小麦TaNAC29和TaNAC2D基因克隆及抗逆功能研究

发布时间：2018-04-12 06:47

本文选题：小麦 + 拟南芥　；参考：《华中科技大学》2016年博士论文

【摘要】：植物经常受到干旱、高盐以及极端温度等各种不利环境因素的影响,对其生长发育产生重大负面效应。当植物处于这些不利环境条件时,其体内许多相关基因会诱导表达。其中,转录因子就是这些重要基因中的一类。在转录因子家族中,NAC(NAM,ATAF与CUC)转录因子通过与其相应的顺式作用元件结合,作为分子开关调控基因的时空表达。近年来,关于NAC转录因子在调控植物生长发育、衰老以及响应非生物胁迫等方面的研究越来越多,但小麦作物中有关NAC转录因子的功能分析还鲜有报道。本文以小麦NAC转录因子为研究对象,首先从普通小麦中国春基因组中鉴定、分离出10个TaNAC基因,在此基础上重点研究了TaNAC29和TaNAC2D两个基因的生物学功能。主要的研究结果如下:1)通过小麦EST数据库搜索,我们成功拼接并克隆得到了10个小麦TaNAC基因,其中有9个为新鉴定的TaNAC基因。2)序列比对表明,新基因TaNAC29定位于小麦2BS染色体。TaNAC29蛋白定位于小麦原生质体的细胞核,并通过酵母单杂交试验证明其具有转录激活活性,酵母实验还揭示了L基序对TaNAC29的转录激活活性是必需的。TaNAC29基因在小麦叶片中具有较高的表达水平,在小麦成熟衰老叶片中的表达远远高于在幼嫩绿色叶片中的表达水平,这一结果暗示TaNAC29基因可能参与小麦叶片的衰老进程。实时荧光定量PCR分析结果显示,小麦幼苗经过高盐、PEG6000、过氧化氢以及ABA处理后,TaNAC29基因在小麦叶片和根中的表达水平均明显增加。为了检验TaNAC29基因的生物学功能,我们获得了TaNAC29过表达的转基因拟南芥。转基因植物发芽和根长实验证明了过表达TaNAC29能显著提高对高盐和脱水胁迫的耐受性,转基因拟南芥还表现出对外源ABA过敏性响应。TaNAC29过表达植株在营养生长和生殖生长期,均增加了对高盐和干旱胁迫的耐受性,并且在生殖生长期间,还表现出延迟抽薹和开花现象。此外,在高盐或脱水胁迫条件下,TaNAC29过表达植物可以积累更少的丙二醛和过氧化氢,具有更高的超氧化物歧化酶和过氧化氢酶活性。在黑暗条件下,TaNAC29转基因小麦初步的实验结果亦显出离体叶片衰老加速的现象,表明TaNAC29基因正调控植物叶片的衰老。3)基因TaNAC2D定位于小麦的5BL染色体上。转录激活实验也证明TaNAC2D是一个转录活化因子,其单独的羧基端(173-327)足以激活报告基因的表达。TaNAC2D也定位于小麦原生质体细胞核中,符合转录因子的特征。TaNAC2D基因在小麦叶片中的表达水平高于其它器官。基因表达模式分析表明,小麦幼苗在高盐、脱水、ABA以及过氧化氢处理下,TaNAC2D基因都上调表达。为了明确TaNAC2D基因的生物学功能,我们获得了TaNAC2D基因过表达的转基因拟南芥。在土壤中生长了35天的转基因拟南芥株系分别经过高盐和干旱胁迫处理后,TaNAC2D过表达植株比野生型植株表现出更高的敏感性响应,但在种子萌发以及萌发后生长期间,相对于野生型植物,TaNAC2D过表达植物则提高了对高盐、脱水以及氧化胁迫的耐受性。为了进一步理解这两种不同的表型反应,我们分析了相关标记基因的表达水平。在1/2 MS培养基上生长了14天的拟南芥幼苗浸泡在高盐中6 h或脱水处理1 h,相对于野生型植物,TaNAC2D过表达植物中的NCED3、RD29A和RD29B基因的转录水平都显著上调,然而,在土壤中生长了35天的植物浸泡在高盐中6 h或脱水处理3 h,相对于野生型植物,这些标记基因在转基因植物中几乎都下调表达。综合上所述,TaNAC29过表达转基因植物在整个生长期间均提高了对盐和干旱胁迫的耐受性。TaNAC2D过表达转基因植物在种子萌发和萌发后生长期间,也提高了对多种非生物胁迫的耐受性,但在土壤中生长了35天的转基因植物却对盐和干旱胁迫表现出更高的敏感性响应。这些结果暗示植物在响应环境胁迫过程中,NAC转录因子复杂的作用机制。
[Abstract]:The plant is often affected by drought, high salt and extreme temperature effect and other unfavorable factors, the growth and development have a significant negative effect. When the plants are in the adverse environmental conditions, the body can induce the expression of many genes. Among them, a class of transcription factors is important. These genes in a family of transcription factors. NAC (NAM, ATAF and CUC) and the corresponding transcription factors by binding to cis elements, as molecular switch spatiotemporal regulation of gene expression. In recent years, a NAC transcription factor in the regulation of plant growth and development, aging and response to abiotic stress research more and more, but the analysis of the NAC transcription factor function wheat crops have rarely been reported. In this paper, the wheat NAC transcription factor as the research object, first from the common wheat genome in spring China identified, isolated 10 TaNAC gene, on the basis of Focus on the biological function of TaNAC29 and TaNAC2D two genes. The main results are as follows: 1) search through the wheat EST database, we successfully spliced and cloned 10 wheat TaNAC genes, 9 of which are new identified TaNAC.2) gene sequence alignment showed that the TaNAC29 gene located on chromosome 2BS of wheat localization of.TaNAC29 protein in wheat protoplast nucleus, and by the yeast one hybrid experiment prove its transcriptional activity, yeast transcription experiment revealed that the L motif of TaNAC29 activation is required for.TaNAC29 with higher expression level in wheat leaves, expressed in mature wheat leaf senescence in much higher expression the level in young green leaves, the results suggest that TaNAC29 gene may be involved in wheat leaf senescence. The results of real-time PCR showed that wheat Seedlings after high salt, PEG6000, hydrogen peroxide and ABA after the treatment, the expression level of TaNAC29 gene in wheat leaves and roots were significantly increased. In order to test the biological function of TaNAC29 gene, we obtained transgenic Arabidopsis overexpressing TaNAC29. Transgenic plants germination and root length experiments demonstrated that overexpression of TaNAC29 can significantly improve the tolerance high salt and dehydration stress, transgenic Arabidopsis showed allergic response to exogenous ABA overexpression of.TaNAC29 in plant growth period of vegetative growth and reproductive, increased tolerance to high salt and drought stress, and during reproductive growth, also showed delayed bolting and flowering phenomenon. In addition, in the high salt or under dehydration stress, TaNAC29 overexpression plants can accumulate less malondialdehyde and hydrogen peroxide, has higher superoxide dismutase and catalase activity in the dark. Under the transgenic wheat TaNAC29 preliminary experimental results also show the in vitro accelerated leaf senescence phenomenon, suggesting that the TaNAC29 gene is the regulation of plant leaf senescence).3 gene TaNAC2D is located on chromosome 5BL of wheat. Transcriptional activation experiments also show that TaNAC2D is a transcriptional activator, its separate C-terminal (173-327) expression of.TaNAC2D to activate the reporter gene is also located in the wheat protoplast nucleus, with expression characteristics of.TaNAC2D gene transcription factor in wheat leaves was higher than that of other organs. Gene expression pattern analysis showed that the dehydration of Wheat Seedlings under high salt, ABA, and H2O2 treatment, the TaNAC2D gene expression in order to clear the biological function of TaNAC2D gene. And we obtained the transgenic Arabidopsis plants overexpressing TaNAC2D gene. Growth in the soil for 35 days in the transgenic Arabidopsis lines respectively after High salt and drought stress treatment, TaNAC2D overexpressing plants than in wild-type plants showed more sensitive response, but in Seed Germination and post germination growth period, compared with wild type plants, TaNAC2D overexpressing plants increased to high salt stress tolerance of dehydration and oxidation. In order to further understand the two different phenotypic responses, we analyzed the expression of marker genes of Arabidopsis thaliana seedlings. Soaking grown in 1/2 MS medium for 14 days in high salt dehydration treatment 6 h or 1 h, compared with wild type plants, over expression of TaNAC2D NCED3 in plants, the transcriptional level of RD29A and RD29B genes were significantly up-regulated however, growth in the soil 35 days plant soaking in high salt in 6 h or 3 h dehydration treatment, compared with wild type plants, these marker genes in transgenic plants. Almost all of the comprehensive expression of TaNAC. 29 overexpression transgenic plants were increased in the whole growth period of salt and drought tolerance of.TaNAC2D over expression transgenic plants during seed germination and post germination growth, but also improve the tolerance to various abiotic stresses in soil, but the growth of transgenic plant was 35 days due to salt and drought the stress response showed a higher sensitivity. These results suggest that plants in response to environmental stress, the mechanism of NAC transcription factor complex.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

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