陆地棉GhNAC3在棉花响应非生物逆境中的功能研究

发布时间：2018-04-12 06:26

本文选题：NAC + 棉花　；参考：《华中农业大学》2017年硕士论文

【摘要】：非生物逆境胁迫严重影响棉花的生长发育、产量及纤维品质,培育抗逆的棉花新品种对棉花生产具有重要的意义。NAC(NAM,ATAF1/2,CUC2)是植物特有的一类转录因子,广泛参与植物对非生物逆境的响应。本研究基于前期棉花干旱胁迫表达谱分离到一个显著诱导表达的NAC基因GhNAC3,对其在棉花中参与响应干旱和盐胁迫的功能进行初步鉴定和机制探索,主要结果如下:我们在棉花中鉴定到232个NAC家族转录因子,并基于公共数据库表达谱数据对其组织表达模式和逆境诱导表达模式进行了分析。同时,我们从陆地棉中克隆到GhNAC3基因,氨基酸序列多重比对结果显示该基因具有保守的NAC结构域;亚细胞定位实验表明GhNAC3编码一个核定位蛋白;酵母反式激活实验揭示GhNAC3 BD结构域(1-160aa)不具有转录激活能力,AD结构域(161-298aa)具有转录激活能力。组织表达模式分析发现GhNAC3在根和花中优势表达;非生物逆境表达模式分析表明,GhNAC3受PEG和NaCl诱导显著上调表达。另外,对GhNAC3启动子的cis-element分析,发现该基因的启动子区域具有响应干旱、盐和ABA的一些顺式作用元件,如ERD1(early responsive to dehydration)、DRE/CRT(dehydration-responsive element/C-repeat)等。在拟南芥中超表达GhNAC3,萌发实验及根长实验表明超表达该基因可以增强转基因拟南芥对渗透压胁迫和盐胁迫的耐受性,对ABA的敏感性降低。利用病毒诱导基因沉默技术(VIGS)沉默GhNAC3的表达,ABA含量在干涉材料中显著降低;干旱处理后,在干涉材料中积累较多的过氧化氢(H2O2)和丙二醛(MDA);离体叶片失水处理、自然干旱和叶盘实验表明在棉花中沉默该基因可以降低棉花对干旱和盐胁迫的耐受性。通过农杆菌侵染棉花下胚轴转化技术,我们获得了稳定转化的超表达材料,与阴性分离材料和野生型一起进行PEG6000模拟的渗透胁迫实验,结果表明,超表达材料较对照材料表现出更好的生长势。以上结果表明,GhNAC3是一个参与调控棉花响应非生物逆境的基因,我们推测该基因可能通过介导ABA信号参与棉花响应干旱和盐胁迫。本研究棉花抗逆育种提供基因资源和理论基础。
[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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S562

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