【作者】 秦付军；

【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， 2010， 博士

【摘要】 组蛋白修饰是表观遗传调控的重要机制之一,包括组蛋白甲基化、乙酰化、磷酸化、泛素化等其它组蛋白修饰,在动植物生长发育和基因表达调控中发挥着重要的作用。水稻是世界上重要的粮食作物,也是单子叶植物中的模式生物,有关水稻表观遗传调控机制的研究还很少报道,因此研究水稻中组蛋白修饰基因的功能具有重要的意义。本研究主要以水稻的组蛋白甲基转移酶基因(Histone methyltransferase, HMTs)和组蛋白去乙酰化酶基因(Histone deacetylase, HDACs)为研究对象,通过生物信息学、克隆和转化等方法研究了HMTs和HDACs基因的结构、表达和功能,阐述了其在水稻表观遗传调控机制中的作用,主要获得以下结果：1.通过同源序列研究发现,水稻中存在12个组蛋白甲基转移酶SUVH(Suppressor of variegation 3-9 homologue)家族基因和18个HDACs基因；对水稻组蛋白甲基转移酶中SUVH家族和HDAC家族基因进行了系统进化分析,并确定拟研究基因；基因结构分析发现,SUVH家族基因都由YDG-SRA、Pre-SET、SET三个保守的结构域组成；对SUVH和HDAC家族基因进行了水稻全生育期芯片的表达模式分析和愈伤、根、小苗、茎、叶、旗叶和幼穗等特异组织的表达谱分析,发现不同的SUVH家族基因具有相似的表达模式,但表达水平不同。2.对SUVH和HDAC家族中拟研究基因SDG703、SDG704、SDG709、SDG710、SDG713、SDG714、SDG715、SDG726、SDG727、SDG728、HDT701、HDA704、HDA706进行了RNAi干涉载体构建,对基因SDG703、SDG704、SDG709、SDG713、SDG714、SDG715、SDG726、SDG728、HDA706、HDT702、HDA711构建了超量表达载体,并获得独立的转基因植株。3.RNA水平检测SUVH和HDAC结果显示,在超量表达和RNAi植株中,各基因的表达量都有不同程度的上升和下降。4.转基因SUVH成员SDG713和SDG728的下调表达分别引起了植株矮小致死和种子粒厚减小的表型；HDAC成员中HDA704的下调表达引起植株矮化和旗叶基部卷曲,外施赤霉素GA3不能互补HDA704的表型。5.亚细胞定位显示SUVH家族SDG713位于细胞核内,与启动子融合的GFP在根尖、幼茎、雌蕊等组织中特异表达。6.SUVH成员SDG714下调表达引起柱头数目增多等花的变态发育,同时引起miRNA159的上升表达和miRNA164的下降表达。7.SUVH成员转基因材料的Western杂交结果显示,与野生型相比,SDG728在超量表达和：RNAi植株中分别引起H3K9me3修饰的上升和下降；SDG704、SDG726、SDG727在RNAi植株中H3K9me3修饰呈下降变化。8.原核表达GST-SDG728和GST-SDG714组蛋白甲基化酶并进行活性实验,结果显示GST-SDG714可以使小牛核心组蛋白和原核表达重组寡聚组蛋白单甲基化和二甲基化；而GST-SDG728仅能使小牛核心组蛋白甲基化,而不能使原核表达重组寡聚组蛋白甲基化,表明SDG728具有三甲基化活性。9.McrBC-PCR方法检测DNA甲基化结果显示,SDG728调控Tos17和一个Tyl-copia类反转座子Os08g03880的DNA甲基化。另外,SDG703、SDG713、SDG715等也影响上述两个反转座子的甲基化水平。10.染色质免疫沉淀结果显示,SDG728调控Tos17和Os08g03880基因区域的H3K9me3水平,在SDG728超量表达植株中,H3K9me3明显增加；在RNAi植株中,H3K9me3则呈显著下降。上述一系列的结果表明,水稻SUVH成员SDG728是一个组蛋白三甲基化酶,通过调控反转座子基因区域的组蛋白三甲基化水平来影响DNA甲基化水平,进而调控反转座子的表达,维持水稻的基因组稳定,揭示了表观遗传调控在调节转座元件基因表达过程中的分子机制。其它SUVH成员和HDAC成员的研究结果表明其在水稻组蛋白修饰和生长发育中起着重要的作用。更多还原

【Abstract】 Histone modifications, including histone methylation, acetylation, phosphorylation, ubiqutination and others, play pivotal roles in plant development and gene regulation. There are few reports on the molecular mechanism of epigenetic regulation in monocots, especially in rice. Therefore it is of great significance to study the function of histone modification gene from rice.This study mainly focused on the histone methyltransferase (HMTs) and histone deacetylase (HDACs). The function and the roles of these genes in epigenetic regulation were investigated by bioinformatics, cloning, transformation and other methods. Main results are shown as following:1.12 SUVHs (Suppressor of variegation 3-9 homologue) histone methyltransferases and 18 HDACs (Histone deacetylase) were identified from rice. Sequence alignment and phylogenetic analysis of SUVHs and HDACs were performed to elucidate the evolution relationship of members within these two families. Gene structure analysis revealed that SUVH proteins contained three conserved domain including YDG (named after 3 conserved amino acids)-SRA (SET-and Ring finger-associated) and pre-SET, SET (named after 3 genes from Drosophila). To study the expression profiles of the rice SUVHs and HDAC genes, a serial Affimetrix microarray analysis of transcripts from various organs at different developmental stages were performed (http://crep.ncpgr.cn). The results revealed that most of the rice SVUH genes showed a similar expression pattern, whereas their expression levels in same tissues or organs were different.2. Over-expression and RNAi vectors of SUVHs and HDACs genes were constructed and transformed into rice to get transgenic plants.3. RNA level analysis indicated that there were the increased expression of each target genes in over-expression and decreased expression in RNAi transgenic plants.4. Down-regulation of SDG713 and SDG728 (SUVHs) led to the lethal dwarf plant and reduced seed thickness, respectively. The dwarf phenotype and twist flag leaf was caused by reduced expression level of HDA704, which were not complemented by treating with gibberellin (GA3).5. The expression pattern of the SDG713-GFP fusion protein showed that SDG713 was a widely expressed nuclear protein with high expressions in root tip, young stem, pistil and others tissues.6. SDG714 RNA interference induced abnormal stigma development and led to the increased miR159 RNA level and decreased miR164 RNA level in rice (indica).7. Western Blot results revealed that SDG728 was required to regulate H3K9me3 and SDG704, SDG726 and SDG727 participated in the regulation of H3K9me3.8. The proteins of GST-SDG728 and GST-SDG714 which were expressed and purified from E.coli were used to study the methyltransferase activity in vitro. Results showed that SDG728 could methylate calf core histones, but not the synthesized non-methylated histones, whereas SDG714 showed a methyltransferase activity on both types of sbustrates. This observation indicated that SDG728 has a trimethyltransferase activity.9. The results of McrBC-PCR denmostrated that DNA methylations of Tos17 and a Ty1-copia like retrotransposon (Os08g03880) were affected by SDG728 and other members such as SDG703, SDG713 and SDG715.10. ChIP (Chromatin immunoprecipitation) data indicated that SDG728 was required for histone H3K9me3 on the Tosl 7 and Os08g03880 loci.All those data demonstrated that SDG728 was a histone trimethyltransferase in rice, which regulated the DNA methylation on the gene region of transposable elements by the changes of histone H3K9me3. The fact that SDG728 could repress the retrotransposons and maintain the genome stability indirectly, suggested its role in regulating the retrotransposon expression of SDG728. Other members of SUVHs and HDACs played important roles in histone modification and rice development.更多还原