本文選題：NAC + 棉花　； 參考：《華中農業(yè)大學》2017年碩士論文

【摘要】：非生物逆境脅迫嚴重影響棉花的生長發(fā)育、產量及纖維品質,培育抗逆的棉花新品種對棉花生產具有重要的意義。NAC(NAM,ATAF1/2,CUC2)是植物特有的一類轉錄因子,廣泛參與植物對非生物逆境的響應。本研究基于前期棉花干旱脅迫表達譜分離到一個顯著誘導表達的NAC基因GhNAC3,對其在棉花中參與響應干旱和鹽脅迫的功能進行初步鑒定和機制探索,主要結果如下:我們在棉花中鑒定到232個NAC家族轉錄因子,并基于公共數據庫表達譜數據對其組織表達模式和逆境誘導表達模式進行了分析。同時,我們從陸地棉中克隆到GhNAC3基因,氨基酸序列多重比對結果顯示該基因具有保守的NAC結構域;亞細胞定位實驗表明GhNAC3編碼一個核定位蛋白;酵母反式激活實驗揭示GhNAC3 BD結構域(1-160aa)不具有轉錄激活能力,AD結構域(161-298aa)具有轉錄激活能力。組織表達模式分析發(fā)現GhNAC3在根和花中優(yōu)勢表達;非生物逆境表達模式分析表明,GhNAC3受PEG和NaCl誘導顯著上調表達。另外,對GhNAC3啟動子的cis-element分析,發(fā)現該基因的啟動子區(qū)域具有響應干旱、鹽和ABA的一些順式作用元件,如ERD1(early responsive to dehydration)、DRE/CRT(dehydration-responsive element/C-repeat)等。在擬南芥中超表達GhNAC3,萌發(fā)實驗及根長實驗表明超表達該基因可以增強轉基因擬南芥對滲透壓脅迫和鹽脅迫的耐受性,對ABA的敏感性降低。利用病毒誘導基因沉默技術(VIGS)沉默GhNAC3的表達,ABA含量在干涉材料中顯著降低;干旱處理后,在干涉材料中積累較多的過氧化氫(H2O2)和丙二醛(MDA);離體葉片失水處理、自然干旱和葉盤實驗表明在棉花中沉默該基因可以降低棉花對干旱和鹽脅迫的耐受性。通過農桿菌侵染棉花下胚軸轉化技術,我們獲得了穩(wěn)定轉化的超表達材料,與陰性分離材料和野生型一起進行PEG6000模擬的滲透脅迫實驗,結果表明,超表達材料較對照材料表現出更好的生長勢。以上結果表明,GhNAC3是一個參與調控棉花響應非生物逆境的基因,我們推測該基因可能通過介導ABA信號參與棉花響應干旱和鹽脅迫。本研究棉花抗逆育種提供基因資源和理論基礎。
[Abstract]:Abiotic stress stress seriously affected cotton growth and development, yield and fiber quality, and the cultivation of new varieties of cotton with resistance to stress was of great significance to cotton production. NACNAMN ATAF1 / 2CUC2) is a kind of plant specific transcription factor.Extensive participation in plant responses to abiotic stresses.In this study, a significant induced expression of NAC gene GhNAC3 was isolated from the expression profile of drought stress in cotton. The function of GhNAC3 in responding to drought and salt stress in cotton was preliminarily identified and the mechanism was explored.The main results are as follows: we identified 232 NAC family transcription factors in cotton and analyzed their tissue expression patterns and stress-induced expression patterns based on the common database expression profile data.At the same time, GhNAC3 gene was cloned from upland cotton. The results of multiple amino acid sequence alignment showed that the gene had conserved NAC domain, and the subcellular localization experiment showed that GhNAC3 encodes a nuclear localization protein.Yeast transactivation assay revealed that GhNAC3 BD domain 1-160aa) had no transcriptional activation ability and AD domain 161-298aa) had transcriptional activation ability.Tissue expression pattern analysis showed that GhNAC3 was predominantly expressed in root and flower, while abiotic stress analysis showed that GhNAC3 was significantly up-regulated by PEG and NaCl.In addition, cis-element analysis of the GhNAC3 promoter showed that the promoter region of the gene had some cis-acting elements such as ERD1(early responsive to dehydration to respond to drought, salt and ABA, such as ERD1(early responsive / CRT dehydration-responsive element / C-repeatation.Overexpression of GhNAC3 in Arabidopsis thaliana, germination and root length experiments showed that overexpression of this gene could enhance tolerance of transgenic Arabidopsis thaliana to osmotic pressure stress and salt stress, and decrease the sensitivity of transgenic Arabidopsis to ABA.Virus induced gene silencing technique (VIGS) was used to silence the expression of GhNAC3 in the interference materials, and after drought treatment, it accumulated more hydrogen peroxide (H _ 2O _ 2) and malondialdehyde (MDAA) in the interference materials, and the leaves lost water in vitro.Natural drought and leaf disc experiments showed that silencing the gene in cotton could reduce the tolerance of cotton to drought and salt stress.By means of Agrobacterium tumefaciens infecting cotton Hypocotyl transformation technique, we obtained stable transformed super-expression materials, and carried out PEG6000 simulated osmotic stress experiments with negative isolates and wild type. The results showed that,The superexpression material showed a better growth potential than the control material.These results suggest that GhNAC3 is a gene involved in the regulation of cotton response to abiotic stress. We speculate that this gene may be involved in cotton response to drought and salt stress by mediating ABA signal.This study provides genetic resources and theoretical basis for cotton stress resistance breeding.
【學位授予單位】：華中農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S562

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本文編號：1738548