拟南芥组蛋白去乙酰化酶基因家族分析及AtSRT2在盐胁迫下调控种子萌发的分子机制

发布时间：2018-02-01 05:41

本文关键词： 拟南芥组蛋白去乙酰化酶高盐胁迫种子萌发表达调控　出处：《中国农业科学院》2015年博士论文　论文类型：学位论文

【摘要】：近年来,随着表观遗传学研究的发展,人们发现DNA甲基化及组蛋白甲基化等表观遗传学修饰在植物生长发育及逆境胁迫应答过程中发挥着重要的调控作用。然而,目前对组蛋白乙酰化修饰的生物学功能及其调控方式缺乏系统深入的研究。本研究以参与维持体内组蛋白乙酰化平衡状态的拟南芥组蛋白去乙酰化酶(Histone deacetylases,HDACs)为研究对象,对其基因家族的系统进化、基因组结构、组织表达特异性、对激素和胁迫处理的应答模式及突变体表型特征进行系统分析,以期对组蛋白乙酰化修饰的生物学功能有更深入的了解。同时,人类Sirtuin类组蛋白去乙酰化酶在染色质沉默、DNA损伤修复、细胞周期调控和自噬等过程发挥着极其重要的功能,而关于植物Sirtuin类蛋白生物学功能的报道不多。因此,本研究重点针对拟南芥其中一个Sirtuin类蛋白成员,At SRT2介导的生物学功能及其作用机制进行了深入的研究。主要获得以下研究结果:1.拟南芥组蛋白去乙酰化酶基因家族特性,基因表达模式及生物学功能分析。对拟南芥组蛋白去乙酰化酶基因(At HDACs)家族进行系统进化树分析,结果表明At HDACs可以分为三大类。At HDACs基因结构中含有大量的内含子,个数多分布在5个以上。At HDACs不均匀的分布在拟南芥5条染色体上,分布最多的是3号和5号染色体。组织特异性表达分析结果表明,At HDACs主要在叶片、荚果和种子中高量表达;At HDACs在激素和胁迫处理下,其表达量不会发生急剧的变化,上下调比例维持在-1.49~3.1。拟南芥突变体表型研究结果表明,与野生型相比较,突变体主要表现为叶形态、花形态和花期异常。2.Sirtuin类蛋白At SRT2参与植物高盐胁迫下种子萌发的调控过程。At SRT2基因突变体srt2在正常条件下,表型与野生型一致,但在高盐条件下,srt2表现出对高盐胁迫敏感。At SRT2有7个可变剪接体,其中At SRT2.7在种子中表达量最高,并且受盐胁迫诱导表达,在srt2背景下过表达At SRT2.7,srt2盐敏感表型部分恢复。这表明At SRT2.7参与了植物盐胁迫下种子萌发的调控。3.At SRT2通过减少盐胁迫下植物基因组的损伤及NAD+含量的积累调控种子的萌发。单细胞凝胶电泳(SCGE)实验表明,在种子萌发过程中,盐胁迫诱导了植物基因组的损伤,且srt2的基因组损伤较野生型更为严重。与野生型比较,srt2对DNA损伤诱导剂甲基磺酸甲酯(MMS)处理更为敏感。另一方面,种子萌发实验表明,NAD+处理可抑制种子萌发,而且srt2对外源NAD+处理较野生型更为敏感。检测发现,在种子萌发过程中,盐胁迫诱导srt2体内NAD+含量积累。这表明DNA损伤和NAD+积累是造成srt2对盐敏感的原因。4.At SRT2通过调控下游盐胁迫相关基因At VAMP714启动子区组蛋白H4K8位点的乙酰化水平影响植物种子萌发期的耐盐性。At SRT2.7定位在植物的细胞核和细胞膜上。与野生型比较,srt2组蛋白H4K8位点乙酰化水平升高。体外酶活实验表明,At SRT2.7是一种NAD+依赖型的组蛋白去乙酰化酶,特异地对H3K9、H3K14、H4K8三个位点进行去乙酰化。在盐胁迫处理下,野生型种子萌发过程中12~48h有明显的H4K8去乙酰化过程,而srt2组蛋白H4K8位点乙酰化水平高于野生型。这些结果说明At SRT2.7通过H4K8乙酰化水平的调控介导盐胁迫下的种子萌发过程。Ch IP-Seq和Ch IP-q PCR结果表明,在盐胁迫下,At SRT2通过降低盐胁迫相关基因At VAMP714启动子区组蛋白H4K8位点的乙酰化水平,抑制At VAMP714的表达,从而减少H2O2对植物体液泡膜的损伤,缓减盐胁迫对种子萌发的影响。以上研究结果表明,拟南芥组蛋白去乙酰化酶广泛参与了植物生长发育和逆境应答过程,且不同的组蛋白去乙酰化酶行使功能不同,表现出调控机制的复杂性。其中,Sirtuin类组蛋白去乙酰化酶At SRT2通过调控组蛋白H4K8位点的乙酰化水平调控盐胁迫下的种子萌发过程,并主要通过减少盐胁迫下植物DNA的损伤以及NAD+的积累行使基因功能。通过本研究增加了我们对植物组蛋白去乙酰化基因家族特性及功能的了解,为进一步揭示植物表观遗传学调控网络创造了条件。
[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.

【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q943.2

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