本文關鍵詞： 擬南芥 組蛋白去乙�；� 高鹽脅迫 種子萌發(fā) 表達調控　出處：《中國農業(yè)科學院》2015年博士論文　論文類型：學位論文

【摘要】：近年來,隨著表觀遺傳學研究的發(fā)展,人們發(fā)現DNA甲基化及組蛋白甲基化等表觀遺傳學修飾在植物生長發(fā)育及逆境脅迫應答過程中發(fā)揮著重要的調控作用。然而,目前對組蛋白乙酰化修飾的生物學功能及其調控方式缺乏系統(tǒng)深入的研究。本研究以參與維持體內組蛋白乙酰化平衡狀態(tài)的擬南芥組蛋白去乙�；�(Histone deacetylases,HDACs)為研究對象,對其基因家族的系統(tǒng)進化、基因組結構、組織表達特異性、對激素和脅迫處理的應答模式及突變體表型特征進行系統(tǒng)分析,以期對組蛋白乙�；揎椀纳飳W功能有更深入的了解。同時,人類Sirtuin類組蛋白去乙酰化酶在染色質沉默、DNA損傷修復、細胞周期調控和自噬等過程發(fā)揮著極其重要的功能,而關于植物Sirtuin類蛋白生物學功能的報道不多。因此,本研究重點針對擬南芥其中一個Sirtuin類蛋白成員,At SRT2介導的生物學功能及其作用機制進行了深入的研究。主要獲得以下研究結果:1.擬南芥組蛋白去乙�；富蚣易逄匦�,基因表達模式及生物學功能分析。對擬南芥組蛋白去乙�；富�(At HDACs)家族進行系統(tǒng)進化樹分析,結果表明At HDACs可以分為三大類。At HDACs基因結構中含有大量的內含子,個數多分布在5個以上。At HDACs不均勻的分布在擬南芥5條染色體上,分布最多的是3號和5號染色體。組織特異性表達分析結果表明,At HDACs主要在葉片、莢果和種子中高量表達;At HDACs在激素和脅迫處理下,其表達量不會發(fā)生急劇的變化,上下調比例維持在-1.49~3.1。擬南芥突變體表型研究結果表明,與野生型相比較,突變體主要表現為葉形態(tài)、花形態(tài)和花期異常。2.Sirtuin類蛋白At SRT2參與植物高鹽脅迫下種子萌發(fā)的調控過程。At SRT2基因突變體srt2在正常條件下,表型與野生型一致,但在高鹽條件下,srt2表現出對高鹽脅迫敏感。At SRT2有7個可變剪接體,其中At SRT2.7在種子中表達量最高,并且受鹽脅迫誘導表達,在srt2背景下過表達At SRT2.7,srt2鹽敏感表型部分恢復。這表明At SRT2.7參與了植物鹽脅迫下種子萌發(fā)的調控。3.At SRT2通過減少鹽脅迫下植物基因組的損傷及NAD+含量的積累調控種子的萌發(fā)。單細胞凝膠電泳(SCGE)實驗表明,在種子萌發(fā)過程中,鹽脅迫誘導了植物基因組的損傷,且srt2的基因組損傷較野生型更為嚴重。與野生型比較,srt2對DNA損傷誘導劑甲基磺酸甲酯(MMS)處理更為敏感。另一方面,種子萌發(fā)實驗表明,NAD+處理可抑制種子萌發(fā),而且srt2對外源NAD+處理較野生型更為敏感。檢測發(fā)現,在種子萌發(fā)過程中,鹽脅迫誘導srt2體內NAD+含量積累。這表明DNA損傷和NAD+積累是造成srt2對鹽敏感的原因。4.At SRT2通過調控下游鹽脅迫相關基因At VAMP714啟動子區(qū)組蛋白H4K8位點的乙�；接绊懼参锓N子萌發(fā)期的耐鹽性。At SRT2.7定位在植物的細胞核和細胞膜上。與野生型比較,srt2組蛋白H4K8位點乙酰化水平升高。體外酶活實驗表明,At SRT2.7是一種NAD+依賴型的組蛋白去乙�；�,特異地對H3K9、H3K14、H4K8三個位點進行去乙酰化。在鹽脅迫處理下,野生型種子萌發(fā)過程中12~48h有明顯的H4K8去乙�；^程,而srt2組蛋白H4K8位點乙酰化水平高于野生型。這些結果說明At SRT2.7通過H4K8乙�；降恼{控介導鹽脅迫下的種子萌發(fā)過程。Ch IP-Seq和Ch IP-q PCR結果表明,在鹽脅迫下,At SRT2通過降低鹽脅迫相關基因At VAMP714啟動子區(qū)組蛋白H4K8位點的乙酰化水平,抑制At VAMP714的表達,從而減少H2O2對植物體液泡膜的損傷,緩減鹽脅迫對種子萌發(fā)的影響。以上研究結果表明,擬南芥組蛋白去乙�；笍V泛參與了植物生長發(fā)育和逆境應答過程,且不同的組蛋白去乙酰化酶行使功能不同,表現出調控機制的復雜性。其中,Sirtuin類組蛋白去乙�；窤t SRT2通過調控組蛋白H4K8位點的乙酰化水平調控鹽脅迫下的種子萌發(fā)過程,并主要通過減少鹽脅迫下植物DNA的損傷以及NAD+的積累行使基因功能。通過本研究增加了我們對植物組蛋白去乙�；蚣易逄匦约肮δ艿牧私�,為進一步揭示植物表觀遺傳學調控網絡創(chuàng)造了條件。
[Abstract]:In recent years, with the development of the concept of table for genetic research, people found that play a crucial role in regulation of DNA methylation and histone methylation and other epigenetic modifications in plant growth and development and stress stress responses. However, at present on histone acetylation the biological function and regulation lack systematic study in this study. Arabidopsis histone histone acetylation is involved in the maintenance of body balance deacetylase (Histone deacetylases, HDACs) as the research object, system evolution, the gene family of genomic structure, tissue specific expression, to analyze the response patterns and mutant phenotypic characteristics in hormone and stress treatment, biology in order to function of histone acetylation has a deeper understanding. At the same time, the human class Sirtuin histone deacetylase in chromatin silencing, DNA damage repair Complex, cell cycle regulation and autophagy process plays a very important function, and on the biological function of plant Sirtuin proteins reported. Therefore, this study focuses on one Arabidopsis Sirtuin protein members, the biological function of At mediated by SRT2 and its mechanism are studied. The main access as follows results: 1. Arabidopsis histone deacetylase gene family characteristics, analysis of gene expression patterns and biological function. Deacetylase gene of Arabidopsis protein group (At HDACs) phylogenetic tree analysis of family, the results show that At HDACs can be divided into three categories:.At HDACs gene structure contains a large number of introns. The distribution of the number distribution in more than 5.At HDACs heterogeneity in Arabidopsis chromosome 5, the largest distribution is 3 and 5 chromosomes. Tissue specific expression analysis showed that At HDACs expression in leaves, pods and seeds in high volume; At HDACs in hormone and stress, the expression will not change dramatically, up and down ratio is maintained at the Arabidopsis -1.49~3.1. mutant phenotype of results showed that compared with the wild type, the mutant showed leaf morphology, floral morphology and flowering of abnormal.2.Sirtuin At SRT2 protein is involved in plant salt stress regulation of.At SRT2 mutant of srt2 seed germination under normal conditions, the phenotype with the wild type, but in high salt conditions, srt2 showed high salt stress sensitive.At SRT2 have 7 alternative splicing, the At SRT2.7 expression was the highest in seed and, under the salt stress induced expression, over expression of At SRT2.7 in the srt2 background, restore srt2 salt sensitive phenotype. This indicates that At SRT2.7 is involved in the regulation of plant salt stress.3.At SRT2 seed germination by reduction Damage and low NAD+ content under salt stress of plant genome accumulation regulation of seed germination. The single cell gel electrophoresis (SCGE) experiments show that during seed germination, salt stress induced the plant genome and srt2 genome damage, damage is more serious than the wild type. Compared with the wild type, srt2 inducing agent methyl methanesulfonate (MMS) treatment of DNA injury is more sensitive. On the other hand, showed that the seed germination experiment, NAD+ treatment could inhibit the seed germination, and srt2 on the treatment of exogenous NAD+ is more sensitive than the wild type. Detected during seed germination, the content of NAD+ in salt stress induced the accumulation of srt2. This indicates that DNA damage the accumulation of NAD+ and srt2 on.4.At SRT2 is caused by the reason of salt sensitive downstream through the regulation of salt stress related genes At VAMP714 started the salt tolerance of.At SR acetylation level of histone H4K8 promoter effect site of plant seed germination T2.7 is located in the plant nucleus and cell membrane. Compared with wild-type, srt2 increased histone H4K8 acetylation sites. The experimental results show that the activity of At, SRT2.7 is a NAD+ dependent histone deacetylase, specifically for H3K9, H3K14, H4K8 three sites of acetylation. Under salt stress, the germination of seeds of wild type 12~48h is obvious in H4K8 deacetylation process, and protein H4K8 histone acetylation level of srt2 group was higher than that of the wild type. These results indicate that At SRT2.7 mediated by regulating the acetylation level of H4K8 guided seed adorable under salt stress of.Ch IP-Seq and Ch IP-q PCR results show that under salt stress, At SRT2 by reducing the salt stress related gene At VAMP714 promoter acetylation level of histone H4K8 promoter sites, inhibit expression of At and VAMP714, thus reducing the H2O2 of plant sap vesicle membrane damage, alleviate the salt stress on Effect of seed germination. The above results show that Arabidopsis histone deacetylase is widely involved in plant growth and development and stress responses, and different histone deacetylase function is different, showing the complexity of regulation mechanisms. Among them, the class Sirtuin histone deacetylase At SRT2 by acetylation the level of regulation of salt regulation of histone H4K8 loci under the stress of seed germination, and by reducing the plants under salt stress DNA damage and the accumulation of NAD+ gene function. We increased exercise deacetylase gene family characteristics and function of plant protein group learned through this study, to further reveal the plant surface to create conditions the concept of genetic regulatory networks.

【學位授予單位】：中國農業(yè)科學院
【學位級別】：博士
【學位授予年份】：2015
【分類號】：Q943.2

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本文編號：1481082