唐古特白刺茉莉酸合成途径关键酶基因NtAOC和NtAOS的克隆及功能分析

发布时间：2018-04-30 08:56

本文选题：唐古特白刺 + 茉莉酸　；参考：《内蒙古大学》2017年硕士论文

【摘要】：唐古特白刺(Nitraria tangutorum Bobr.)为蒺藜科白刺属灌木,通常分布在中国西北部的沙漠地区,具有抵抗高温、干旱、严寒、盐碱化以及防风固沙的能力,对维持沙漠生态平衡和防止土壤盐碱化具有重要意义。已有研究表明茉莉酸作为新型植物激素在植物抗逆方面发挥积极作用。本研究克隆了唐古特白刺茉莉酸合成途径关键酶基因丙二烯氧化物环化酶NtAOC和丙二烯氧化物合酶NtAOS,分析其表达特性,同时构建真核表达载体,将其转入拟南芥中,通过模拟盐胁迫验证NtAOC基因在拟南芥盐胁迫应答中发挥的作用。开展本项研究对探究唐古特白刺中茉莉酸对植物抵抗盐胁迫的调控作用具有重要意义。主要研究结果如下:1.依据转录组数据信息克隆了唐古特白刺NtAOC和NtAOS基因ORF。生物信息学分析表明,NtAOC含有774 bp的开放阅读框,编码257个氨基酸,NtAOS含有1566 bp的开放阅读框,编码521个氨基酸。NtAOC与长春花、豌豆、蒺藜状苜蓿的AOC亲缘关系较近,NtAOS与甜橙、桑树以及野茶树的AOS亲缘关系较近。2.定量RT-PCR结果显示,NtAOC和NtAOS基因在根、茎、叶、花、果实中均表达,且野外采集植物的根茎叶中的表达量均高于室内培养植物根、茎、叶中表达量。NtAOC在花中表达量最高,茎中最低,野外采集植物中表达量大小依次为:花果实叶根茎。而NtAOS在根中表达量最高,在果实中最低,表达量大小依次为:根茎花叶果实。低温、PEG、NaCl、H202、ABA、MeJA处理唐古特白刺幼苗,NtAOC和NtAOS基因的表达量均增加,证明各种非生物胁迫及ABA、茉莉酸激素本身都能诱导唐古特白刺NtAOC和NtAOS基因的表达。3.将NtAOC和NtAOS基因转入拟南芥过表达,获得T3代转基因植株。对NtAOC功能验证结果显示,转基因植株对盐胁迫敏感性增加,随盐胁迫浓度增加,转NtAOC基因拟南芥植株的生长受到抑制的程度亦增加。150mMNaCl处理下,转NtAOC基因拟南芥的鲜重、叶绿素、脯氨酸、ABA含量以及抗氧化酶(POD、CAT)活性均显著低于野生型,丙二醛含量显著高于野生型。4.qPCR检测盐胁迫条件下转基因拟南芥中茉莉酸类合成相关基因AtAOC2、AtOPR3、AtJAZ1、抗氧化系统相关基因AtSOD1、AtC4T1、AtAPX1、脯氨酸合成关键基因AtP5CS1、离子转运相关基因AtSOS1、AtNHX1、抗逆相关转录因子AtABF4、AtDREB2A、AtMYB2的表达量,发现茉莉酸合成基因表达量显著升高,而各类胁迫相关基因及茉莉合成抑制蛋白基因在转基因植株中表达量均降低,且显著低于野生型。说明盐胁迫下NTaOC基因在拟南芥中的超表达诱导了拟南芥自身茉莉酸合成途径关键酶基因的表达,同时负调控了抗逆相关转录因子以及抗逆相关基因的表达,从而使转基因植株表现出对盐胁迫的敏感性。
[Abstract]:Nitraria tangutorum Bobr.) Tribulus terrestris is usually distributed in the desert area of northwestern China. It has the ability of resisting high temperature, drought, cold, salinization and sand fixation. It is of great significance to maintain the ecological balance of desert and prevent soil salinization. Studies have shown that jasmonic acid as a new plant hormone plays an active role in plant stress resistance. In this study, the key enzyme genes of jasmonic acid biosynthesis pathway, NtAOC and NtAOSs, were cloned, their expression characteristics were analyzed, and the eukaryotic expression vector was constructed, which was transferred into Arabidopsis thaliana (Arabidopsis thaliana). The role of NtAOC gene in salt stress response of Arabidopsis thaliana was verified by simulating salt stress. This study is of great significance in exploring the regulation of jasmonic acid on plant salt resistance. The main results are as follows: 1. NtAOC and NtAOS genes were cloned from transcriptional data. Bioinformatics analysis showed that NtAOC contained 774bp open reading frame, encoding 257 amino acids NtAOS containing 1566 BP open reading frame, encoding 521 amino acids. NtAOC was closely related to AOC of Catharanthus roseus, pea and Tribula terrestris. The AOS phylogeny of mulberry and wild tea was close to. 2. The results of quantitative RT-PCR showed that NtAOC and NtAOS genes were expressed in roots, stems, leaves, flowers and fruits. The expression of NtAOC in stems and leaves was higher than that in indoor cultured plants, and the expression of NtAOC in stems and leaves was the highest in flowers and the lowest in stems. The order of expression in field collected plants was: flower, fruit, leaf, root and stem. However, the expression of NtAOS was the highest in root and lowest in fruit, and the order of expression was as follows: rhizomatous mosaic fruit. The expression of NtAOC and NtAOS genes in Spurs chinensis seedlings treated with ABAJA treated with NaCl-H202AJA at low temperature showed that the expression of NtAOC and NtAOS genes could be induced by various abiotic stresses and ABA and jasmonic acid hormones. NtAOC and NtAOS genes were transferred into Arabidopsis thaliana and T 3 transgenic plants were obtained. The results of functional verification of NtAOC showed that the sensitivity of transgenic plants to salt stress increased, and the growth inhibition degree of transgenic Arabidopsis thaliana plants with NtAOC gene increased with the increase of salt stress concentration. The fresh weight and chlorophyll of Arabidopsis thaliana transgenic with NtAOC gene were also increased under the treatment of .150mm NaCl. The content of proline ABA and the activity of antioxidant enzyme POD CAT were significantly lower than those of wild type. The content of malondialdehyde in transgenic Arabidopsis thaliana under salt stress was significantly higher than that in wild type. 4. QPCR was used to detect the biosynthesis of jasmonates in Arabidopsis thaliana. AtAOC 2, AtOPR3, AtJAZ1, AtSOD1, AtC4T1, AtP5CS1, AtSOS1 and AtNHX1, respectively, were detected in Arabidopsis thaliana under salt stress. The expression of AtABF4, AtDREB2AN, AtMYB2, a related transcription factor, It was found that the expression of jasmonic acid biosynthesis genes was significantly increased, while the expression of various stress related genes and jasmine biosynthesis inhibitor genes in transgenic plants was significantly lower than that of wild-type plants. These results suggest that the overexpression of NTaOC gene in Arabidopsis thaliana induced the expression of key enzymes in Arabidopsis autojasmonic acid biosynthesis pathway, and negatively regulated the expression of stress related transcription factors and stress resistance related genes in Arabidopsis thaliana. The transgenic plants showed sensitivity to salt stress.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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