【作者】 李秀娟；

【导师】 仪慧兰；

【作者基本信息】 山西大学 ， 植物学， 2009， 硕士

【摘要】 拟南芥（Arabidopsis thaliana.L）是植物生物学研究中遗传背景最清楚的模式植物,其基因组测序工作已经完成,并且全基因组DNA甲基化图谱绘制已有报道。利用拟南芥丰富的遗传资源和相对先进的技术平台,可以为植物发育及抗性研究提供有力的手段和理论支持。二氧化硫（SO₂）属于全球性大气污染物,SO₂污染不仅会造成局部或全球气候反常,而且会对动植物的生长发育造成直接危害。如果空气中SO₂浓度超出植物的耐受范围,就会对植物产生毒害作用。目前为止,植物的抗逆机理还不清楚。本文首次对SO₂诱导拟南芥差异表达基因的DNA甲基化特征进行了分析,为研究植物抗SO₂胁迫机制提供实验依据。本实验以拟南芥为研究对象,以SO₂诱导拟南芥基因表达谱分析结果为基础,使用CpG岛数据库,对SO₂熏气后差异表达基因进行了CpG岛的预测与分析,分析结果表明:（1）在R>1的差异表达基因中,细胞代谢、结合、转录调控、信号转导和物质运输5类功能基因中含CpG岛的基因数目较多,推测这些基因表达水平的改变可能与DNA甲基化修饰相关。含CpG岛的转录调控基因在整个SO₂诱导拟南芥基因表达谱中大部分表达下调,而且在自然状态下大多处于非甲基化状态,推测转录调控基因的下调表达可能与基因内部CpG岛发生甲基化相关。（2）由诱导表达基因和沉默基因所含CpG岛数目和位置分布,推测SO₂胁迫可能主要通过诱导表达基因和沉默基因转录区的CpG岛甲基化修饰来调控基因的表达。与抗逆相关的诱导表达基因可能通过转录区发生去甲基化诱导基因的表达,沉默基因可能通过转录区的DNA甲基化而抑制基因的转录。ORF区CpG岛分析结果表明,沉默基因在ORF区发生甲基化修饰的几率更大。（3）根据SO₂熏气后拟南芥基因表达谱分析结果及CpG岛预测结果,选定MLH1、ACS-6、NIT2三个差异表达基因作为DNA甲基化分析的目的基因。采用甲基化敏感限制性内切酶（MS-RE）-PCR技术对与逆境胁迫密切相关的三个基因（MHL1、ACS-6和NIT2）进行了启动子区CCGG序列甲基化状态分析,结果表明30 mg·m^-3的SO₂连续熏气3 d后未引起MHL1启动子区CCGG序列甲基化位点的改变,对照组和实验组MHL1启动子区CCGG均为全甲基化的^5mCCGG;也未引起ACS-6启动子区CCGG甲基化位点的改变,对照组和实验组ACS-6启动子区CCGG均处于非甲基化状态;但30 mg·m^-3的SO₂连续熏气3 d后引起了NIT2启动子CCGG另一条链的第一个C发生甲基化,CCGG序列从对照组半甲基化^5mCCGG转变为全甲基化^5mCCGG。本论文通过对SO₂连续熏气3 d后拟南芥差异表达基因的CpG岛分析,推测SO₂胁迫下DNA甲基化可能调控了基因的差异性表达。使用（MS-RE）-PCR技术检测MLH1、ACS-6、NIT2三个差异表达基因启动子区CCGG位点的甲基化状态,结果表明,SO₂胁迫引起了拟南芥差异表达基因甲基化状态的改变。更多还原

【Abstract】 Arabidopsis thaliana is considered to be a good model for plant researchbecause of genetic background. The whole genome of Arabidopsis thalianahas been sequenced and its genome-scale DNA methylation maps haverecently reported. We can provide powerful methods and theoretical supportfor plant development and resistance research by using abundant geneticresources and relatively advanced technology platform.Sulfur dioxide （SO₂） is one of common air pollutants. SO₂ pollution notonly causes local or global abnormal climate, but also exerts adverse effectson plants. The growth and development of most plant species are inhibitedafter exposure to high concentrations of SO₂. Up to now, the mechanism ofplants tolerance to environment stresses is unclear. To further understand themechanism of plants tolerance to environment stresses, analysis of DNAmethylation was firstly carried out in differential expression genes ofArabidopsis thaliana exposure to SO₂.We analyzed the differential expression genes by using CpG islanddatabase based on gene profiles in 4-week-old Arabidopsis seedlings exposedto SO₂(30 mg ? m^-3, for 3 days). The results showed that: （1） Transcriptionalgenes played an important role in regulation of gene expression in bothup-regulated and down-regulated genes. The down-regulation oftranscriptional gene expression might be caused by methylation of CpGislands. （2） CpG islands distributed mainly in transcribed regions of inducedgenes and silent genes, we predicted that the methylation status of thetranscribed region CpG islands might play an important role in regulation ofinduced genes and silent genes. （3） From CpG islands in ORF, we knew thatsilent genes were more inclined to be methylated.Methylation-sensitive restriction endonuclease-PCR, named （MS-RE）-PCR, were used to reveal the CCGG methylation status in the promoters ofMHL1、ACS-6 and NIT2. The results showed that there were no methylationchanges at CCGG sites in MHL1 and ACS-6 promoters after Arabidopsis seedlings exposed to SO₂ (30 mg ? m^-3, for 3 days). The CCGG methylationstatus of MHL1 promoters was ^5mCCGG and the CCGG methylation status ofACS-6 promoters was CCGG in both SO₂ fumigation group and the control.But CCGG methylation status of NIT2 promoter changed after SO₂fumigation. The first cytosine of CCGG in NIT2 promoter washemimethylated （5’C^mCGG in single strand） in the control group andchanged to be full methylation （5’C^mCGG in double strands） after exposureto SO₂.更多还原