稻曲病菌休眠与非休眠厚垣孢子的MSAP分析
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摘要
本研究以稻曲病菌Ustilaginoidea virens黄色(非休眠)厚垣孢子、黑色(休眠)厚垣孢子、厚垣孢子萌发后产生的分生孢子(非休眠)3种孢子为材料,采用DNA甲基化敏感扩增多态性技术对供试材料基因组DNA进行甲基化分析,探究稻曲病菌休眠与非休眠厚垣孢子DNA甲基化的水平和模式。结果显示,3种形态孢子的基因组DNA甲基化水平差异明显,分生孢子基因组DNA中CCGG序列的总甲基化率为20.56%,黑色休眠厚垣孢子为25.63%,黄色非休眠厚垣孢子为33.52%。黄色和黑色的厚垣孢子主要是全甲基化模式,其全甲基化率高于半甲基化率,其中黄色厚垣孢子全甲基化率高达17.68%;分生孢子则是以半甲基化模式为主。结果对揭示稻曲病菌厚垣孢子休眠的分子机制,探究真核生物孢子的分子休眠机制有普遍意义。
Methylation sensitive amplification polymorphism(MSAP) analysis was performed to profile the DNA methylation levels and pattern of three forms of dormant and non-dormant chlamydospores and conidia in Ustilaginoidea virens. The results showed that the difference of DNA methylation was obvious among them. The total methylation rate of CCGG sequence in genomic DNA of conidia was 20.56%; the black chlamydospores(dormant) was 25.63%, and the yellow chlamydospores(non-dormant) was 33.52%. The fully-methylated ratio of yellow chlamydospores was up to 17.68%, the highest among the two kinds of chlamydospores and conidia. The main pattern was full-methylation in dormant and non-dormant chlamydospores of U.virens, while conidia DNA was dominated by hemi-methylation pattern. Therefore,this study would be helpful for the understanding the molecular mechanism of dormancy in chlamydospores of U.virens. It has a universal and theoretical value for the study of the molecular mechanism of dormancy in eucaryotes.
Methylation sensitive amplification polymorphism(MSAP) analysis was performed to profile the DNA methylation levels and pattern of three forms of dormant and non-dormant chlamydospores and conidia in Ustilaginoidea virens. The results showed that the difference of DNA methylation was obvious among them. The total methylation rate of CCGG sequence in genomic DNA of conidia was 20.56%; the black chlamydospores(dormant) was 25.63%, and the yellow chlamydospores(non-dormant) was 33.52%. The fully-methylated ratio of yellow chlamydospores was up to 17.68%, the highest among the two kinds of chlamydospores and conidia. The main pattern was full-methylation in dormant and non-dormant chlamydospores of U.virens, while conidia DNA was dominated by hemi-methylation pattern. Therefore,this study would be helpful for the understanding the molecular mechanism of dormancy in chlamydospores of U.virens. It has a universal and theoretical value for the study of the molecular mechanism of dormancy in eucaryotes.
引文
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[19] ZEMACH A, MCDANIEL I E, SILVA P, et al. Genome-wide evolutionary analysis of eukaryotic DNA methylation [J]. Science, 2010, 328: 916-919.
[20] HONDA S, LEWIS Z A, HUARTE M, et al. The DMM complex prevents spreading of DNA methylation from transposons to nearby genes in Neurospora crassa [J]. Genes & Development, 2010, 24(5): 443-454.
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[23] IKEDA K, VAN VU B, KADOTANI N, et al. Is the fungus Magnaporthe losing DNA methylation?[J]. Genetics, 2013, 195: 845-855.
[24] JEON J, CHOI J, LEE G W, et al. Genome-wide profiling of DNA methylation provides insights into epigenetic regulation of fungal development in a plant pathogenic fungus, Magnaporthe oryzae [J]. Scientific Reports, 2015, 5:8567.
[25] JAROSLAV F, ALES K. How to interpret methylation sensitive amplified polymorphism (MSAP) profiles?[J]. BMC Genetics, 2014, 15:2. DOI: 10.1186/1471 -2156-15-2.
[26] NAKAYASHIKI H, IKEDA K, HASHIMOTO Y, et al. Methylation is not the main force repressing the retrotransposon MAGGY in Magnaporthe grisea[J]. Nucleic Acids Research, 2001, 29(6): 1278-1284.
[27] SALMON A, AINOUCHE M L, WENDEL J F,et al. Genetic and epigenetic consequences of recent hybridization and polyploidy in Spartina (Poaceae) [J].Molecular Ecology,2005,14:1163-1175.
[28] 李卫国, 常天俊, 龚红梅. MSAP技术及其在植物遗传学研究中的应用[J]. 生物技术, 2008, 18(1): 83-87.
[29] 黄韫宇, 张海军, 邢燕霞, 等. NaCl 胁迫对黄瓜种子萌发的影响及DNA甲基化的MSAP分析[J]. 中国农业科学, 2013, 46(8):1646-1656.
[30] MISHRA P K, BAUM M, CARBON J. DNA methylation regulates phenotype-dependent transcriptional activity in Candida albicans [J]. PNAS, 2011, 108(29): 11965-11970.
[31] 褚会娟, 张今今, 王喆, 等. 丹参MSAP分子标记技术体系的优化及其应用[J]. 西北植物学报, 2011(5): 920-928.
[32] SANTOS D, FEVERIRO P. Loss of DNA methylation affects somatic embryogenesis in Medicago truncatula [J].Plant Cell,Tissue and Organ Culture,2002,70(2):155-161.
[33] XIA Ran, WANG Junguo, LIU Chunyan, et al. ROR1/RPA2A, a putative replication protein A2, functions in epigenetic gene silencing and in regulation of meristem development in Arabidopsis [J].The Plant Cell,2006,18(1):85-103.
[34] 丛建民, 陈凤清, 沈海龙, 等. 水曲柳胚后熟时期MSAP分析[J].南京林业大学学报, 2015, 39(3): 39-44.
[2] 王金辉,陈越华.湖南省2004年中、晚稻稻曲病流行情况调查[J].中国植保导刊,2005,25(8):14-15.
[3] 姜慎,唐春生,谭志琼.国内外稻曲病研究现状[J].热带农业科学,2010,30(3):62-66.
[4] 邓根生.国内稻曲病研究现状[J].植物保护,1989,15(6):39-40.
[5] 吕世琼,刘浩,赵江林,等.稻曲菌素研究进展[J].中国农学通报,2010,26(14):265-268.
[6] SUN Xianyun, KANG Shu, ZHANG Yongjie, et al. Genetic diversity and population structure of rice pathogen Ustilaginoidea virens in China [J/OL].PLoS ONE,2013,8(9):e76879.
[7] GUO Xiaoyi, LI Yan, FAN Jing, et al. Progress in the study of false smut disease in Rice [J]. Journal of Agricultural Science and Technology, 2012:1211-1217.
[8] 刘安国,何念杰,汪金莲.稻曲病菌[Ustilaginoidea virens (Cooke) Takahashi]厚垣孢子发芽力的研究[J].江西农业大学学报,1989,11(3):29-34.
[9] 王疏,白元俊,周永力,等.稻曲病菌的病原学[J].植物病理学报,1998,28(1):19-24.
[10] 王国良.稻曲病菌厚垣孢子侵染时期和侵染途径的研究[J].植物保护学报,1992,19(2):97-100.
[11] 陆凡,陈志谊,陈毓苓,等.稻曲病菌的生物学特性及其侵染循环中某些未确定要点的研究[J].江苏农业学报,1996,12(4):35-40.
[12] 左广胜,冉西京,杜生茂,等.稻曲病菌初侵染源研究[J].中国农学通报,1996,12(5):17-18.
[13] SCHAEFER C B, OOI S K T, BESTOR T H, et al. Epigenetic decisions in mammalian germ cells [J]. Science, 2007, 316: 398-399.
[14] REIK W. Stability and flexibility of epigenetic gene regulation in mammalian development [J].Nature,2007,447: 425-432.
[15] TAMAME M, ANTEQUERA F, VILLANUEVA J R, et al. High-frequency conversion to a “fluffy” developmental phenotype in Aspergillus spp. by 5-azacytidine treatment: evidence for involvement of a single nuclear gene [J]. Molecular and Cellular Biology, 1983, 3(12): 2287-2297.
[16] ANTEQUERA F, TAMAME M, VILLANUEVA J R, et al. DNA methylation in the fungi [J]. The Journal of Biological Chemistry, 1984, 259(13): 8033-8036.
[17] DHILLON B, CAVALETTO J R, WOOD K V, et al. Accidental amplification and inactivation of a methyltransferase gene eliminates cytosine methylation in Mycosphaerella graminicola [J]. Genetics, 2010, 186: 67-77.
[18] LIU Siyang, LIN Jianqing, WU Honglong, et al. Bisulfite sequencing reveals that Aspergillus flavus holds a hollow in DNA methylation [J/OL]. PLoS ONE, 2012, 7(1): e30349.
[19] ZEMACH A, MCDANIEL I E, SILVA P, et al. Genome-wide evolutionary analysis of eukaryotic DNA methylation [J]. Science, 2010, 328: 916-919.
[20] HONDA S, LEWIS Z A, HUARTE M, et al. The DMM complex prevents spreading of DNA methylation from transposons to nearby genes in Neurospora crassa [J]. Genes & Development, 2010, 24(5): 443-454.
[21] LEWIS Z A, ADHVARYU K K, HONDA S, et al. DNA methylation and normal chromosome behavior in Neurospora depend on five components of a histone methyltransferase complex, DCDC [J/OL].PLoS Genetics,2010,6(11): e1001196.
[22] REYNA-LOPEZ G E, SIMPSON J, RUIZ-HERRERA J. Differences in DNA methylation patterns are detectable during the dimorphic transition of fungi by amplification of restriction polymorphism [J].Molecular Genetics and Genomics,1997,253:703-710.
[23] IKEDA K, VAN VU B, KADOTANI N, et al. Is the fungus Magnaporthe losing DNA methylation?[J]. Genetics, 2013, 195: 845-855.
[24] JEON J, CHOI J, LEE G W, et al. Genome-wide profiling of DNA methylation provides insights into epigenetic regulation of fungal development in a plant pathogenic fungus, Magnaporthe oryzae [J]. Scientific Reports, 2015, 5:8567.
[25] JAROSLAV F, ALES K. How to interpret methylation sensitive amplified polymorphism (MSAP) profiles?[J]. BMC Genetics, 2014, 15:2. DOI: 10.1186/1471 -2156-15-2.
[26] NAKAYASHIKI H, IKEDA K, HASHIMOTO Y, et al. Methylation is not the main force repressing the retrotransposon MAGGY in Magnaporthe grisea[J]. Nucleic Acids Research, 2001, 29(6): 1278-1284.
[27] SALMON A, AINOUCHE M L, WENDEL J F,et al. Genetic and epigenetic consequences of recent hybridization and polyploidy in Spartina (Poaceae) [J].Molecular Ecology,2005,14:1163-1175.
[28] 李卫国, 常天俊, 龚红梅. MSAP技术及其在植物遗传学研究中的应用[J]. 生物技术, 2008, 18(1): 83-87.
[29] 黄韫宇, 张海军, 邢燕霞, 等. NaCl 胁迫对黄瓜种子萌发的影响及DNA甲基化的MSAP分析[J]. 中国农业科学, 2013, 46(8):1646-1656.
[30] MISHRA P K, BAUM M, CARBON J. DNA methylation regulates phenotype-dependent transcriptional activity in Candida albicans [J]. PNAS, 2011, 108(29): 11965-11970.
[31] 褚会娟, 张今今, 王喆, 等. 丹参MSAP分子标记技术体系的优化及其应用[J]. 西北植物学报, 2011(5): 920-928.
[32] SANTOS D, FEVERIRO P. Loss of DNA methylation affects somatic embryogenesis in Medicago truncatula [J].Plant Cell,Tissue and Organ Culture,2002,70(2):155-161.
[33] XIA Ran, WANG Junguo, LIU Chunyan, et al. ROR1/RPA2A, a putative replication protein A2, functions in epigenetic gene silencing and in regulation of meristem development in Arabidopsis [J].The Plant Cell,2006,18(1):85-103.
[34] 丛建民, 陈凤清, 沈海龙, 等. 水曲柳胚后熟时期MSAP分析[J].南京林业大学学报, 2015, 39(3): 39-44.