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DNA甲基化的分子机制
A DNA molecule consists of two strands, each strand = polynucleotide.
植物表观遗传学
巩志忠 中国农业大学生物学院 Tel: 6273-3733 Email: gongzz@
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： Paramutation
WS, hypermethylation
Col hypomethylation
1 2
350 bp
I top
X
3
ATG TAG PAI2 gene is silenced
3 2 Top, V Middle, I
PAI3, no activity
F1
Genes Dev. 2003 17:2036-47. Melquist S, Bender J. Genetics. 2004 166:437-48.
Strands held together by hydrogen bonds between complementary nucleotide pairs: Adenine with Thymine, Cytosine with Guanine.
CH3
double-helix structure
植物DNA甲基化的形式
Max-Planck-Institut für 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.
DsRNA
Inverted DNA R
DCL3
siRNAs ~24bp
Pol II
AGO4
DRD1
RNA POLYMERASE 2 (RDR2), DICER-LIKE 3 (DCL3), RNA POLYMERASE D1 (RPD1) and ARGONAUTE 4 (AGO4)
CG CNG CHH
WT ddm1
ddm1突变体：整体基因组 DNA甲基化与野生型相比 减少了70%。
rDNA
Kakutani et al PNAS, 1996, 93: 12406-12411
DDM1 encodes a SWI2/SNF2-like protein
Mouse: Lsh
Jeddeloh et al. Nature Genetics 22 :94-971999
DOMAINS REARRANGED METHYLASE
???
Chan et al., Nat Rev Genet. 2005 6:351-60
TGS: RNA-directed DNA methylation: Establishing DNA methylation DNA
Me Me Me RNA pol IV (NRDP2,NRPD1A) RDR2
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, Sänger HL. RNA-directed de novo methylation of genomic sequences in plants Cell. 1994 Feb 11;76(3):567-76
suvh4/hda6 cmt3
Superman（clark kent alleles ） (hypermethylated )
Me Me Me
promoter
Marker gene
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, Superman
Four classes of DNA methyltransferase in Arabidopsis thaliana
3.遗传学方法
Me Me Me
promoter
Marker gene
Mutant screening
Me Me Me
Promoter
Marker gene
promoter
Marker gene
MOM1
HOG1, KYP1/SUVH4, CMT3,AGO4 RTS1/ HDA6, DRD1 ,2,3, NRPD1a, DDM1, MET1 ----
CH3
动植物共有
CG
CH3 CNG CH3
CHH (C/A/T)
DNA甲基化的生物学意义：
1.调控转座子的活性，保护基因组DNA
2.调控基因的表达
如何研究植物的DNA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
1 F2 2 Top, V
I top 3 Middle, I Low gene expression
hypermethylated
Some plants revert to hypomethylation status
WS pai1
pai1 strain accumulates fluorescent tryptophan pathway intermediates, as well as displaying yellow-green leaf pigmentation, reduced size, increased bushiness, and reduced fertility.
Dorweiler et al, Plant cell, 2000 Alleman et al., Nature, 2006, 442:295-8.
853-bp repeats
In B-I: more methylation, but more open chromatin structure In B-P: less methylation, more dense chromatin structure
Chandler et al, PMB, 2000
Chandler et al, PMB, 2000
With activating mutator
Chandler et al, PMB, 2000
Paramutation 是由基因控制的
mop1: mediator of paramutation1， RNA dependent RNA Pol IV
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, 299–334.
Hinton, T., and Goodsmith, W. (1950). An analysis of phenotypic reversions at the brown locus in Drosophila. J. Exp. Zool. 114, 103–114.
植物甲基化DNA分子机制研究的 遗传学方法
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 ） 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.
Arabidopsis Wassilewskija strain
tryptophan enzyme phosphoribosylanthranilate isomerase (PAI)
PAI1-4: 350bp+ORF: hypermethylation hypomethylation
hypomethylation S15a promoter+ first exon
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.Me来自AGO4DRD1