發(fā)布時間：2018-04-12 06:47

  本文選題：小麥 + 擬南芥��； 參考：《華中科技大學》2016年博士論文

【摘要】：植物經常受到干旱、高鹽以及極端溫度等各種不利環(huán)境因素的影響,對其生長發(fā)育產生重大負面效應。當植物處于這些不利環(huán)境條件時,其體內許多相關基因會誘導表達。其中,轉錄因子就是這些重要基因中的一類。在轉錄因子家族中,NAC(NAM,ATAF與CUC)轉錄因子通過與其相應的順式作用元件結合,作為分子開關調控基因的時空表達。近年來,關于NAC轉錄因子在調控植物生長發(fā)育、衰老以及響應非生物脅迫等方面的研究越來越多,但小麥作物中有關NAC轉錄因子的功能分析還鮮有報道。本文以小麥NAC轉錄因子為研究對象,首先從普通小麥中國春基因組中鑒定、分離出10個TaNAC基因,在此基礎上重點研究了TaNAC29和TaNAC2D兩個基因的生物學功能。主要的研究結果如下:1)通過小麥EST數(shù)據庫搜索,我們成功拼接并克隆得到了10個小麥TaNAC基因,其中有9個為新鑒定的TaNAC基因。2)序列比對表明,新基因TaNAC29定位于小麥2BS染色體。TaNAC29蛋白定位于小麥原生質體的細胞核,并通過酵母單雜交試驗證明其具有轉錄激活活性,酵母實驗還揭示了L基序對TaNAC29的轉錄激活活性是必需的。TaNAC29基因在小麥葉片中具有較高的表達水平,在小麥成熟衰老葉片中的表達遠遠高于在幼嫩綠色葉片中的表達水平,這一結果暗示TaNAC29基因可能參與小麥葉片的衰老進程。實時熒光定量PCR分析結果顯示,小麥幼苗經過高鹽、PEG6000、過氧化氫以及ABA處理后,TaNAC29基因在小麥葉片和根中的表達水平均明顯增加。為了檢驗TaNAC29基因的生物學功能,我們獲得了TaNAC29過表達的轉基因擬南芥。轉基因植物發(fā)芽和根長實驗證明了過表達TaNAC29能顯著提高對高鹽和脫水脅迫的耐受性,轉基因擬南芥還表現(xiàn)出對外源ABA過敏性響應。TaNAC29過表達植株在營養(yǎng)生長和生殖生長期,均增加了對高鹽和干旱脅迫的耐受性,并且在生殖生長期間,還表現(xiàn)出延遲抽薹和開花現(xiàn)象。此外,在高鹽或脫水脅迫條件下,TaNAC29過表達植物可以積累更少的丙二醛和過氧化氫,具有更高的超氧化物歧化酶和過氧化氫酶活性。在黑暗條件下,TaNAC29轉基因小麥初步的實驗結果亦顯出離體葉片衰老加速的現(xiàn)象,表明TaNAC29基因正調控植物葉片的衰老。3)基因TaNAC2D定位于小麥的5BL染色體上。轉錄激活實驗也證明TaNAC2D是一個轉錄活化因子,其單獨的羧基端(173-327)足以激活報告基因的表達。TaNAC2D也定位于小麥原生質體細胞核中,符合轉錄因子的特征。TaNAC2D基因在小麥葉片中的表達水平高于其它器官。基因表達模式分析表明,小麥幼苗在高鹽、脫水、ABA以及過氧化氫處理下,TaNAC2D基因都上調表達。為了明確TaNAC2D基因的生物學功能,我們獲得了TaNAC2D基因過表達的轉基因擬南芥。在土壤中生長了35天的轉基因擬南芥株系分別經過高鹽和干旱脅迫處理后,TaNAC2D過表達植株比野生型植株表現(xiàn)出更高的敏感性響應,但在種子萌發(fā)以及萌發(fā)后生長期間,相對于野生型植物,TaNAC2D過表達植物則提高了對高鹽、脫水以及氧化脅迫的耐受性。為了進一步理解這兩種不同的表型反應,我們分析了相關標記基因的表達水平。在1/2 MS培養(yǎng)基上生長了14天的擬南芥幼苗浸泡在高鹽中6 h或脫水處理1 h,相對于野生型植物,TaNAC2D過表達植物中的NCED3、RD29A和RD29B基因的轉錄水平都顯著上調,然而,在土壤中生長了35天的植物浸泡在高鹽中6 h或脫水處理3 h,相對于野生型植物,這些標記基因在轉基因植物中幾乎都下調表達。綜合上所述,TaNAC29過表達轉基因植物在整個生長期間均提高了對鹽和干旱脅迫的耐受性。TaNAC2D過表達轉基因植物在種子萌發(fā)和萌發(fā)后生長期間,也提高了對多種非生物脅迫的耐受性,但在土壤中生長了35天的轉基因植物卻對鹽和干旱脅迫表現(xiàn)出更高的敏感性響應。這些結果暗示植物在響應環(huán)境脅迫過程中,NAC轉錄因子復雜的作用機制。
[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.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q943.2

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1 黃權軍;小麥TaNAC29和TaNAC2D基因克隆及抗逆功能研究[D];華中科技大學;2016年

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本文編號：1738616