【作者】 黄利民；

【导师】 周道绣；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 表观遗传学是指在DNA序列不改变的情况下,碱基序列以外的各种修饰和与之相关的各种蛋白质或RNA协同作用下,调控基因的表达,以完成生命周期或适应环境变化,且可以在世代之间传递。表观遗传学涉及的机制包括DNA甲基化、组蛋白修饰和染色质变构等方面。组蛋白乙酰化是真核生物表观遗传调控的重要组成部分。水稻是世界上重要的粮食作物,有关水稻表观遗传学的研究报道目前很少。本研究主要以水稻中的组蛋白去乙酰化酶（HDAC）基因为对象,通过生物信息学、基因克隆和转化等方法研究了水稻HDAC基因的结构、表达及其生理功能,阐述了HDAC基因在植物的表观遗传调控及其环境适应中的重要意义。本研究主要有以下结果:1.通过同源序列法研究发现,水稻基因组数据库中存在着19个组蛋白去乙酰化酶的基因,其中RPD3/HDA1超家族的组蛋白去乙酰化酶基因14个,HD2家族3个,SIR2家族2个。2.对水稻HDAC的三个不同家族基因成员分别进行了序列分析比较和系统进化分析。3.共构建了10个基因的RNAi抑制表达载体,5个基因的超量表达载体,并获得转基因植株。4.以SIR2家族的OsSRT1基因为重点展开研究。通过Northern杂交对其表达模式研究发现,OsSRT1在快速分化的器官和组织中高水平表达。OsSRT1蛋白定位于细胞核内。5.Western杂交证据显示,OsSRT1抑制表达家系的组蛋白H3K9（lysine-9 of H3）的乙酰化程度增加,而H3K9的双甲基化程度降低。OsSRT1 RNAi转基因植株中发生H₂O₂的产生,DNA片段化和细胞死亡,产生假病斑表型变异,属于超敏反应类型的程序性细胞死亡。6.OsSRT1的超量表达不产生明显的形态变异,但能增强植株对于百草枯的耐受能力,提高超量表达转基因家系对氧化胁迫的抗性。7.为了进一步分析OsSRT1所调控的下游基因以及其调控方式,本研究采用Affymetrix水稻全基因组生物芯片对抑制表达植株中基因表达状况进行了分析。结果表明,抑制表达植株中转座子和反转座子以及超敏反应相关的基因和细胞程序性死亡有关的基因的转录被激活。8.对抑制表达植株芯片分析中所获得的差异表达基因进行了深入的分析。差异表达基因的Gene Ontology（GO）分类显示OsSRT1正调控防御反应、DNA修复和苯丙醇代谢过程,这些基因大都涉及胁迫相关细胞程序性死亡信号途径相关的基因。9.通过RT-PCR验证了RNAi植株中一些衰老相关基因、病程相关蛋白基因等标志基因的表达,进一步证实了其与PCD的关系。10.通过染色质免疫沉淀技术和Real-time PCR检测发现,这些上升表达的转座子和反转座子以及细胞程序性死亡相关基因的启动子区域组蛋白H3K9乙酰化程度明显上升,因此推测这些基因是OsSRT1的直接靶基因。11.DNA甲基化分析结果暗示,OsSRT1的下降表达很可能是以一种随机的方式作用于DNA甲基化。12.分别对HD2家族的OsHDT702基因和RPD3/HDA1超家族的OsHDA710基因的RNAi转基因家系进行了初步观察,发现它们可能分别与植株叶片形态和植株高度有关。通过对上述问题的研究,揭示了水稻组蛋白去乙酰化酶基因OsSRT1在保护基因组稳定和防止DNA损伤,维持植物细胞的正常生长方面具有重要的作用。加深了表观遗传调控网络对生物发育、疾病、植物抗逆等许多重要生命现象调控的理解和认识。更多还原

【Abstract】 The term "epigenetics" defines heritable states of gene activity not encoded in the DNA sequence.Any given epigenetic state appears to be correlated with histone modifications,DNA methylation,and chromatin remodeling.Histone modification is an integral component of the epigenetic network in many eukaryotes.Research over the past few years has focused on yeast, animal,human and Arabidopsis.The study of epigentics in monocots,especially in rice lagged behind.The objective of this study is to isolate and characterize genes encoding histone modification enzyme,which are involved in the developmental regulation and epigenetic inheritance.The main results are as follows.1.A total of 19 HDAC genes were identified in rice genome.Of the 19 HDACs,14 belong to the RPD3/HDA1 superfamily,three belong to the HD2 family,and two belong to the SIR2 family.2.Sequence alignments and phylogenetic analysis were performed to elucidate the relationship of different members in the three HDAC families.3.A total of 10 genes were constructed for RNAi suppression,and 5 genes were constructed for over-expression.The constructs were used to transform an indica rice variety ’Minghui63’.4.This study focused on OsSRT1,a member of SIR2 family,which is NAD⁺-dependent histone deacetylase.We studied the expression profile of OsSRT1.The results showed that OsSRT1 was a widely expressed nuclear protein with higher levels in rapidly dividing tissues.5.OsSRT1 RNA interference induced an increase of histone H3K9（lysine-9 of H3） acetylation and a decrease of H3K9 dimethylation,leading to H₂O₂ production,DNA fragmentation,cell death,and lesions mimicking plant hypersensitive responses during incompatible interactions with pathogens.6.No particular visible or morphological phenotypic changes were observed in OsSRT1 over-expression plants.But over-expression of OsSRT1 gene in transgenic rice resulted in enhanced tolerance to oxidative stress compared to the wild type. 7.To determine more downstream OsSRT1-regulated genes and its regulation mechanism in OsSRT1 RNAi plants,we performed microarray analysis using the Affymetrix oligonucletide rice chip array.The results revealed that the transcription of many transposons and retrotransposons in addition to genes related to hypersensitive response and/or programmed cell death was activated.8.The results indicated that the analysis of GO classification in differential expressed genes showed that OsSRT1 actively regulated defense response,DNA repair,and phenylpropanoid metabolism,those of which always related to the stress-responsive and stress-related or PCD signaling pathways,respectively.9.To confirm the microarray data,we performed semiquantitative RT-PCR analysis of RNA isolated from OsSRT1 RNAi young leaves harvested at 7,11,and 21 days after germination to compare with wild-type and OsSRT1 overexpression plants.10.ChIP assays showed that OsSRT1 down-regulation induced histone H3K9 acetylation on the transposable elements and some of the hypersensitive response-related genes, suggesting that these genes may be among the primary targets of deacetylation regulated by OsSRT1.11.DNA methylation analysis suggested that down-regulation of OsSRT1 was likely to have a stochastic effect on DNA methylation.12.Narrow leaves were produced in OsHDT702 RNAi plants,and dwarf phenotype was observed in OsHDA710 RNAi plants,further analysis is needed.Our data together suggest that the rice SIR2-1ike gene is required for safeguard against genome instability and cell damage to ensure plant cell growth,but likely implicates different molecular mechanisms than yeast and animal homologs.更多还原