【作者】 张娅；

【导师】 陈耀锋；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2007， 博士

【摘要】 对于开花植物来说,在适宜的条件,适宜的时间实现从营养生长阶段向生殖生长阶段的转变,对植物发育有着重要的意义。通过对大量突变体的遗传分析,在拟南芥中已经发现了四条主要途径通过调节体内因素和外界环境影响开花时间,自主途径、春化途径、赤霉素途径和光周期途径。植物的开花发育受表观遗传调控。表观遗传是一门新学科,主要研究染色质修饰和染色质重塑等对基因转录表达的调控机制。组蛋白的共价修饰包括赖氨酸的乙酰化、甲基化和精氨酸的甲基化等。组蛋白末端氨基酸共价修饰对植物开花发育的调控作用的研究表明FLC的抑制因子和促进因子通过改变组蛋白氨基酸的共价修饰,影响FLC基因所在区域的染色质重塑,调控FLC的转录表达水平,从而调控开花时间。哺乳动物SKB1(Shk1 kinase-bingding protein 1,又名PRMT5,Protein Arginine methyltransferases 5)在调控染色质重塑、基因转录、RNA剪切和细胞增殖与分化过程中起重要作用。拟南芥SKB1基因(At4g31120)与其序列高度同源,在拟南芥基因组仅含有单拷贝SKB1基因。目前关于蛋白质精氨酸甲基转移酶及其催化的精氨酸甲基化修饰在植物发育中的作用还没有任何报道。本论文集中研究了拟南芥精氨酸甲基转移酶SKB1基因调控植物开花发育的遗传和生化机理,获得了如下结果:1.通过对拟南芥精氨酸甲基转移酶SKB1的序列分析,发现SKB1含有精氨酸甲基转移酶必需的5个保守序列,并且与哺乳动物的同源蛋白PRMT5在序列上具有很高的相似性。利用原核表达系统纯化His-SKB1重组蛋白,运用切胶回收的方法免疫制备SKB1特异性抗体,研究了SKB1的生化功能。纯化GST-SKB1融合蛋白,利用3H标记的甲基供体S-腺苷甲硫氨酸(3H- SAM)分析SKB1的甲基转移酶活性发现组蛋白H4是SKB1的特异底物。2.获得了两个SKB1 T-DNA插入突变体株系,突变体表现出晚花的表型,同时叶色深绿,叶片弯曲。通过转化SKB1 cDNA可以回复突变体晚花的表型,超表达SKB1引起拟南芥早花,同时拟南芥开花时间与SKB1的表达量呈正相关,说明SKB1促进植物开花,是拟南芥开花发育的正调控因子。3.分析了在不同生理条件下skb1突变体的表型,发现不同光周期处理后,突变体都表现晚花的表型,表明突变体对光周期不敏感;而春化和赤霉素处理则可以部分恢复突变体晚花的表型,由此确定SKB1属于开花自主途径。4.RT-PCR分析发现在skb1突变体中FLC的表达量明显高于野生型,同时通过杂交发现flc突变体可以部分恢复skb1突变体晚花的表型,skb1×flc双突变体的开花时间介于野生型和skb1突变体之间。表明FLC过量表达是skb1晚花的原因。5.SKB1::GUS融合基因分析,原位杂交以及Western检测SKB1的时空表达模式表明SKB1主要在幼苗的茎顶端分生组织和幼叶中表达,与开花自主途径基因很相似。6.利用组蛋白特异位点修饰的抗体,Western检测表明在体内和体外SKB1都可以特异地对称性双甲基化修饰H4R3(H4R3sme2)。7.染色质免疫沉淀(ChIP)技术分析表明SKB1特异的结合在FLC基因组DNA启动子区,通过对称性双甲基化修饰组蛋白H4R3调控开花时间。综上所述,拟南芥精氨酸甲基转移酶SKB1通过对称性双甲基化目标基因FLC染色质的启动子区域,抑制FLC的表达,从而促进植物开花。本论文不仅阐明了蛋白质精氨酸甲基转移酶SKB1调控开花发育的分子机制,而且还可能揭示表观遗传修饰调控植物发育的新途径。更多还原

【Abstract】 Plant flowering is a crucial developmental transition from the vegetative to reproductive phase and is properly timed by a number of intrinsic and environmental cues. Investigations in Arabidopsis have identified four major pathways (photoperiod, vernalization, gibberellin (GA), and autonomous) involved in regulating flowering time. Plant flowering is influenced by epigenetic factors. Epigenetic regulations mainly involve the mechanism of histone modification and chromatin remodeling regulating gene transcription. Chromatin covalent modification included acetylation and methylation of lysine and arginine. The recent characterization of FLC repressors and activators has shown that some of these regulatory proteins are involved in the covalent modification of FLC chromatin and controlling the flowering time.Mammalian SKB1(Shk1 kinase-bingding protein 1),also called PRMT5( Protein Arginine methyltransferases 5)has been shown to regulate chromatin remodelling, gene transcription, RNA splicing, and cell proliferation and differentiation. The Arabidopsis genome contains a single-copy SKB1 gene (At4g31120) shows high homology with the human PRMT5. However, little is known about the functions of histone arginine methylation in plants. In this dissertation, we focused on the genetic and biochemical mechanism of SKB1 controlling flowering time in Arabidopsis and obtained the following results:1. By comparison of Arabidopsis SKB1 amino acid sequence with homologous proteins of diverse organisms, we found that SKB1 contains five conserved motifs of protein arginine methyltransferases. The sequence of SKB1 is highly aligned with PRMT5, which is the homologue of mammalian. To analyze biochemical characterization of SKB1, the His-SKB1 recombinannt protein was purified in E.coli cells. The antibody to SKB1 was produced by using of the method of cutting and reclaiming gels. We assayed in vitro a GST-SKB1 fusion protein for methylation activity with 3H-SAM. Only H4 was methylated.2. We identified two T-DNA insertional mutants. SKB1 lesion results in late flowering. The skb1 mutant plants also formed leaves slightly more curled and darker than wild-type plants. We introduced SKB1 cDNA into skb1mutant plants. 35S::SKB1 could rescue the skb1-1 mutant, resulting in transgenic plants with a wild-type phenotype. Plants overexpressing SKB1 had early flowering feature and SKB1-promoted flowering time showed a stoichiometric relation with SKB1 protein level. So SKB1 is a positive regulator of floral initiation and prevents late flowering.3. We sought to identify the pathway that SKB1 was involved in by treating skb1 mutant plants with the different conditions. Under long-day or short-day photoperiods, skb1 mutant displayed later flowering than wild-type plants, which indicates that skb1 mutants were sensitive to photoperiod. Vernalization and GA treatment reversed in part the delayed flowering of skb1 mutant plants. Taken together, these results suggest that SKB1 is a new member of the autonomous pathway.4. RT-PCR result indicated that the expression of the flowering repressor gene FLC was significantly up-regulated in skb1 mutant. We introduced skb1 into FLC-null mutant. The late-flowering phenotype of skb1 was suppressed by flc mutant. Thus, the flowering time control by SKB1 is targeted towards FLC expression.5. The temporal and spatial expression pattern of SKB1 was analyzed with use of a SKB1 promoter driving GUS, in situ hybridization and Western with a specific antibody against SKB1. SKB1 was more abundant in shoot apex and young leaves in seedlings, which was similar to that of genes in autonomous pathway.6. We used specific antibodies modificated histone of specific site to examine whether SKB1 methylates H4R3. The results suggested that SKB1 can methylate H4R3 symmetrically in vitro and in vivo.7. Chromatin immunoprecipitation (ChIP) assay revealed that SKB1 binds to the FLC promoter and mediates histone H4R3 symmetric dimethylation (H4R3sme2) to control flowering time.Thus, SKB1 affects flowering development by alterating FLC expression via symmetric dimethylation of H4R3 in its promoter.SKB1-mediated H4R3sme2 is a novel histone mark required for repression of FLC expression and flowering time control. Base on these results of this dissertation, we not only clarified the molecular mechanism of aginine methyltransferse SKB1 controlling flowering time in Arabidopsis, but the new histone code of epigenetic inheritance to regulate plant development.更多还原