本文關(guān)鍵詞： 長葉紅砂 谷氧還蛋白 超氧化物歧化酶 非生物脅迫 轉(zhuǎn)基因擬南芥　出處：《內(nèi)蒙古大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：長葉紅砂(Reaumuriatrigyna)是東阿拉善-西鄂爾多斯特有的強(qiáng)旱生泌鹽鹽生植物,對鹽漬荒漠環(huán)境具有極強(qiáng)的適應(yīng)性。轉(zhuǎn)錄組深度測序結(jié)果顯示,該植物在鹽脅迫條件下,抗氧化系統(tǒng)相關(guān)基因差異表達(dá)明顯,對植物響應(yīng)逆境脅迫具有積極作用。研究表明超氧化物歧化酶SOD和谷氧還蛋白GRX參與了植物逆境脅迫下的活性氧清除及蛋白質(zhì)氧化還原狀態(tài)的調(diào)節(jié)。本研究從長葉紅砂中克隆了RtGRX和RtSOD基因,對其進(jìn)行組織特異性分析及不同脅迫下表達(dá)特性分析,將該基因轉(zhuǎn)入擬南芥,驗(yàn)證其在不同脅迫條件下的功能,為深入了解RtGRX及RtSOD基因在植物抵抗逆境脅迫過程中的調(diào)控功能奠定了基礎(chǔ)。主要結(jié)果如下:1.依據(jù)轉(zhuǎn)錄組數(shù)據(jù)庫信息擴(kuò)增得到長葉紅砂谷氧還蛋白基因,命名為RtGRX;長葉紅砂超氧化物歧化酶基因,命名為RtSOD。RtGRX基因的開放閱讀框大小為807bp,編碼268個(gè)氨基酸,預(yù)測分子量66.98kDa,理論等電點(diǎn)5.17。RtSOD基因的開放閱讀框大小為663bp,編碼220個(gè)氨基酸,預(yù)測分子量55.90kDa理論等電點(diǎn)5.11。氨基酸序列比對分析顯示,RtGRX屬于GRX蛋白家族,RtSOD屬于Cu/ZnSOD蛋白家族。系統(tǒng)進(jìn)化樹分析顯示長葉紅砂的RtGRX與番茄和美花煙草的GRX親緣關(guān)系較近,而RtSOD與剛毛檉柳的SOD親緣關(guān)系最近。2.組織特異性分析結(jié)果顯示,RtGRX和RtSOD基因在長葉紅砂根、莖、葉中均表達(dá),且在莖中表達(dá)量顯著高于根和葉�；虮磉_(dá)特性分析結(jié)果顯示,采用400mM NaCl、4℃、PEG、H2O2、ABA等脅迫處理長葉紅砂,RtGRX和RtSOD基因的表達(dá)量均上調(diào);在不同濃度NaCl脅迫下,兩基因均在400mM NaCl下表達(dá)量最高。3.構(gòu)建RtGRX和RtSOD真核表達(dá)載體,將其轉(zhuǎn)化到擬南芥中,結(jié)果發(fā)現(xiàn):鹽、干旱脅迫條件下轉(zhuǎn)RtGRX和RtSOD基因擬南芥的生長狀況(根長、鮮重、葉綠素含量)均優(yōu)于野生型,抗氧化酶活性(SOD、POD、CAT)和脯氨酸含量較野生型顯著升高,H2O2及MDA含量較野生型顯著降低,說明以上兩基因提高了轉(zhuǎn)基因植株的抗氧化酶活性,減少了體內(nèi)活性氧含量,減輕了膜損傷程度,從而提高了轉(zhuǎn)基因植物在鹽和干旱脅迫環(huán)境下的生長發(fā)育、光合作用以及抵抗氧化脅迫的能力,增強(qiáng)了轉(zhuǎn)基因植株的耐受性。4.qPCR檢測轉(zhuǎn)基因擬南芥中響應(yīng)逆境脅迫相關(guān)基因的表達(dá)量,如抗氧化系統(tǒng)相關(guān)基因AtSOD1、AtPOD1、AtAPX1、AtCAT1,脯氨酸合成關(guān)鍵基因AtP5CS1,離子轉(zhuǎn)運(yùn)蛋白基因AtSOS1,結(jié)果顯示,轉(zhuǎn)基因植株中以上基因的表達(dá)量均顯著高于野生型,說明RtGRX和RtSOD基因在擬南芥中的超表達(dá)提高了轉(zhuǎn)基因株系參與植物氧化平衡、滲透平衡以及離子平衡等方面的基因活性,進(jìn)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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