珍稀泌盐植物长叶红砂RtGRX与RtSOD基因的克隆及功能分析

发布时间：2018-02-08 12:52

  本文关键词： 长叶红砂 谷氧还蛋白 超氧化物歧化酶 非生物胁迫 转基因拟南芥　出处：《内蒙古大学》2017年硕士论文　论文类型：学位论文

【摘要】：长叶红砂(Reaumuriatrigyna)是东阿拉善-西鄂尔多斯特有的强旱生泌盐盐生植物,对盐渍荒漠环境具有极强的适应性。转录组深度测序结果显示,该植物在盐胁迫条件下,抗氧化系统相关基因差异表达明显,对植物响应逆境胁迫具有积极作用。研究表明超氧化物歧化酶SOD和谷氧还蛋白GRX参与了植物逆境胁迫下的活性氧清除及蛋白质氧化还原状态的调节。本研究从长叶红砂中克隆了RtGRX和RtSOD基因,对其进行组织特异性分析及不同胁迫下表达特性分析,将该基因转入拟南芥,验证其在不同胁迫条件下的功能,为深入了解RtGRX及RtSOD基因在植物抵抗逆境胁迫过程中的调控功能奠定了基础。主要结果如下:1.依据转录组数据库信息扩增得到长叶红砂谷氧还蛋白基因,命名为RtGRX;长叶红砂超氧化物歧化酶基因,命名为RtSOD。RtGRX基因的开放阅读框大小为807bp,编码268个氨基酸,预测分子量66.98kDa,理论等电点5.17。RtSOD基因的开放阅读框大小为663bp,编码220个氨基酸,预测分子量55.90kDa理论等电点5.11。氨基酸序列比对分析显示,RtGRX属于GRX蛋白家族,RtSOD属于Cu/ZnSOD蛋白家族。系统进化树分析显示长叶红砂的RtGRX与番茄和美花烟草的GRX亲缘关系较近,而RtSOD与刚毛柽柳的SOD亲缘关系最近。2.组织特异性分析结果显示,RtGRX和RtSOD基因在长叶红砂根、茎、叶中均表达,且在茎中表达量显著高于根和叶。基因表达特性分析结果显示,采用400mM NaCl、4℃、PEG、H2O2、ABA等胁迫处理长叶红砂,RtGRX和RtSOD基因的表达量均上调;在不同浓度NaCl胁迫下,两基因均在400mM NaCl下表达量最高。3.构建RtGRX和RtSOD真核表达载体,将其转化到拟南芥中,结果发现:盐、干旱胁迫条件下转RtGRX和RtSOD基因拟南芥的生长状况(根长、鲜重、叶绿素含量)均优于野生型,抗氧化酶活性(SOD、POD、CAT)和脯氨酸含量较野生型显著升高,H2O2及MDA含量较野生型显著降低,说明以上两基因提高了转基因植株的抗氧化酶活性,减少了体内活性氧含量,减轻了膜损伤程度,从而提高了转基因植物在盐和干旱胁迫环境下的生长发育、光合作用以及抵抗氧化胁迫的能力,增强了转基因植株的耐受性。4.qPCR检测转基因拟南芥中响应逆境胁迫相关基因的表达量,如抗氧化系统相关基因AtSOD1、AtPOD1、AtAPX1、AtCAT1,脯氨酸合成关键基因AtP5CS1,离子转运蛋白基因AtSOS1,结果显示,转基因植株中以上基因的表达量均显著高于野生型,说明RtGRX和RtSOD基因在拟南芥中的超表达提高了转基因株系参与植物氧化平衡、渗透平衡以及离子平衡等方面的基因活性,进而提高其抗逆性。
[Abstract]:Reaumuriatrigyna) is a strong xerophytic halophyte plant in East Alashan-West Ordos, which has a strong adaptability to saline desert environment. The results of transcriptome deep sequencing showed that the plant was under salt stress. The differential expression of genes related to antioxidant system was obvious. The study showed that superoxide dismutase (SOD) and glutathione (GRX) were involved in reactive oxygen species (Ros) scavenging and protein redox regulation under plant stress. The RtGRX and RtSOD genes were cloned from Yehongsha. The gene was transformed into Arabidopsis thaliana by tissue specific analysis and expression characteristic analysis under different stress conditions to verify its function under different stress conditions. The main results are as follows: 1. According to the information of transcriptional database, the long leaf red glutenin gene was obtained. The open reading frame named RtSOD.RtGRX gene was 807bp, encoding 268 amino acids, the predicted molecular weight was 66.98kDa. the open reading frame size of theoretical isoelectric point 5.17.RtSOD gene was 663bpand encoding 220 amino acids. The predicted molecular weight was 55.90 kDa theoretical isoelectric point 5.11.The amino acid sequence alignment analysis showed that RtGRX belonged to the GRX protein family and Cu/ZnSOD protein family. Phylogenetic tree analysis showed that the phylogenetic tree analysis showed that the RtGRX of longleaf red sand was closely related to the GRX of tomato and American flower tobacco. The results of tissue specific analysis showed that RtGRX and RtSOD genes were expressed in root, stem and leaf of Tamarix chinensis, and the expression level in stem was significantly higher than that in root and leaf. The expression of RtGRX and RtSOD genes were upregulated by 400mm NaCl-4 鈩，

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