短柄草甲硫氨酸亚砜还原酶基因家族及MSRB1.1基因的功能分析

发布时间：2018-06-02 12:17

本文选题：短柄草 + 甲硫氨酸亚砜还原酶　；参考：《山东大学》2017年硕士论文

【摘要】：盐、旱等非生物胁迫会诱发细胞内产生过量活性氧(Reactive Oxygen Species,ROS),致使蛋白质等生物大分子发生氧化损伤。其中,蛋白质表面含有甲硫氨酸(methionine,Met)和半胱氨酸等残基的蛋白最易受到这种氧化损伤。机体内进化的甲硫氨酸亚砜还原酶(methionine sulfoxide reductase,MSR)可以将氧化的甲硫氨酸还原,保护细胞免受氧化损伤。尽管研究表明生物中MSR基因参与了多种氧化还原反应,但它们在短柄草(B.distachyon)中的作用未见报道。我们从短柄草测序公共数据库中发掘并克隆出6个短柄草MSR基因,系统研究了其应对非生物胁迫的功能与作用机制。主要研究内容包括:1短柄草MSR基因家族及转基因酵母的抗逆分析短柄草MSRR基因家族包括6个基因,其中3个MSRR4(MSRA2,-A4,-A5)和 3 个 MSRB(MSRB1,-B3,-B5);8 个 cDNA,其中BdMSRB 和BdMSRB5分别有一对不同的剪接产物(BdMSRB1.1/-B1.2和BdMSRB5.1/-B5.2)。BdMS和家族成员的蛋白序列中都含有同源的保守结构域,催化性的半胱氨酸分布于其中,具有典型的MSR结构特点。这些基因在短柄草的根、茎、叶以及不同发育时期的小穗中都有不同丰度的表达。启动子序列分析结果显示它们的启动子上有多种已知功能的胁迫响应的顺式作用元件,诱导实验也证明BdMSRR家族成员受到如盐、旱、低温、CdCl2、过氧化氢以及ABA的诱导。底物特异性分析结果显示,短柄草MSRB1.1能够特异还原R型的甲硫氨酸亚砜(Met-R-SO),属于B亚家族。转化酵母实验结果显示短柄草MSR可以增强酵母对盐胁迫、渗透胁迫以及氧化胁迫的耐性。酶活分析表明,在盐胁迫条件下,BdMSRA4,BdMSRB1.1和BdMSRB5.1还原游离型和肽型MetSO成Met的能力显著增强。2 BdMRB1.1在拟南芥中的抗逆功能与作用机制生物信息学分析显示BdMSRB1.1编码的蛋白包含一个高度保守的N端低复杂性区域和C端的SelR催化结构域,其C端包含两个保守的半胱氨酸和组氨酸,且最后的硒代半胱氨酸可结合硒和锌,结合酶学性质结果显示该蛋白具有甲硫氨酸-R-亚砜还原酶活性。亚细胞定位结果显示BdMSRB1.1定位在叶绿体。酶活测定结果表明过表达系的MSR酶活比野生系高,而突变系则低于野生系。在NaCl、Mannitol、H2O2以及ABA处理下,转基因株系的抗胁迫能力明显优于野生株系Col0,而突变体株系则相反。测量各株系的丙二醛(MDA)、ROS和可溶性糖的含量,结果显示,过表达BdMSRB1.1基因可降低转基因拟南芥的MDA和ROS含量,提高可溶性糖的含量。通过对相关代谢过程Marker基因分析,结果表明BdMSRB1.1一方面提高了 ROS清除系统关键酶的表达;另一方面,还促进了可溶性糖合成途径中两个关键酶蔗糖合成酶(Sucrose Synthase,SUS)和蔗糖磷酸合成酶(SucrosePhosphate Synthase,SPS)的上调表达。以上结果显示,短柄草BdMSRB1.1基因参与了植物抵抗非生物胁迫的过程。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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