植物表观遗传学.ppt

植物表观遗传学植物表观遗传学Epigenetics（表观遗传学）:是指以不涉及到DNA序列的改变、但可以通过有丝分裂和减数分裂进行遗传的生物现象。自然界中的表观遗传学现象：自然界中的表观遗传学现象：Paramutation最早的例子来自果蝇的变化。Muller,H.J.(1930).Types of visible variations induced by x-rays in Drosophila.J Genet.22,299334.Hinton,T.,and Goodsmith,W.(1950).An analysis of phenotypic reversions at the brown locus in Drosophila.J.Exp.Zool.114,103114.Paramutation has been extensively characterized at three maize loci:r1,b1（helixloop-helix(bHLH)factors）,and pl1(myb)Brink(1956,1958)首先描述了r1基因的 Paramutation 现象Chandler et al,PMB,2000Chandler et al,PMB,2000Chandler et al,PMB,2000With activating mutatorParamutation 是由基因控制的Dorweiler et al,Plant cell,2000Alleman et al.,Nature,2006,442:295-8.mop1:mediator of paramutation1，RNA dependent RNA Pol IVIn B-I:more methylation,but more open chromatin structure High expression In B-P:less methylation,more dense chromatin structureLow expressionStam et al.,2002,Gene&Dev853-bp repeats Henderson IR,Jacobsen SE.Nature,2007447:418-24 目前表观遗传学研究概况目前表观遗传学研究概况拟南芥作为模式植物的优点:1.个体小，易于管理2.生长周期短，种子量大3.易于转化，进行基因功能研究4.基因组小，重复序列少，完成测序表观遗传学的分子生物学机制包括：DNA甲基化组蛋白修饰染色质重组RNA干扰植物植物DNADNA甲基化的分子机制甲基化的分子机制A DNA molecule consists of two strands,each strand=polynucleotide.Strands held together by hydrogen bonds between complementary nucleotide pairs:Adenine with Thymine,Cytosine with Guanine.double-helix structureCH3植物植物DNADNA甲基化的形式甲基化的形式CGCH3CNGCH3CHH(C/A/T)CH3动植物共有DNA甲基化的生物学意义：1.调控转座子的活性，保护基因组DNA2.调控基因的表达如何研究植物的如何研究植物的DNADNA甲基化甲基化1.DNA甲基化敏感的限制性内切酶2.亚硫酸氢钠测序：重亚硫酸盐使DNA中未发生甲基化的胞嘧啶脱氨基转变成尿嘧啶,而甲基化的胞嘧啶保持不变，PCR扩增所需片段,则尿嘧啶全部转化成胸腺嘧啶，最后,对PCR产物进行测序 3.HPLC：整体基因组甲基化水平,4.免疫化学法：利用特异的5mC抗体，结合整体基因组芯片，测定DNA甲基化区域RNA介导的DNA甲基化最初发现Wassenegger M,Heimes S,Riedel L,S nger HL.RNA-directed de novo methylation of genomic sequences in plants Cell.1994 Feb 11;76(3):567-76 Max-Planck-Institut fr Biochemie,Abteilung Viroidforschung,Martinsried,Federal Republic of Germany.One monomeric and three oligomeric potato spindle tuber viroid(PSTVd)cDNA units were introduced into the tobacco genome via the Agrobacterium-mediated leaf-disc transformation.Mette MF,van der Winden J,Matzke MA,Matzke AJ.Production of aberrant promoter transcripts contributes to methylation and silencing of unlinked homologous promoters in trans.EMBO J.1999 18:241-8.Mette MF,Aufsatz W,van der Winden J,Matzke MA,Matzke AJ.Transcriptional silencing and promoter methylation triggered by double-stranded RNA.EMBO J.2000 19:5194-201.植物甲基化植物甲基化DNADNA分子机制研究的分子机制研究的遗传学方法遗传学方法拟南芥DDM1（decrease in DNA methylation 1)基因Vongs A,Kakutani T,Martienssen RA,Richards EJ.Arabidopsis thaliana DNA methylation mutants.Science.1993，260:1926-8.1.利用DNA甲基化敏感的酶，酶切基因组DNA，检测甲基化程度的变化（centromeric repetitive DNA）WTddm1ddm1突变体：整体基因组DNA甲基化与野生型相比减少了70%。rDNAKakutani et al PNAS,1996,93:12406-12411DDM1 encodes a SWI2/SNF2-like proteinJeddeloh et al.Nature Genetics 22:94-971999Mouse:LshMET1/DDM2:Cytosine MethyltransferaseAntisense-Met1:reduce 32-71%cytosine DNA methylation Anti-Met1 WTDNAmethylation site:CG dinucleotides 2.转基因法Finnegan et al,PNAS 1996 93:8449-54 3.3.遗传学方法遗传学方法promoterMarker geneMe Me MeMutant screeningPromoterMarker geneMe Me MeMOM1promoterMarker geneHOG1,KYP1/SUVH4,CMT3,AGO4RTS1/HDA6,DRD1,2,3,NRPD1a,DDM1,MET1-promoterMarker geneMe Me Me外源沉默基因:带有标记基因的T-DNA插入；在基因组的某处产生dsRNA,沉默基因组同源序列。内源沉默基因：PAI,SupermanChan et al.,Nat Rev Genet.2005 6:351-60DOMAINS REARRANGED METHYLASE Four classes of DNA methyltransferase in Arabidopsis thaliana?DsRNAsiRNAsDCL324bpTGS:RNA-directed DNA methylation:Establishing DNA methylationInverted DNA RPol IIMe MeMeMET1?DRM2DNARNA pol IV(NRDP2,NRPD1A)RDR2Me MeMeCG CNG CHHAGO4DRD1RNA POLYMERASE 2(RDR2),DICER-LIKE 3(DCL3),RNA POLYMERASE D1(RPD1)and ARGONAUTE 4(AGO4)DRM2:DOMAINS REARRANGED METHYLASEMaintenance of CG DNA methylationMET1HDA6(HISTONE DEACETYLASE 6)DDM1Maintenance of CNG methylationCMT3,KYP(KRYPTONITE)/SU(VAR)3-9 HOMOLOG 4(SUVH4)AGO1DsRNAAGO1KYPCMT3CNGMeCHHMesiRNAsDCL324bpTGS:RNA-directed DNA methylationInverted DNA RPol IIMe MeMeMET1?DRM2MET1HDA6CGMeDDM1DNARNA pol IVRDR2Me MeMeCG CNG CHHAGO4DRD1DRM2Specific DNA methylation loci in Arabidopsis Chan et al.,Nat Rev Genet.2005 6:351-60.(pathogen related)Wassilewskija strainArabidopsis tryptophan enzyme phosphoribosylanthranilate isomerase(PAI)S15a promoter+first exonPAI1-4:350bp+ORF:hypermethylationhypomethylationhypomethylation23123I topTop,VMiddle,ICol hypomethylationF1PAI2 gene is silencedXPAI3,no activityMelquist S,Bender J.Genetics.2004 166:437-48.Genes Dev.2003 17:2036-47.ATG350 bpTAGWS,hypermethylation23I topTop,VMiddle,I1F2hypermethylatedLow gene expressionSome plants revert to hypomethylation statussuvh4/hda6 cmt3WS pai1pai1 strain accumulates fluorescent tryptophan pathway intermediates,as well as displaying yellow-green leaf pigmentation,reduced size,increased bushiness,and reduced fertility.Superman（clark kent alleles）(hypermethylated)Suppressorago4,cmt3,kyp,在基因组水平上，DNA甲基化多发现于位于着丝粒及附近的DNA重复区、转座子。拟南芥多于5%的表达基因，其启动子区域有DNA甲基化,大约1/3以上的基因在编码区有DNA甲基化,但生物学意义不清楚。一般编码区甲基化程度高的地方，基因转录水平也高。但启动子区域甲基化高的基因，转录水平较低，且多表现基因表达的组织特异性。