【作者】 戴艳；

【导师】 孙其信； 倪中福；

【作者基本信息】 中国农业大学 ， 作物遗传育种， 2005， 博士

【摘要】 表观遗传学是指在不改变DNA序列的情况下,在碱基序列外的各种修饰和与之相关的各种蛋白质或RNA的协同作用下,调控基因的表达,以完成生命周期或适应环境变化,而且这套系统还能在世代之间传递。就目前的研究来看,表观遗传学主要包括组蛋白密码、DNA甲基化、RNA干涉、基因组印记等多个方面。小麦是世界第二大粮食作物,关于小麦表观遗传学的研究鲜有报道。本文采用简并性引物同源扩增及电子克隆的方法,分析了小麦组蛋白修饰酶基因及DNA甲基转移酶基因的结构及其表达,以期从转录水平增加对小麦生长发育调控的认识。此外,采用转基因的方法在拟南芥中超表达了一个组蛋白乙酰转移酶基因和一个组蛋白脱乙酰化酶基因,以期探索它们的生物学功能,主要结果如下: 1) 利用电子克隆方法,结合RT-PCR分析,从小麦中克隆了包含完整ORF的2个不同类型的组蛋白乙酰转移酶基因(MYST类型,GNAT类型)和4个代表不同类型的(SIR-2类型,HD2类型,RPD3类型,HDA1类型)组蛋白脱乙酰化酶基因,分别命名为TaHAT1-2,TaHD1-4。结构分析表明它们具有组蛋白乙酰转移酶及脱乙酰化酶的典型的结构特征。亚细胞定位结果表明TaHAT1定位在细胞核中,TaHD3属于核质穿梭型的组蛋白脱乙酰化酶。本研究是有关小麦组蛋白乙酰转移酶及脱乙酰化酶基因的首次报道。 2) 采用同源序列扩增法,分离克隆了9个小麦的SET蛋白基因片段,同源进化分析表明它们均属于SU(VAR)3-9类型的SET蛋白基因。同时利用电子克隆结合RT-PCR方法分离了两个具有完整ORF的SET蛋白基因,分别命名为TaSET1和TaSET2。同源进化分析表明它们分别属于ASH和SU(VAR)3-9类型的SET蛋白基因。 3) 采用生物信息学手段及结合RT-PCR方法获得了4条代表不同类型(Dnmt1,Dnmt2,CMT,Dnmt3)的小麦DNA甲基转移酶基因的片段,分别命名为TaMET1,TaMET2b,TaCMT和TaMET3;同时采用电子克隆结合RT-PCR方法获得一个具有完整ORF的Dnmt2类型基因,命名为TaMET2a。此外,也获得了该序列相应于ORF区的基因组序列,序列分析表明在该基因ORF区域含有10个内含子。 4) 以小麦3338水浸泡4-28小时(每隔4小时取一次样)的种子的种胚,水浸泡48小时的胚根、胚芽,不同发育时期的叶片、根系及干种子、授粉6天、12天的种子为材料检测了所获得基因的表达情况。结果表明所检测的基因在大部分材料中均表达。有趣的是只有TaMET1和TaMET3在干种子中表达,其它所检测的基因均不表达。此外,以拔节期的小麦3338,2463及其组配的强优势杂交种的叶片、茎、根系为材料,采用半定量RT-PCR方法检测了所获得基因的表达情况,结果表明它们在小麦杂交种与亲本之间存在明显的表达差异,并且差异表达模式因基因类型和所检测的组织器官不同而变化。 5) 在拟南芥中分别超表达MYST类型的小麦组蛋白乙酰转移酶基因TaHAT1及RPD3类型的脱乙酰化酶基因TaHD3,结果发现它们均出现了叶片、花的畸形发育,我们推测这两个基因与小麦生长发育的调控有关。本研究是对推测的小麦组蛋白乙酰转移酶及脱乙酰化酶基因功能研究的首次报道。更多还原

【Abstract】 Epigenetic regulation is a major aspect of gene control by which heritable changes in gene expression occur without an alteration in DNA sequence, which mainly includes histone code, DNA methylation, RNA interference and genomic imprinting.The study of epigentics in wheat lagged behind. In this article, genes encoding histone modification enzyme and DNA methltransferase were isolated using degenerate primer PCR and in silico cloning.The main results are as follows:1) Two histone acetyltransferase genes and four histone deacetylase genes which belonged to different types were isolated using in silico cloning and RT-PCR method, designated TaHAT1-2, TaHD1-4 respectively. Amino acid sequence analysis suggested that they all contained typical structrue characteristics of histone acetyltransferase and deacetylase. Subcellular localization indicated that TaHATl protein became concentrated in the nucleus and TaHD3 displayed nuclear-cytoplasmic distribution.To our knowledge, no genes encoding histone acetyltransferase and deacylase has been reported in wheat2) Nine SET domain protein gene fragments were obtained by degenerate-primer PCR. Phylogenetic analysis suggested that they belonged to SU (VAR) 3-9 class SET domain protein.ln addition, two SET domain protein genes were obtained using in silico cloning and RT-PCR method, named TaSET1-2 respectively. Phylogenetic analysis indicated that they belonged to ASH and SU (VAR) 3-9 class SET domain protein respectively.3) We isolated four wheat cDNA fragments and one cDNA with open reading frame encoding DNA methyltransferase and designated TaMETl, TaMET2a, TaMET2b, TaCMT and TaMET3, respectively. BLAST searches and phylogenetic analysis suggested that 5 cDNAs belonged to four classes (Dnmt1, Dnmt2, CMT and DnmG) of DNA methyltransferase genes. TaMET2a encoded a protein of 376 aa and contained eight of ten conserved motifs characteristic of DNA methyltransferase. Genomic sequence of TaMET2a was obtained and found to contain ten introns and eleven exons.4) Expression analysis of these genes obtained in this article revealed that they were expressed in developing seed, during germination and various vegetative tissues, but in quite different abundance. It was interesting to note that TaMETl and TaMET3 involved in maintenance methylation and de novo methylation were clearly detected in dry seeds and other genes had not been detected. Moreover, differential expression patterns of these genes were observed between wheat hybrid and its parents in leaf, stem and root of jointing stage, some were up-regulated while some others were down-regulated in hybrid.5) Overexpression of TaHATl and TaHD3 genes in Arabidopsis suggested that they were both associated with various developmental abnormalities, mainly including leaf and flower defect which suggested that TaHATl and TaHD3 may be involved in a general regulatory mechanism responsible for plant plasticity and variation in nature.This is a first report about function study of histone acetyltransferase and deacetylase in wheat.更多还原