàn)結(jié)果顯示短柄草MSR可以增強(qiáng)酵母對(duì)鹽脅迫、滲透脅迫以及氧化脅迫的耐性。酶活分析表明,在鹽脅迫條件下,BdMSRA4,BdMSRB1.1和BdMSRB5.1還原游離型和肽型MetSO成Met的能力顯著增強(qiáng)。2 BdMRB1.1在擬南芥中的抗逆功能與作用機(jī)制生物信息學(xué)分析顯示BdMSRB1.1編碼的蛋白包含一個(gè)高度保守的N端低復(fù)雜性區(qū)域和C端的SelR催化結(jié)構(gòu)域,其C端包含兩個(gè)保守的半胱氨酸和組氨酸,且最后的硒代半胱氨酸可結(jié)合硒和鋅,結(jié)合酶學(xué)性質(zhì)結(jié)果顯示該蛋白具有甲硫氨酸-R-亞砜還原酶活性。亞細(xì)胞定位結(jié)果顯示BdMSRB1.1定位在葉綠體。酶活測(cè)定結(jié)果表明過(guò)表達(dá)系的MSR酶活比野生系高,而突變系則低于野生系。在NaCl、Mannitol、H2O2以及ABA處理下,轉(zhuǎn)基因株系的抗脅迫能力明顯優(yōu)于野生株系Col0,而突變體株系則相反。測(cè)量各株系的丙二醛(MDA)、ROS和可溶性糖的含量,結(jié)果顯示,過(guò)表達(dá)BdMSRB1.1基因可降低轉(zhuǎn)基因擬南芥的MDA和ROS含量,提高可溶性糖的含量。通過(guò)對(duì)相關(guān)代謝過(guò)程Marker基因分析,結(jié)果表明BdMSRB1.1一方面提高了 ROS清除系統(tǒng)關(guān)鍵酶的表達(dá);另一方面,還促進(jìn)了可溶性糖合成途徑中兩個(gè)關(guān)鍵酶蔗糖合成酶(Sucrose Synthase,SUS)和蔗糖磷酸合成酶(SucrosePhosphate Synthase,SPS)的上調(diào)表達(dá)。以上結(jié)果顯示,短柄草BdMSRB1.1基因參與了植物抵抗非生物脅迫的過(guò)程。
[Abstract]:Salt, drought and other abiotic stresses induce the production of excess active oxygen (Reactive Oxygen Species, ROS) in cells, causing oxidative damage to biological macromolecules such as protein, including methionine (methionine, Met) and cysteine residues, which are most vulnerable to this oxidative damage. Methionine sulfoxide reductase (MSR) can restore the oxidized methionine to protect cells from oxidative damage. Although studies have shown that the MSR gene is involved in a variety of redox reactions, their role in B.distachyon has not been reported. 6 MSR genes were cloned, and the function and mechanism of their response to abiotic stress were systematically studied. The main contents included: 1 the MSR gene family and the resistance analysis of the transgenic yeast, including 6 genes, of which 3 MSRR4 (MSRA2, -A4, -A5) and 3 MSRB (MSRB1, -B3, -B5); 8 cDNA. DMSRB and BdMSRB5 have a conservative domain in the protein sequences of different splice products (BdMSRB1.1/-B1.2 and BdMSRB5.1/-B5.2).BdMS and family members. The catalytic cysteine is distributed in it and has typical MSR structure characteristics. These genes are in the roots, stems, leaves and different developmental stages of the short stalks. The results of promoter sequence analysis showed that there were a variety of known functional cis acting elements on the promoter of the promoter. The induction experiments also showed that the BdMSRR family members were induced by salt, drought, low temperature, CdCl2, hydrogen peroxide and ABA. The substrate specificity analysis showed that the MSRB1.1 could be used as the substrate specific analysis. The specific reduction of R type methionine sulfoxide (Met-R-SO) belongs to the B subfamily. The transformation yeast experiment showed that sb MSR could enhance the tolerance of yeast to salt stress, osmotic stress and oxidative stress. Enzyme activity analysis showed that the ability of BdMSRA4, BdMSRB1.1 and BdMSRB5.1 to reduce free type and peptide MetSO to Met under salt stress showed that the ability to become Met was obvious. Bioinformatics analysis of the anti inverse function and mechanism of.2 BdMRB1.1 in Arabidopsis thaliana shows that the BdMSRB1.1 encoded protein contains a highly conservative N terminal low complex region and C terminal SelR catalytic domain, which contains two conserved cysteine and histidine at the C end, and the final selenocysteine can be combined with selenium and zinc. The results showed that the protein had the activity of methionine -R- sulfoxide reductase. The subcellular localization results showed that BdMSRB1.1 was located in the chloroplast. The results of enzyme activity assay showed that the MSR enzyme activity of the overexpressed line was higher than that of the wild line, but the mutant line was lower than that of the wild line. Under the treatment of NaCl, Mannitol, H2O2 and ABA, the resistance to stress of the transgenic lines was resistant to stress. The ability was obviously superior to the wild strain Col0, but the mutant line was opposite. The content of malondialdehyde (MDA), ROS and soluble sugar were measured. The results showed that overexpression of BdMSRB1.1 gene could reduce the content of MDA and ROS in transgenic Arabidopsis and increase the content of soluble sugar. The results of Marker gene analysis of related metabolic processes showed BdMSRB. 1.1 increased the expression of key enzymes in the ROS scavenging system; on the other hand, it also promoted the up-regulated expression of two key enzymes, sucrose synthase (Sucrose Synthase, SUS) and sucrose phosphate synthetase (SucrosePhosphate Synthase, SPS) in the soluble sugar synthesis pathway. The above results showed that the scavenger BdMSRB1.1 gene was involved in plant resistance. The process of biological stress.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q943.2

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