本文選題：唐古特白刺 + 茉莉酸�。� 參考：《內(nèi)蒙古大學(xué)》2017年碩士論文

【摘要】：唐古特白刺(Nitraria tangutorum Bobr.)為蒺藜科白刺屬灌木,通常分布在中國(guó)西北部的沙漠地區(qū),具有抵抗高溫、干旱、嚴(yán)寒、鹽堿化以及防風(fēng)固沙的能力,對(duì)維持沙漠生態(tài)平衡和防止土壤鹽堿化具有重要意義。已有研究表明茉莉酸作為新型植物激素在植物抗逆方面發(fā)揮積極作用。本研究克隆了唐古特白刺茉莉酸合成途徑關(guān)鍵酶基因丙二烯氧化物環(huán)化酶NtAOC和丙二烯氧化物合酶NtAOS,分析其表達(dá)特性,同時(shí)構(gòu)建真核表達(dá)載體,將其轉(zhuǎn)入擬南芥中,通過(guò)模擬鹽脅迫驗(yàn)證NtAOC基因在擬南芥鹽脅迫應(yīng)答中發(fā)揮的作用。開(kāi)展本項(xiàng)研究對(duì)探究唐古特白刺中茉莉酸對(duì)植物抵抗鹽脅迫的調(diào)控作用具有重要意義。主要研究結(jié)果如下:1.依據(jù)轉(zhuǎn)錄組數(shù)據(jù)信息克隆了唐古特白刺N(yùn)tAOC和NtAOS基因ORF。生物信息學(xué)分析表明,NtAOC含有774 bp的開(kāi)放閱讀框,編碼257個(gè)氨基酸,NtAOS含有1566 bp的開(kāi)放閱讀框,編碼521個(gè)氨基酸。NtAOC與長(zhǎng)春花、豌豆、蒺藜狀苜蓿的AOC親緣關(guān)系較近,NtAOS與甜橙、桑樹(shù)以及野茶樹(shù)的AOS親緣關(guān)系較近。2.定量RT-PCR結(jié)果顯示,NtAOC和NtAOS基因在根、莖、葉、花、果實(shí)中均表達(dá),且野外采集植物的根莖葉中的表達(dá)量均高于室內(nèi)培養(yǎng)植物根、莖、葉中表達(dá)量。NtAOC在花中表達(dá)量最高,莖中最低,野外采集植物中表達(dá)量大小依次為:花果實(shí)葉根莖。而NtAOS在根中表達(dá)量最高,在果實(shí)中最低,表達(dá)量大小依次為:根莖花葉果實(shí)。低溫、PEG、NaCl、H202、ABA、MeJA處理唐古特白刺幼苗,NtAOC和NtAOS基因的表達(dá)量均增加,證明各種非生物脅迫及ABA、茉莉酸激素本身都能誘導(dǎo)唐古特白刺N(yùn)tAOC和NtAOS基因的表達(dá)。3.將NtAOC和NtAOS基因轉(zhuǎn)入擬南芥過(guò)表達(dá),獲得T3代轉(zhuǎn)基因植株。對(duì)NtAOC功能驗(yàn)證結(jié)果顯示,轉(zhuǎn)基因植株對(duì)鹽脅迫敏感性增加,隨鹽脅迫濃度增加,轉(zhuǎn)NtAOC基因擬南芥植株的生長(zhǎng)受到抑制的程度亦增加。150mMNaCl處理下,轉(zhuǎn)NtAOC基因擬南芥的鮮重、葉綠素、脯氨酸、ABA含量以及抗氧化酶(POD、CAT)活性均顯著低于野生型,丙二醛含量顯著高于野生型。4.qPCR檢測(cè)鹽脅迫條件下轉(zhuǎn)基因擬南芥中茉莉酸類(lèi)合成相關(guān)基因AtAOC2、AtOPR3、AtJAZ1、抗氧化系統(tǒng)相關(guān)基因AtSOD1、AtC4T1、AtAPX1、脯氨酸合成關(guān)鍵基因AtP5CS1、離子轉(zhuǎn)運(yùn)相關(guān)基因AtSOS1、AtNHX1、抗逆相關(guān)轉(zhuǎn)錄因子AtABF4、AtDREB2A、AtMYB2的表達(dá)量,發(fā)現(xiàn)茉莉酸合成基因表達(dá)量顯著升高,而各類(lèi)脅迫相關(guān)基因及茉莉合成抑制蛋白基因在轉(zhuǎn)基因植株中表達(dá)量均降低,且顯著低于野生型。說(shuō)明鹽脅迫下NTaOC基因在擬南芥中的超表達(dá)誘導(dǎo)了擬南芥自身茉莉酸合成途徑關(guān)鍵酶基因的表達(dá),同時(shí)負(fù)調(diào)控了抗逆相關(guān)轉(zhuǎn)錄因子以及抗逆相關(guān)基因的表達(dá),從而使轉(zhuǎn)基因植株表現(xiàn)出對(duì)鹽脅迫的敏感性。
[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.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：Q943.2

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