5-azaC对菠菜生长发育及DNA甲基化的影响
摘要
菠菜(Spinacia oleracea L.)为藜科(Chenopodiaceae)菠菜属(Spinacia)一年生或两年生草本植物。菠菜作为一种常见的园艺蔬菜作物已经被广泛栽培,且菠菜一般为雌雄异株,极少雌雄同株,其染色体组为2n=2x=24(XY)。因此,对于菠菜的研究目前主要集中在新品种选育、栽培技术优化以及性别决定及分化机制三个方面,对其他方面研究尤其是菠菜表观遗传特征尤其是性别发育的表观调控机制的研究鲜有报道。DNA甲基化作为最早发现的基因组表观遗传修饰途径之一,已有证据表明该机制可以植物基因的表达进行表观调控,从而影响植物的生长发育。为了探索DNA甲基化在菠菜营养生长阶段及性别发育阶段的作用,本文以甲基化抑制剂5-azaC处理及未处理的菠菜为材料,通过比较观察,分析菠菜的生长发育过程及性比的变化,同时,采用MS-ISSR、MS-RAPD和HPLC法对基因组DNA甲基化水平进行检测,分析菠菜表型变化和基因组DNA甲基化水平之间的相关关系,从而尝试揭示DNA甲基化对于菠菜生长发育及性别过程中的作用机制。主要研究结果如下:
1.使用不同浓度的5-azaC对菠菜种子进行处理,分析甲基化抑制剂5-azaC对于菠菜发芽势、发芽率、株高、叶长、根长、开花时间及性比的影响。结果表明,5-15μM的5-azaC对菠菜处理后,使发芽率、发芽势、根长、苗长、叶长、株高与对照相比,随着浓度的增高而降低,开花时间提前,但是与对照相比相差不显著;菠菜对30μM浓度处理不敏感,各项生长指标与对照相差很少;50-1000μM的5-azaC对菠菜处理后,随着浓度的增加与对照相比各项生长指标都降低,其中高浓度有明显的抑制作用,如株高与对照相比明显变矮,开花时间内提前1d以上。但对菠菜性比和千粒重影响不大,表现不显著。
2.以浓度为30、100、500μM的甲基化抑制剂5-azaC处理和未处理的菠菜单株叶片基因组为材料,经过甲基化限制性内切酶HpaII和MspI进行酶切后,采用MS-RAPD和MS-ISSR技术对基因组DNA变化进行分析。结果发现,从400条RAPD引物和65条ISSR引物中筛选出20条MS-RAPD特异引物和35条MS-ISSR特异引物,从12个样品共扩增出667条可统计的清晰条带,多态性位点的百分率分别为86.88%和86.24%。通过对特异酶切位点的分析发现,在30、100、500μM处理中发生了甲基化和去甲基化作用,去甲基化作用稍占主导地位。
3.以浓度为30、100、500μM的甲基化抑制剂5-azaC处理和未处理的菠菜单株基因组为材料,利用高效液相色谱法检测不同器官组织的菠菜总DNA甲基化的水平。结果表明,经过不同浓度处理,当5-azaC浓度超过30μM后,随着浓度的增加基因组甲基化水平显著性的降低,当5-azaC浓度超过500μM时,基因组甲基化水平变化不显著。在不同器官组织检测中,成熟叶片的DNA甲基化水平最高,其次是幼叶、子叶、下胚轴,胚根最低(平均值在4.61%)。同样可以看出随着处理浓度的增加DNA甲基化水平降低。
Spinach (Spinacia oleracea L , 2n=2x=24 (XY)) is an annual (rarely biennial) flowering plant in the family of Amaranthaceae. It was cultivated widely as a common vegetable gardening. Spinach is a diploid dioecious plant, which possesses a pair of heteromorphic sex chromosome X and Y. Breeding of new variety, optimizing cultivation technology and the mechanism of sex determination were being studied in the past several decades. The research that focused on epigenetics characters especicially involved in epigenetics regulating mechanism of sexual development was rarely been reported. Plant DNA methylation was one of the first discovering genome epigenetic modification ways, and it was proved to succeed in regulating the plant gene expression. In order to explore the affection on DNA methylation in spinach growth and sex development stage, the following experiments were performed. The epigenetics phenotype and sex ratio were been investigated after treating use the methylation inhibitor 5-azaC. Meanwhile, the DNA methylation pattern and methylation level were detected by MS-ISSR、MS-RAPD and HPLC methods. The result showed as follow:
1. Different 5-azaC concentration was used to treat the spinach feed and its affection on germination energy, germination percentage, Plant height, leaf length, root length, flowering time and sex ratio of spinach was explored. The result showed germination energy, germination percentage, root length, Plant height and leaf length are negative correlated with the 5-azaC concentration among 5-15μM compared with the control plants, and the flowering a little earlier than control plants. The spinach was not sensitive to 30μM 5-azaC and every growth indices was almost same as the control group. The spinach was treated by 5-azaC whose concentration was 50-1000μM and its growth indices decreased as the concentration increased. The high concentration inhibited obviously growth indices, for example the plant height was lower than the control group and the the flowering earlier more than one day. However it has little affection on spinach sex ratio and 1000- grain weight.
2. The spinach was treated by 30、100、500μM methylation inhibitor, then its leaf and the normal one gene group were digested by methylation-sensitive restriction endonuclease. The change between them was investigated by MS-RAPD and MS-ISSR technology. The result showed that 20 polymorphism MS-RAPD primers were selected from 400 primers and 35 polymorphism MS-ISSR primers from 65 primers. 667distinct bands were obtained from 12 samples. The percentage of polymorphic loci were respectively 86.88% and 86.24%, respectively.Through the analysis on specific endonuelease sites, it found methylation and demethylation appeared during 30、100、500μM treat and demethylation play a important role.
3. The extend of total DNA methylation of different spinach tissue were detected by HPLC method, the material was obtained from the treat plant by 30、100、500μM methylation inhibitor and the untreated control plants. The result showed that extend of gene methylation decreased obviously with the concentration increased in 30μM. However the changed was not obviously when the concentration exceed 500μM. Among the different tissue detection, the mature leaf had the highest DNA methylation extend, then spire, cotyledon and hypocotyl in sequence, and radicle has the lowest (the average value was 4.61%).The same trend that the gene methylation extend decreased as the concentration increased were observed.
1.使用不同浓度的5-azaC对菠菜种子进行处理,分析甲基化抑制剂5-azaC对于菠菜发芽势、发芽率、株高、叶长、根长、开花时间及性比的影响。结果表明,5-15μM的5-azaC对菠菜处理后,使发芽率、发芽势、根长、苗长、叶长、株高与对照相比,随着浓度的增高而降低,开花时间提前,但是与对照相比相差不显著;菠菜对30μM浓度处理不敏感,各项生长指标与对照相差很少;50-1000μM的5-azaC对菠菜处理后,随着浓度的增加与对照相比各项生长指标都降低,其中高浓度有明显的抑制作用,如株高与对照相比明显变矮,开花时间内提前1d以上。但对菠菜性比和千粒重影响不大,表现不显著。
2.以浓度为30、100、500μM的甲基化抑制剂5-azaC处理和未处理的菠菜单株叶片基因组为材料,经过甲基化限制性内切酶HpaII和MspI进行酶切后,采用MS-RAPD和MS-ISSR技术对基因组DNA变化进行分析。结果发现,从400条RAPD引物和65条ISSR引物中筛选出20条MS-RAPD特异引物和35条MS-ISSR特异引物,从12个样品共扩增出667条可统计的清晰条带,多态性位点的百分率分别为86.88%和86.24%。通过对特异酶切位点的分析发现,在30、100、500μM处理中发生了甲基化和去甲基化作用,去甲基化作用稍占主导地位。
3.以浓度为30、100、500μM的甲基化抑制剂5-azaC处理和未处理的菠菜单株基因组为材料,利用高效液相色谱法检测不同器官组织的菠菜总DNA甲基化的水平。结果表明,经过不同浓度处理,当5-azaC浓度超过30μM后,随着浓度的增加基因组甲基化水平显著性的降低,当5-azaC浓度超过500μM时,基因组甲基化水平变化不显著。在不同器官组织检测中,成熟叶片的DNA甲基化水平最高,其次是幼叶、子叶、下胚轴,胚根最低(平均值在4.61%)。同样可以看出随着处理浓度的增加DNA甲基化水平降低。
Spinach (Spinacia oleracea L , 2n=2x=24 (XY)) is an annual (rarely biennial) flowering plant in the family of Amaranthaceae. It was cultivated widely as a common vegetable gardening. Spinach is a diploid dioecious plant, which possesses a pair of heteromorphic sex chromosome X and Y. Breeding of new variety, optimizing cultivation technology and the mechanism of sex determination were being studied in the past several decades. The research that focused on epigenetics characters especicially involved in epigenetics regulating mechanism of sexual development was rarely been reported. Plant DNA methylation was one of the first discovering genome epigenetic modification ways, and it was proved to succeed in regulating the plant gene expression. In order to explore the affection on DNA methylation in spinach growth and sex development stage, the following experiments were performed. The epigenetics phenotype and sex ratio were been investigated after treating use the methylation inhibitor 5-azaC. Meanwhile, the DNA methylation pattern and methylation level were detected by MS-ISSR、MS-RAPD and HPLC methods. The result showed as follow:
1. Different 5-azaC concentration was used to treat the spinach feed and its affection on germination energy, germination percentage, Plant height, leaf length, root length, flowering time and sex ratio of spinach was explored. The result showed germination energy, germination percentage, root length, Plant height and leaf length are negative correlated with the 5-azaC concentration among 5-15μM compared with the control plants, and the flowering a little earlier than control plants. The spinach was not sensitive to 30μM 5-azaC and every growth indices was almost same as the control group. The spinach was treated by 5-azaC whose concentration was 50-1000μM and its growth indices decreased as the concentration increased. The high concentration inhibited obviously growth indices, for example the plant height was lower than the control group and the the flowering earlier more than one day. However it has little affection on spinach sex ratio and 1000- grain weight.
2. The spinach was treated by 30、100、500μM methylation inhibitor, then its leaf and the normal one gene group were digested by methylation-sensitive restriction endonuclease. The change between them was investigated by MS-RAPD and MS-ISSR technology. The result showed that 20 polymorphism MS-RAPD primers were selected from 400 primers and 35 polymorphism MS-ISSR primers from 65 primers. 667distinct bands were obtained from 12 samples. The percentage of polymorphic loci were respectively 86.88% and 86.24%, respectively.Through the analysis on specific endonuelease sites, it found methylation and demethylation appeared during 30、100、500μM treat and demethylation play a important role.
3. The extend of total DNA methylation of different spinach tissue were detected by HPLC method, the material was obtained from the treat plant by 30、100、500μM methylation inhibitor and the untreated control plants. The result showed that extend of gene methylation decreased obviously with the concentration increased in 30μM. However the changed was not obviously when the concentration exceed 500μM. Among the different tissue detection, the mature leaf had the highest DNA methylation extend, then spire, cotyledon and hypocotyl in sequence, and radicle has the lowest (the average value was 4.61%).The same trend that the gene methylation extend decreased as the concentration increased were observed.
引文
[1]丁宝章,王遂义,高增义.河南植物志[M].郑州:河南科学技术出版社,1988,353.
[2]桂明珠,王承林,林金莲,等.菠菜(Spinacia oleracea Mill.)营养器官内部桔构的初步观察[J].东北农学院学报,1979,1:83-91.
[3] Lan T, Zhang S, Liu B, et al. Differentiating sex chromosomes of the dioecious Spinacia oleracea L. (spinach) by FISH of 45S rDNA [J]. Cytogenet Genome Res, 2006, 114(2): 175-177.
[4]吴爱忠,王玉林,林观捷,等.菠菜杂交种及其亲本品种的染色体核型分析[J].上海农学院学报,1993,11(1):1-6.
[5] Hiaga,T.Sex and chromosones in Spinacia oleracea L [J]. Jap.Jour. Genetics, 1935, 10:218-222.
[6]徐杰,吴燕燕.菠菜叶片中乙醇酸氧化酶3种同工酶的生化特性[J].中国生物化学与分子生物学报,2001,17(4):537-540.
[7] D.Noah.Sather,Amber York,Kevin J.Pobursky,et.al.Sequence evolution and sex-specific expression patterns of the C class floral indentity gene,SpAGAMOUS,in dioecious Spinacia oleracea L [J]. Planta,2005, 222:284-292
[8]丛娟,李晓玲,宫莉,等.菠菜基因组DNA提取方法与条件的研究[J].长春工业大学学报(自然科学版),2007,28(4):467-472.
[9]梅燚,侯雷平,崔艳玲,等.菠菜AFLP反应体系初探[J].华北农学报,2010,25(4):111-115.
[10]杨金华,邓传良,张准超,等.菠菜性别相关RAPD分子标记体系的建立与优化[J].江苏农业科学,2009,2:40-42.
[11]屠发志,彭世清.植物表观遗传与DNA甲基化[J].生物技术通讯,2007,18(5):155-158.
[12]陈秀莉,马利兵.DNA甲基化与基因表达调节[J].生物技术通讯,2010,5:7-10.
[13] Monk M. Epigenetic programing of differential gene in development and evolution [J]. De Gent, 1995, 17(3):188-197.
[14] Soares J, Pinto A E, Cunha C V, et al. Global DNA hypomethylation in breast carcinoma: correlation with prognostic factors and tumor ptogression [J]. Cancer, 1999, 85: 112-118.
[15] Kakutani T, Jeddeloh J.A , Flowera S.K et al . Developmental abnormalities and epimutations ass- ociated with DNA hypomethylation mutations [J]. Proc Natl Acad Sci USA , 1996 , 93 : 12406 -12 411
[16] Nakano Y, Steward N, Sekine M, et al. A tobacoo NtMET1 cDNA encoding a DNA methylthansferase: molecular characterization and abnormal phenotype of transgene to- bacoo plants [J]. Plant Cell Phy- siol, 2000, 41(4): 448-457.
[17] Burn J E, Bagnall D J, Metzger J D,et al. DNA mehylation,vernalization and the initiatio n of flower- ing[J]. Proc Natl Acad Sci USA, 1993, 90(1) : 287-291.
[18] Siemonsma, J.S., K. Piluek . Plant Resources of South-East Asia [M].Wageningen: Pudoc Scientific Publishers, 1993:266-268.
[19]金同铭.菠菜的营养与药用[J].蔬菜,2000,1:17-18.
[20]王迪轩,夏鹤高.介绍几个菠菜优良品种[J].北京农业:实用技术,2010,5:13-14.
[21]迪文.夏菠菜品种介绍[J].当代蔬菜,2004,4:8.
[22]孔庆凤,王进兴,张爱华.菠菜种子剥壳发芽技术[J].种子,2004,23(5):88.
[23]曹军.菠菜的栽培技术[J].青海农林科技,2005,1:67-68.
[24]胡正华,索福喜,赵晓莉,等.UV-B辐射增加与酸雨复合处理对菠菜种子萌发和幼苗生长的影响[J].中国农学通报,2005,21(6):284-285.
[25] Fang L, Shiliang L, Xiaolei J, et al. Influence of P and Cd Interaction on Spinach Growth and Uptake P and Cd in Calcareous Soil [J].Chinaese Agricultural Science Bulletin, 200 5, 21(4): 310.
[26]洪法水,王雪峰,刘超,等,Ce3+对菠菜D1/D2/Cytb559复合物影响的光谱研究[J].中国科学B辑:化学,2002,32(6):519-526.
[27] Komai F, Okuse I, Harada T. Somatic embryogenesis and plantregeneration in culture of root segmen- ts of spinach (Spinacia oleracea L.) [J]. Plant Sci, 1996, 113:203–208.
[28] Sasaki H. Callus and organ formation in tissue cultures of spinach (Spinacia oleracea L.) [J]. J. Japan Soc. Hort. Sci., 1989, 58: 149-153.
[29] Al-Khayri JM, Huang FH, Morelock TE, et al. Stimulation of shoot regeneration in spinach callus by gibberellic acid. [J].Hort. Sci, 1992, 27: 1046.
[30] T.R Shojaei,V. Salari,D Ramazan,et al.The effect of plant growth regulators, explants and cultivars on spinach (Spinacia oleracea L.) tissue culture [J]. African Journal of Biotechnology , 2010, 9(27):4179-4185.
[31]汪俏梅,寿森炎.蔬菜作物的性别分化及其在生产上的应用[J].植物学通讯,1999:411-416.
[32] Sherry R A, Eckard K J, Lord E M. Flower development in dioecious Spinacia oleracea (Chenopodia- ceae) [J]. Amer J Bot, 1993, 80: 283-291.
[33] Bemis W P, Wilson G B. A new hypothesis explaining the genetics of sex determination in Spinacea oleracea [J]. J.Hered, 1953, 44: 91-95.
[34] Khattak JZK, Torp AM, Andersen SB.A genetic linkage map of Spinacia oleracea and localization of a sex determination locus [J]. Euphytica, 2006, 148: 311-318.
[35] Jamilena M, Mariotti B, Manzano S. Plant sex chromosomes: molecular structure and function [J]. Cy- togenet Genome Res, 2008, 120: 255-264.
[36] Ellis J R, Janick J. The chromosomes of spinacia oleracea [J]. Am. J. Bot, 1960, 47: 210-214.
[37] Bose S, and Janick J. Karyo-races in Spinacia oleracea [J]. Am. J. Bot, 1961, 48:238-241.
[38] Muneo I, Janick J. Induced tetraploidy in philodendron [J]. J Hered, 1963 54: 37-38.
[39] Dressler, O. Erfarungen bei der Vermehrung and Züchtung mon?zischer Spinatsorten (Spinacia oleracea L.) [J]. Z PflZücht ,1973,70:108-128.
[40] Penel C L, Greppin H. Evolution of the auxin-oxidase and peroxidase activity during the spinach’s photoperiodic induction and sexualistion [J]. Plant Cell Physiol, 1972, 13(1): 151-156.
[41]曹宗巽,梅慧生,杨中汉,等.赤霉素和乙烯利对菠菜性别表现的控制及其与同工酶的关系[J].植物生理学报,1980,6(2):150-156.
[42]高武军,肖理会,卢龙斗,等.菠菜的性别相关同工酶标记分析[J].河南师范大学学报(自然科学版) ,2006,34(4):147-150.
[43] Gizawy A M, Oksh I, Sharaf A. Effect of gibberellic acid and Alar on flower and seed yield of spinach [J]. Egyptian Jour of Horticul, 1993, 19(2): 191-200.
[44] Chalakhyan M, Khrianin V N. Sexuality in Plants and Its Hormonal Regulation [M]. New York: Sprin- ger-Verlag, 1987: 48-58.
[45] Freeman D C, Vitale J J. The influence of environmant on the sex ratio and fitness of spinach [J]. Bot. Gaz, 1985, 146: 137-142.
[46] Buryanova Y , Shevchuk T. The use of prokaryotic DNA methyltransferases as experimental and ana- lytical tools in modern biology [J]. Anal Biochem, 2005, 338(1): 1-11.
[47] Finnegan EJ,Kovac KA,Plant DNA methyhransferases[J].Plant Mol Biol,2000, 43: 189-201.
[48] Kankel MW, Ramsey DE, Stokes TL, et al. Arabidopsis MET1 cytosine methyltransferase mutants [J]. Genetics, 2003, 163(3): 1109-1122.
[49] Finnegan EJ, Peacock WJ, Dennis ES. Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development [J].Proc Natl Acad Sci USA, 1996, 93(16): 8449?8454.
[50] Jacobsen SE, Meyerowitz EM. Hypermethylated SUPER- MAN epigenetic alleles in Arabidopsis [J]. Science, 1997, 277(5329): 1100-1103.
[51] Henikoff S, Comai L. A DNA methyltransferase homolog with a chromodomain exists in multiple polymorphic forms in Arabidopsis [J]. Genetics, 1998, 149(1): 307-318.
[52] Cao X, Springer NM, Muszynski MG, et al. Conserved plant genes with similarity to mammalian de novo DNA methyltransferases [J]. Proc Natl Acad Sci USA, 2000 97(9): 4979-4984.
[53]夏晗,刘美芹,尹伟伦,等.植物DNA甲基化调控因子研究进展[J].遗传,2008,30(4):426-432.
[54] Freitag M, Selke r EU . Controlling DNA methylation: many roads to one modification [J]. Curr Opin Genet Dev 2005,15(2), 191-199.
[55] Flavell,R.B. Inactivation of gene expression in plants as a on sequence of specific sequence duplicat- ion [J]. Proc,Natl,Acid,Sci ,1994, 91:3490-3496.
[56] Ronemus M,Galbiati M,Ticknor C, et al. Demethylation-induced developmental plieotropy in Arabid- opsis [J]. Science, 1996, 2:654-657.
[57] Lizal P, Relichova J. The effect of day length, vernalization and DNA demethylation on the flowering time in Arabidopsis thaliana [J]. Physiologia Plantarum,2001,113: 121-127.
[58] Sano H,Kamada I,Youssefian S, et al. A Single treatment of rice seedlings with 5-azacytidine induces heritable dwarfism and undermethylation of genomic DNA [J]. Mol Gen Genet,1990,220:441-447.
[59] King G J. Morphological development in brassica oleraceais modulated by in vivo treatment with5-azacytidine [J]. Jourm Horticul Sci, 1995, 70(2):333-342.
[60] Fieldes M.A, Schaeffer S.M, Krech M.J, et al. DNA hypomethylation in 5-azacytidine induced early- flwoering lines of flax [J]. Theor Appl Genet. 2005,111(1):136-149.
[61]汪炳良,李水凤,曾广文,等.5-azaC对萝卜茎尖DNA甲基化和开花的影响[J].核农学报,2005,19 (4):265-268.
[62]李梅兰,曾广文,朱祝军.5-氮杂胞苷促进白菜开花的效应分析[J].浙江大学学报(农业与生命科学版),2003,29:287-29.
[63] Horváth E , Szalai G, Janda T,et al . Effect of vernalization and 5-azacytidine on the methylation level of DNA in wheat ( Triticum aestivum L. , cv.Martonvasar15) [J]. Plant Science , 2003 , 165 : 689- 692
[64]陈芳.5-氮杂胞苷对小麦生长发育及DNA甲基化的影响[D].开封:河南大学,2009
[65] Finnegan E J, Genger R K, et al. DNA methylation and the promotion of flowering by vernalization [J]. Plant Biology, 1998, 95: 5824-5829
[66] Matzke M A, Mctte M F, et al. Transgene silencing by the host Genome defense: implification for the evolution of epigenetic control mechanisms in plants and vertebrates [J] . Plant Mol Biol , 2000, 43: 401-415
[67] King G J. Morphological development in brassica oleraceais modulated by in vivo treatment with 5- azacy tidine [J]. Jourm Horticul Sci, 1995, 70(2):333-342
[68] Jesse D. Hollister and Brandon S. Gaut1. Epigenetic silencing of transposable elements: A trade-off between reduced transposition and deleterious effects on neighboring gene expression [J]. Genome Res. 2009 19: 1419-1428
[69] Nan X et al. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex [J]. Nature1998,393:386-389
[70] Kapoor A, Agarwal M, Andreucci A, et al. Epigenetic transcriptional silencing and 5-Azacytidine mediated reactivation of a complex transgene in rice [J]. Plant Physiol, 1997, 115: 361-373.
[71] Buryanov Y.I,Shevchuk T.V. DNA methyltransferases and structural-functional specificity of eukar- yotic DNA modification [J]. Biochemistry (Moscow), 2005, 70: 730-742.
[72] Kinoshita T, Miura A, Choi Y, et al. One-way control of FWA imprinting in Arabidopsis endosperm by DNA methylation [J]. Science, 2004,303(5657): 521–523.
[73] Zhu Zhen,Woodlbury K,Huang L,.Effects of 5-azacytidine on transformation and gene expression in Nicotiana tabacum [J]. In Virtro Cell Dev Biol, 1991,27 :77-83
[74] Mittelsten Scheid O,Paszkowski J,Potrykus I,.Reversible inactivation of a transgene in Arabidopsis thaliana [J].Mol Gen Genet, 1991,228:104-112
[75] El-Maarri O.Becker T.Junen J Gender specific differences in levels of DNA methylation at selected loci from human total blood:a tendency toward higher methylation levels in males [J]. Human Genet, 2007, 122: 5055-514.
[76] Fuke C,Shimabukuro M,Petronis A,et al.Age related changes in 5-methylcytosine content in humanperipheral leukeeytes and placentas: an HPLC-based study [J]. Ann Hum Genet, 2004, 68(3): 196-204.
[77] Shimabukuro M.Sasaki T.Imamura A Global hypomethylation of peripheral leukocyte DNA in male patients with schizophrenia:a potentiallink between epigenetics and schizophrenia [J]. J Psychiatr Res. 2006,41(12):1042-1046.
[78]白小青,王金勇,陈英,等.荣昌猪仔猪性别间DNA甲基化水平的差异研究[J].遗传育种,2010,46(13):12-14. [79 ] Wenli Zhang, Xiue Wang, Qingyi Yu, et al. DNA methylation and heterochromatinization in the male-specific region of the primitive Y chromosome of papaya [J]. Genome Res. 2008, 18: 1938- 1943
[80] Jiri Siroky, Monica Ruffini Castiglione ,Boris Vyskot. DNA methylation patterns of Melandrium album chromosomes [J].Chromosome Research, 1998, 6, 441-446
[81] Bohuslav Janou?ek , Jiri Siroky , Boris Vyskot. Epigenetic control of sexual phenotype in a dioecious plant Melandrium album [J]. Mol Gen Genet , 1996, 250:483-490
[82] Kuo K.C, McCune R A, Gehrke C W, et al. Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA [J]. Nucleic Acids Res, 1980, 8: 4763-4776.
[83] Wu J, Issa J, Hermen J, et al. Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIN3T3 ceils [J]. Proc Natl Acad Sci USA, 1993, 90(19): 8891–8895.
[84] Oakeley E J, Podesta A, Jost J P. Developmental changes in DNA methylation of the two tobacco pollen nuclei during maturation [J]. Proc Natl Acad Sci USA, 1997, 94: 11721–11725.
[85] Oakeley E J, Schmitt F, Jost J P. Quantification of 5-methylcytosine in DNA by the chloroacetaldehyde reaction [J]. Biotechniques, 1999, 27(4): 744-6,748-50,752.
[86] Fraga M F, Uriol E, Borja D L, et al. High-performance capillary electrophoretic method for the qu- antification of 5-methyl 2-deoxycytidine in genomic DNA: application to plant, animal and human cancer tissues [J]. Electrophoresis, 2002, 23: 1677-1681.
[87] Oefner, P J, Bonn G K, Huber C G, et al. Comparative study of capillary zone electrophoresis and high-performance liquid chromatography in the analysis of oligonucleotides and DNA [J]. Chro- matogr, 1992, 625(2): 331-3401.
[88]房静远,萧树东,李蓉蓉,等.HPLC检测胃癌DNA甲基化的初步研究[J].中国肿瘤临床,1997,24(7):538-540
[89]钟兰,王建波.DNA超甲基化在小麦耐盐胁迫中的作用[J].武汉植物学研究,2007,25(1):102-104
[90]丁明丽.小麦成熟胚脱分化过程的DNA甲基化研究[D].郑州:河南农业大学,2008
[91]陆光远,伍晓明,等.油菜种子萌发过程中DNA甲基化的MSAP分析[J].科学通报,2005,50(24):2750-2756
[92] Epigenomics AG. Biocompare(http://www. biocompare.com), 2003
[93] Zhang X, Yazaki J, Sundaresan A , et al.Genome-wide High-Resolution Mapping and Functional Analysis of DNA Methylation in Arabidopsis [J]. Cell, 2006,126: 1189-1201
[94] Xiang H, Zhu JD, Chen Q, et al. Single base-resolution methylome of the silkworm reveals a sparse epigenomic map [J]. Nature Biotechnology , 2010,28(5): 516-U181
[95]谭建新,孙玉洁.表观基因组学研究方法进展与评价[J].遗传,2009,31(1):3-12
[96] Chen ZJ, Pikaard CS Epigenetic silencing of RNA polymerase I transcription: a role for DNA methy- lation and histone modification in nucleolar dominance [J]. Genes Dev 1997, 11:2124–2136
[97] Alleman M, Doctor J. Genomic imprinting in plants:observations and evolutionary implications [J]. Plant Mol Biol,2000, 43:147-161
[98]胡春燕,周建华.DNA甲基化修饰异常与肺癌[J].国际病理科学与临床杂志,2009,29(5):398-402
[99] Herman J G, Graff J R, Myohanen S, et al. Methylation-specific PCR: a novel PCR assay for methyl- tion status of CpG islands [J]. Proc Natl Acad Sci USA, 1996, 93(18), 9821 -9826.
[100]南楠,曾凡锁,詹亚光.植物DNA甲基化及其研究策略[J].植物学通报,2008,25(1):102-111
[101] Shiraishi M, Sekiguchi A, Oates A J, et al. Methyl-CpG binding domain column chromatography as a tool for the analysis of genomic DNA methylation [J]. Ana Biochem, 2004, 329(1): 1-10.
[102]聂丽娟,王子成.DNA甲基化抑制剂作用机理及其在植物发育生物学研究中的应用[J].核农学报,2007,21 (4):362～365
[103] Zakharov A F, Egolina N A. Differential spiralization along mammalian mitotic chromosomes. I. BUdR-revealed differentiation in Chinese hamster chromosomes [J]. Chromosoma, 1972, 38: 341-365
[104]李艳,梁前进,陶伟.伴随X染色体失活的甲基化修饰[J].高技术通讯,2007,17(12):1307-1311
[105] Tsaftaris AS, Kafka M. Mechanisms of heterosis in crop plants [M].J. Crop Prod,1998, 1: 95-111
[106] Xiong L Z, Xu C G, Saghai Maroof M A,et.al. Patterns of cytosine methylation in an elite rice hybrid and its parental lines detected by a methylation-sensitive amplification polymorphism technique [J]. Mol Gen Gen,1999, 261(3):439-446.
[107]仪治本,孙毅,牛天堂,等.高粱基因组DNA胞嘧啶甲基化在杂交种和亲本间差异研究[J] .作物学报,2005,31(9):1138-1143
[108]肖理彗,高武军,张小莉,等.葎草DNA的提取及AFLP标记反应体系的建立[J].分子植物育种,2008,6(2):393-398.
[109]姚焕玲.怀地黄转基因和组织培养品系鉴定和基因克隆[D].新乡:河南师范大学,2010.
[110]卢龙斗.木犀属微型态特征和系统发育研究(博士学位论文)[D].南京:南京林业大学,2007
[111]布朗著,魏群译.基因克隆和DNA分析(第5版)(中文版)[M].北京:高等教育出版社,2007
[112]赖国毅,陈超.SPSS17.0中文版常用功能与应用实例精讲[M].北京:电子工业出版社,2010
[113] GB/T 3543.4-1995,农作物种子检验规程发芽试验[S].北京:中国标准出版社,1996.
[114] Nelson, M. and M. McClelland. Site-specitic methylation:effect on DNA modification methyltrans- ferases and restriction endonucleases [J]. Nucleic Acid Res., (Suppl.), 198- 9 ,17: 389-415
[115] Mc Clelland, M., M. Nelson and E. Raschke. Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases [J]. Nucleic Acids Res., 1994, 22: 3640 -3659
[116] Nelson, M. and M. McClelland. Site-specific methylation: effect on DNA modification methyltrans- ferases and restriction endo nucleases [J]. Nucleic Acids Res., (Suppl.), 1991 ,19: 2045-2071.
[117] Demeulemeester AC,Van Stallen N. Degree of DNA methylation in chicory (Cichorium intybus L.): influence of plant age and vernalization [J]. Plant Science, 1999, 142: 101- 108.
[118] Debasis K.W,Yu I.C.Y. Detection of DNA methylation changes during somatic embryogenesis of Siberian ginseng(Elcuterococcns senticosus) [J]. Plant Science, 2003, 165: 61-68.
[119] Issa J.P,Ottavino Y.L, Celano P, et al . Methylation of the oestrogen receptor CpG Island links ageing and neoplasia in human colon [J]. Nature Genetics, 1994,7: 536-540.
[120] Messeguer R, Ganal M W, Steffens J C, et al. Characterization of the level, target sites and inheri- tance of cytosine methylation in tomato nuclear DNA [J]. Plant Mol Boil, 1991, 16(5): 753-770
[121] Lund G, Messing J, Viotti A. Endosperm specific demethylation and activation of specific alleles of alpha-tubulin genes of Zea may [J]. Mol Gen Genet, 1995, 246(6): 716-722
[122] Dhar M.S, Pethe V.V, Gupta V.S, et al. Predominance and tissue specificity of adenine methylation in rice [J]. Theor Appl Genet, 1990, 80(3): 402-408
[123]董亚娟.人参(Panax ginseng C.A.Meyer)年龄的鉴定研究[D].上海:复旦大学,2007
[124] Bird A .DNA methylation patterns and epigenetic memory.Genes Dev [J]. 2002,16: 6-21.
[125] Johnston J W, Harding K HPLC analysis of plant DNA methylation:a study of critical methodological factors [J].Plant Physiology and Biochemistry,2005(43):844-853
[126] Jorg Tost ,DNA methylatio Methods and Protocols [M].Humana Press,2009,23-34
[127] Chakrabarty D, Yu K W, Paek K Y. Detection of DNA methylation changes during somatic embryo- genesis of Siberian ginseng (Eleu-terococcus senticosus) [J]. Plant Sci, 2003, 165(1): 61-68
[128] C.W. Gehrke, R.A. McCune, M.A. Gamasosa, et al.Quantitative reversed-phase- high-performance liquid- chromatographyof major and modified nucleosides in DNA [J]. J. Chromatogr, 1984, 301: 199–219.
[129] BENDER J. DNA methylation and Epigenetics [J].Annual Review of Plant Biology, 2004,55:41-68
[130] Messagure R, Ganal MW, Steffens JC. Characterization of the level, target sites and inheritance of cytosine methylation in tomato nuclear DNA [J]. Plant Mol Biol 1991, 16, 753-770.
[131] Xiong LZ, Xu CG, Saghai-Maroof MA, et al.Patterns of cytosine methylation in an elite rice hybrid and its parental lines by a methylation-sensitive amplification polymorphism technique [J]. Mol Gen Genet 1999,261, 439-446.
[132] Ruiz-Gaarcia L,Cervera M T,Martinez-Zapater J M. DNA methylation increases throu-ghhout Arabi- dopsis Development [J]. Planta,2005,222:301-306
[133] Demeulemeester M A C, Stallen N V,Proft M P D. Degree of DNA methylation in chicory: influenceof plant age and vemalization [J].Plant Sci,1999,142:101-108.
[2]桂明珠,王承林,林金莲,等.菠菜(Spinacia oleracea Mill.)营养器官内部桔构的初步观察[J].东北农学院学报,1979,1:83-91.
[3] Lan T, Zhang S, Liu B, et al. Differentiating sex chromosomes of the dioecious Spinacia oleracea L. (spinach) by FISH of 45S rDNA [J]. Cytogenet Genome Res, 2006, 114(2): 175-177.
[4]吴爱忠,王玉林,林观捷,等.菠菜杂交种及其亲本品种的染色体核型分析[J].上海农学院学报,1993,11(1):1-6.
[5] Hiaga,T.Sex and chromosones in Spinacia oleracea L [J]. Jap.Jour. Genetics, 1935, 10:218-222.
[6]徐杰,吴燕燕.菠菜叶片中乙醇酸氧化酶3种同工酶的生化特性[J].中国生物化学与分子生物学报,2001,17(4):537-540.
[7] D.Noah.Sather,Amber York,Kevin J.Pobursky,et.al.Sequence evolution and sex-specific expression patterns of the C class floral indentity gene,SpAGAMOUS,in dioecious Spinacia oleracea L [J]. Planta,2005, 222:284-292
[8]丛娟,李晓玲,宫莉,等.菠菜基因组DNA提取方法与条件的研究[J].长春工业大学学报(自然科学版),2007,28(4):467-472.
[9]梅燚,侯雷平,崔艳玲,等.菠菜AFLP反应体系初探[J].华北农学报,2010,25(4):111-115.
[10]杨金华,邓传良,张准超,等.菠菜性别相关RAPD分子标记体系的建立与优化[J].江苏农业科学,2009,2:40-42.
[11]屠发志,彭世清.植物表观遗传与DNA甲基化[J].生物技术通讯,2007,18(5):155-158.
[12]陈秀莉,马利兵.DNA甲基化与基因表达调节[J].生物技术通讯,2010,5:7-10.
[13] Monk M. Epigenetic programing of differential gene in development and evolution [J]. De Gent, 1995, 17(3):188-197.
[14] Soares J, Pinto A E, Cunha C V, et al. Global DNA hypomethylation in breast carcinoma: correlation with prognostic factors and tumor ptogression [J]. Cancer, 1999, 85: 112-118.
[15] Kakutani T, Jeddeloh J.A , Flowera S.K et al . Developmental abnormalities and epimutations ass- ociated with DNA hypomethylation mutations [J]. Proc Natl Acad Sci USA , 1996 , 93 : 12406 -12 411
[16] Nakano Y, Steward N, Sekine M, et al. A tobacoo NtMET1 cDNA encoding a DNA methylthansferase: molecular characterization and abnormal phenotype of transgene to- bacoo plants [J]. Plant Cell Phy- siol, 2000, 41(4): 448-457.
[17] Burn J E, Bagnall D J, Metzger J D,et al. DNA mehylation,vernalization and the initiatio n of flower- ing[J]. Proc Natl Acad Sci USA, 1993, 90(1) : 287-291.
[18] Siemonsma, J.S., K. Piluek . Plant Resources of South-East Asia [M].Wageningen: Pudoc Scientific Publishers, 1993:266-268.
[19]金同铭.菠菜的营养与药用[J].蔬菜,2000,1:17-18.
[20]王迪轩,夏鹤高.介绍几个菠菜优良品种[J].北京农业:实用技术,2010,5:13-14.
[21]迪文.夏菠菜品种介绍[J].当代蔬菜,2004,4:8.
[22]孔庆凤,王进兴,张爱华.菠菜种子剥壳发芽技术[J].种子,2004,23(5):88.
[23]曹军.菠菜的栽培技术[J].青海农林科技,2005,1:67-68.
[24]胡正华,索福喜,赵晓莉,等.UV-B辐射增加与酸雨复合处理对菠菜种子萌发和幼苗生长的影响[J].中国农学通报,2005,21(6):284-285.
[25] Fang L, Shiliang L, Xiaolei J, et al. Influence of P and Cd Interaction on Spinach Growth and Uptake P and Cd in Calcareous Soil [J].Chinaese Agricultural Science Bulletin, 200 5, 21(4): 310.
[26]洪法水,王雪峰,刘超,等,Ce3+对菠菜D1/D2/Cytb559复合物影响的光谱研究[J].中国科学B辑:化学,2002,32(6):519-526.
[27] Komai F, Okuse I, Harada T. Somatic embryogenesis and plantregeneration in culture of root segmen- ts of spinach (Spinacia oleracea L.) [J]. Plant Sci, 1996, 113:203–208.
[28] Sasaki H. Callus and organ formation in tissue cultures of spinach (Spinacia oleracea L.) [J]. J. Japan Soc. Hort. Sci., 1989, 58: 149-153.
[29] Al-Khayri JM, Huang FH, Morelock TE, et al. Stimulation of shoot regeneration in spinach callus by gibberellic acid. [J].Hort. Sci, 1992, 27: 1046.
[30] T.R Shojaei,V. Salari,D Ramazan,et al.The effect of plant growth regulators, explants and cultivars on spinach (Spinacia oleracea L.) tissue culture [J]. African Journal of Biotechnology , 2010, 9(27):4179-4185.
[31]汪俏梅,寿森炎.蔬菜作物的性别分化及其在生产上的应用[J].植物学通讯,1999:411-416.
[32] Sherry R A, Eckard K J, Lord E M. Flower development in dioecious Spinacia oleracea (Chenopodia- ceae) [J]. Amer J Bot, 1993, 80: 283-291.
[33] Bemis W P, Wilson G B. A new hypothesis explaining the genetics of sex determination in Spinacea oleracea [J]. J.Hered, 1953, 44: 91-95.
[34] Khattak JZK, Torp AM, Andersen SB.A genetic linkage map of Spinacia oleracea and localization of a sex determination locus [J]. Euphytica, 2006, 148: 311-318.
[35] Jamilena M, Mariotti B, Manzano S. Plant sex chromosomes: molecular structure and function [J]. Cy- togenet Genome Res, 2008, 120: 255-264.
[36] Ellis J R, Janick J. The chromosomes of spinacia oleracea [J]. Am. J. Bot, 1960, 47: 210-214.
[37] Bose S, and Janick J. Karyo-races in Spinacia oleracea [J]. Am. J. Bot, 1961, 48:238-241.
[38] Muneo I, Janick J. Induced tetraploidy in philodendron [J]. J Hered, 1963 54: 37-38.
[39] Dressler, O. Erfarungen bei der Vermehrung and Züchtung mon?zischer Spinatsorten (Spinacia oleracea L.) [J]. Z PflZücht ,1973,70:108-128.
[40] Penel C L, Greppin H. Evolution of the auxin-oxidase and peroxidase activity during the spinach’s photoperiodic induction and sexualistion [J]. Plant Cell Physiol, 1972, 13(1): 151-156.
[41]曹宗巽,梅慧生,杨中汉,等.赤霉素和乙烯利对菠菜性别表现的控制及其与同工酶的关系[J].植物生理学报,1980,6(2):150-156.
[42]高武军,肖理会,卢龙斗,等.菠菜的性别相关同工酶标记分析[J].河南师范大学学报(自然科学版) ,2006,34(4):147-150.
[43] Gizawy A M, Oksh I, Sharaf A. Effect of gibberellic acid and Alar on flower and seed yield of spinach [J]. Egyptian Jour of Horticul, 1993, 19(2): 191-200.
[44] Chalakhyan M, Khrianin V N. Sexuality in Plants and Its Hormonal Regulation [M]. New York: Sprin- ger-Verlag, 1987: 48-58.
[45] Freeman D C, Vitale J J. The influence of environmant on the sex ratio and fitness of spinach [J]. Bot. Gaz, 1985, 146: 137-142.
[46] Buryanova Y , Shevchuk T. The use of prokaryotic DNA methyltransferases as experimental and ana- lytical tools in modern biology [J]. Anal Biochem, 2005, 338(1): 1-11.
[47] Finnegan EJ,Kovac KA,Plant DNA methyhransferases[J].Plant Mol Biol,2000, 43: 189-201.
[48] Kankel MW, Ramsey DE, Stokes TL, et al. Arabidopsis MET1 cytosine methyltransferase mutants [J]. Genetics, 2003, 163(3): 1109-1122.
[49] Finnegan EJ, Peacock WJ, Dennis ES. Reduced DNA methylation in Arabidopsis thaliana results in abnormal plant development [J].Proc Natl Acad Sci USA, 1996, 93(16): 8449?8454.
[50] Jacobsen SE, Meyerowitz EM. Hypermethylated SUPER- MAN epigenetic alleles in Arabidopsis [J]. Science, 1997, 277(5329): 1100-1103.
[51] Henikoff S, Comai L. A DNA methyltransferase homolog with a chromodomain exists in multiple polymorphic forms in Arabidopsis [J]. Genetics, 1998, 149(1): 307-318.
[52] Cao X, Springer NM, Muszynski MG, et al. Conserved plant genes with similarity to mammalian de novo DNA methyltransferases [J]. Proc Natl Acad Sci USA, 2000 97(9): 4979-4984.
[53]夏晗,刘美芹,尹伟伦,等.植物DNA甲基化调控因子研究进展[J].遗传,2008,30(4):426-432.
[54] Freitag M, Selke r EU . Controlling DNA methylation: many roads to one modification [J]. Curr Opin Genet Dev 2005,15(2), 191-199.
[55] Flavell,R.B. Inactivation of gene expression in plants as a on sequence of specific sequence duplicat- ion [J]. Proc,Natl,Acid,Sci ,1994, 91:3490-3496.
[56] Ronemus M,Galbiati M,Ticknor C, et al. Demethylation-induced developmental plieotropy in Arabid- opsis [J]. Science, 1996, 2:654-657.
[57] Lizal P, Relichova J. The effect of day length, vernalization and DNA demethylation on the flowering time in Arabidopsis thaliana [J]. Physiologia Plantarum,2001,113: 121-127.
[58] Sano H,Kamada I,Youssefian S, et al. A Single treatment of rice seedlings with 5-azacytidine induces heritable dwarfism and undermethylation of genomic DNA [J]. Mol Gen Genet,1990,220:441-447.
[59] King G J. Morphological development in brassica oleraceais modulated by in vivo treatment with5-azacytidine [J]. Jourm Horticul Sci, 1995, 70(2):333-342.
[60] Fieldes M.A, Schaeffer S.M, Krech M.J, et al. DNA hypomethylation in 5-azacytidine induced early- flwoering lines of flax [J]. Theor Appl Genet. 2005,111(1):136-149.
[61]汪炳良,李水凤,曾广文,等.5-azaC对萝卜茎尖DNA甲基化和开花的影响[J].核农学报,2005,19 (4):265-268.
[62]李梅兰,曾广文,朱祝军.5-氮杂胞苷促进白菜开花的效应分析[J].浙江大学学报(农业与生命科学版),2003,29:287-29.
[63] Horváth E , Szalai G, Janda T,et al . Effect of vernalization and 5-azacytidine on the methylation level of DNA in wheat ( Triticum aestivum L. , cv.Martonvasar15) [J]. Plant Science , 2003 , 165 : 689- 692
[64]陈芳.5-氮杂胞苷对小麦生长发育及DNA甲基化的影响[D].开封:河南大学,2009
[65] Finnegan E J, Genger R K, et al. DNA methylation and the promotion of flowering by vernalization [J]. Plant Biology, 1998, 95: 5824-5829
[66] Matzke M A, Mctte M F, et al. Transgene silencing by the host Genome defense: implification for the evolution of epigenetic control mechanisms in plants and vertebrates [J] . Plant Mol Biol , 2000, 43: 401-415
[67] King G J. Morphological development in brassica oleraceais modulated by in vivo treatment with 5- azacy tidine [J]. Jourm Horticul Sci, 1995, 70(2):333-342
[68] Jesse D. Hollister and Brandon S. Gaut1. Epigenetic silencing of transposable elements: A trade-off between reduced transposition and deleterious effects on neighboring gene expression [J]. Genome Res. 2009 19: 1419-1428
[69] Nan X et al. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex [J]. Nature1998,393:386-389
[70] Kapoor A, Agarwal M, Andreucci A, et al. Epigenetic transcriptional silencing and 5-Azacytidine mediated reactivation of a complex transgene in rice [J]. Plant Physiol, 1997, 115: 361-373.
[71] Buryanov Y.I,Shevchuk T.V. DNA methyltransferases and structural-functional specificity of eukar- yotic DNA modification [J]. Biochemistry (Moscow), 2005, 70: 730-742.
[72] Kinoshita T, Miura A, Choi Y, et al. One-way control of FWA imprinting in Arabidopsis endosperm by DNA methylation [J]. Science, 2004,303(5657): 521–523.
[73] Zhu Zhen,Woodlbury K,Huang L,.Effects of 5-azacytidine on transformation and gene expression in Nicotiana tabacum [J]. In Virtro Cell Dev Biol, 1991,27 :77-83
[74] Mittelsten Scheid O,Paszkowski J,Potrykus I,.Reversible inactivation of a transgene in Arabidopsis thaliana [J].Mol Gen Genet, 1991,228:104-112
[75] El-Maarri O.Becker T.Junen J Gender specific differences in levels of DNA methylation at selected loci from human total blood:a tendency toward higher methylation levels in males [J]. Human Genet, 2007, 122: 5055-514.
[76] Fuke C,Shimabukuro M,Petronis A,et al.Age related changes in 5-methylcytosine content in humanperipheral leukeeytes and placentas: an HPLC-based study [J]. Ann Hum Genet, 2004, 68(3): 196-204.
[77] Shimabukuro M.Sasaki T.Imamura A Global hypomethylation of peripheral leukocyte DNA in male patients with schizophrenia:a potentiallink between epigenetics and schizophrenia [J]. J Psychiatr Res. 2006,41(12):1042-1046.
[78]白小青,王金勇,陈英,等.荣昌猪仔猪性别间DNA甲基化水平的差异研究[J].遗传育种,2010,46(13):12-14. [79 ] Wenli Zhang, Xiue Wang, Qingyi Yu, et al. DNA methylation and heterochromatinization in the male-specific region of the primitive Y chromosome of papaya [J]. Genome Res. 2008, 18: 1938- 1943
[80] Jiri Siroky, Monica Ruffini Castiglione ,Boris Vyskot. DNA methylation patterns of Melandrium album chromosomes [J].Chromosome Research, 1998, 6, 441-446
[81] Bohuslav Janou?ek , Jiri Siroky , Boris Vyskot. Epigenetic control of sexual phenotype in a dioecious plant Melandrium album [J]. Mol Gen Genet , 1996, 250:483-490
[82] Kuo K.C, McCune R A, Gehrke C W, et al. Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA [J]. Nucleic Acids Res, 1980, 8: 4763-4776.
[83] Wu J, Issa J, Hermen J, et al. Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIN3T3 ceils [J]. Proc Natl Acad Sci USA, 1993, 90(19): 8891–8895.
[84] Oakeley E J, Podesta A, Jost J P. Developmental changes in DNA methylation of the two tobacco pollen nuclei during maturation [J]. Proc Natl Acad Sci USA, 1997, 94: 11721–11725.
[85] Oakeley E J, Schmitt F, Jost J P. Quantification of 5-methylcytosine in DNA by the chloroacetaldehyde reaction [J]. Biotechniques, 1999, 27(4): 744-6,748-50,752.
[86] Fraga M F, Uriol E, Borja D L, et al. High-performance capillary electrophoretic method for the qu- antification of 5-methyl 2-deoxycytidine in genomic DNA: application to plant, animal and human cancer tissues [J]. Electrophoresis, 2002, 23: 1677-1681.
[87] Oefner, P J, Bonn G K, Huber C G, et al. Comparative study of capillary zone electrophoresis and high-performance liquid chromatography in the analysis of oligonucleotides and DNA [J]. Chro- matogr, 1992, 625(2): 331-3401.
[88]房静远,萧树东,李蓉蓉,等.HPLC检测胃癌DNA甲基化的初步研究[J].中国肿瘤临床,1997,24(7):538-540
[89]钟兰,王建波.DNA超甲基化在小麦耐盐胁迫中的作用[J].武汉植物学研究,2007,25(1):102-104
[90]丁明丽.小麦成熟胚脱分化过程的DNA甲基化研究[D].郑州:河南农业大学,2008
[91]陆光远,伍晓明,等.油菜种子萌发过程中DNA甲基化的MSAP分析[J].科学通报,2005,50(24):2750-2756
[92] Epigenomics AG. Biocompare(http://www. biocompare.com), 2003
[93] Zhang X, Yazaki J, Sundaresan A , et al.Genome-wide High-Resolution Mapping and Functional Analysis of DNA Methylation in Arabidopsis [J]. Cell, 2006,126: 1189-1201
[94] Xiang H, Zhu JD, Chen Q, et al. Single base-resolution methylome of the silkworm reveals a sparse epigenomic map [J]. Nature Biotechnology , 2010,28(5): 516-U181
[95]谭建新,孙玉洁.表观基因组学研究方法进展与评价[J].遗传,2009,31(1):3-12
[96] Chen ZJ, Pikaard CS Epigenetic silencing of RNA polymerase I transcription: a role for DNA methy- lation and histone modification in nucleolar dominance [J]. Genes Dev 1997, 11:2124–2136
[97] Alleman M, Doctor J. Genomic imprinting in plants:observations and evolutionary implications [J]. Plant Mol Biol,2000, 43:147-161
[98]胡春燕,周建华.DNA甲基化修饰异常与肺癌[J].国际病理科学与临床杂志,2009,29(5):398-402
[99] Herman J G, Graff J R, Myohanen S, et al. Methylation-specific PCR: a novel PCR assay for methyl- tion status of CpG islands [J]. Proc Natl Acad Sci USA, 1996, 93(18), 9821 -9826.
[100]南楠,曾凡锁,詹亚光.植物DNA甲基化及其研究策略[J].植物学通报,2008,25(1):102-111
[101] Shiraishi M, Sekiguchi A, Oates A J, et al. Methyl-CpG binding domain column chromatography as a tool for the analysis of genomic DNA methylation [J]. Ana Biochem, 2004, 329(1): 1-10.
[102]聂丽娟,王子成.DNA甲基化抑制剂作用机理及其在植物发育生物学研究中的应用[J].核农学报,2007,21 (4):362～365
[103] Zakharov A F, Egolina N A. Differential spiralization along mammalian mitotic chromosomes. I. BUdR-revealed differentiation in Chinese hamster chromosomes [J]. Chromosoma, 1972, 38: 341-365
[104]李艳,梁前进,陶伟.伴随X染色体失活的甲基化修饰[J].高技术通讯,2007,17(12):1307-1311
[105] Tsaftaris AS, Kafka M. Mechanisms of heterosis in crop plants [M].J. Crop Prod,1998, 1: 95-111
[106] Xiong L Z, Xu C G, Saghai Maroof M A,et.al. Patterns of cytosine methylation in an elite rice hybrid and its parental lines detected by a methylation-sensitive amplification polymorphism technique [J]. Mol Gen Gen,1999, 261(3):439-446.
[107]仪治本,孙毅,牛天堂,等.高粱基因组DNA胞嘧啶甲基化在杂交种和亲本间差异研究[J] .作物学报,2005,31(9):1138-1143
[108]肖理彗,高武军,张小莉,等.葎草DNA的提取及AFLP标记反应体系的建立[J].分子植物育种,2008,6(2):393-398.
[109]姚焕玲.怀地黄转基因和组织培养品系鉴定和基因克隆[D].新乡:河南师范大学,2010.
[110]卢龙斗.木犀属微型态特征和系统发育研究(博士学位论文)[D].南京:南京林业大学,2007
[111]布朗著,魏群译.基因克隆和DNA分析(第5版)(中文版)[M].北京:高等教育出版社,2007
[112]赖国毅,陈超.SPSS17.0中文版常用功能与应用实例精讲[M].北京:电子工业出版社,2010
[113] GB/T 3543.4-1995,农作物种子检验规程发芽试验[S].北京:中国标准出版社,1996.
[114] Nelson, M. and M. McClelland. Site-specitic methylation:effect on DNA modification methyltrans- ferases and restriction endonucleases [J]. Nucleic Acid Res., (Suppl.), 198- 9 ,17: 389-415
[115] Mc Clelland, M., M. Nelson and E. Raschke. Effect of site-specific modification on restriction endonucleases and DNA modification methyltransferases [J]. Nucleic Acids Res., 1994, 22: 3640 -3659
[116] Nelson, M. and M. McClelland. Site-specific methylation: effect on DNA modification methyltrans- ferases and restriction endo nucleases [J]. Nucleic Acids Res., (Suppl.), 1991 ,19: 2045-2071.
[117] Demeulemeester AC,Van Stallen N. Degree of DNA methylation in chicory (Cichorium intybus L.): influence of plant age and vernalization [J]. Plant Science, 1999, 142: 101- 108.
[118] Debasis K.W,Yu I.C.Y. Detection of DNA methylation changes during somatic embryogenesis of Siberian ginseng(Elcuterococcns senticosus) [J]. Plant Science, 2003, 165: 61-68.
[119] Issa J.P,Ottavino Y.L, Celano P, et al . Methylation of the oestrogen receptor CpG Island links ageing and neoplasia in human colon [J]. Nature Genetics, 1994,7: 536-540.
[120] Messeguer R, Ganal M W, Steffens J C, et al. Characterization of the level, target sites and inheri- tance of cytosine methylation in tomato nuclear DNA [J]. Plant Mol Boil, 1991, 16(5): 753-770
[121] Lund G, Messing J, Viotti A. Endosperm specific demethylation and activation of specific alleles of alpha-tubulin genes of Zea may [J]. Mol Gen Genet, 1995, 246(6): 716-722
[122] Dhar M.S, Pethe V.V, Gupta V.S, et al. Predominance and tissue specificity of adenine methylation in rice [J]. Theor Appl Genet, 1990, 80(3): 402-408
[123]董亚娟.人参(Panax ginseng C.A.Meyer)年龄的鉴定研究[D].上海:复旦大学,2007
[124] Bird A .DNA methylation patterns and epigenetic memory.Genes Dev [J]. 2002,16: 6-21.
[125] Johnston J W, Harding K HPLC analysis of plant DNA methylation:a study of critical methodological factors [J].Plant Physiology and Biochemistry,2005(43):844-853
[126] Jorg Tost ,DNA methylatio Methods and Protocols [M].Humana Press,2009,23-34
[127] Chakrabarty D, Yu K W, Paek K Y. Detection of DNA methylation changes during somatic embryo- genesis of Siberian ginseng (Eleu-terococcus senticosus) [J]. Plant Sci, 2003, 165(1): 61-68
[128] C.W. Gehrke, R.A. McCune, M.A. Gamasosa, et al.Quantitative reversed-phase- high-performance liquid- chromatographyof major and modified nucleosides in DNA [J]. J. Chromatogr, 1984, 301: 199–219.
[129] BENDER J. DNA methylation and Epigenetics [J].Annual Review of Plant Biology, 2004,55:41-68
[130] Messagure R, Ganal MW, Steffens JC. Characterization of the level, target sites and inheritance of cytosine methylation in tomato nuclear DNA [J]. Plant Mol Biol 1991, 16, 753-770.
[131] Xiong LZ, Xu CG, Saghai-Maroof MA, et al.Patterns of cytosine methylation in an elite rice hybrid and its parental lines by a methylation-sensitive amplification polymorphism technique [J]. Mol Gen Genet 1999,261, 439-446.
[132] Ruiz-Gaarcia L,Cervera M T,Martinez-Zapater J M. DNA methylation increases throu-ghhout Arabi- dopsis Development [J]. Planta,2005,222:301-306
[133] Demeulemeester M A C, Stallen N V,Proft M P D. Degree of DNA methylation in chicory: influenceof plant age and vemalization [J].Plant Sci,1999,142:101-108.