| DNA methylation,an important form of biological epigenetic modification,refers to a methyl group covalent modification which occurs on part of the cytosine bases in the DNA sequence.DNA methylation exists universally in animals, plants and fungi,playing an important role in eukaryotic gene expression,cell differentiation and development.Yunnan is the largest province in the production of tobacco; Yunnan tobacco occupies a decisive positionthroughout the whole nation.The flue-cured tobacco Nicotiana tabacum L. var. Honghuadajinyuan,is a main cultival in Yunnan because of its superior quality,strong resistance,low fertilizer requriement and eurytopicity.In this study,we used Nicotiana tabacum L. var. Honghuadajinyuan planted in two counties of Dali,Yunnan province,including Weishan(25'13'37.96"N,100'18'25.83"W,2061m above the sea level),and Yunlong (25'53'08.38"N,99'122'15.77"E,1957m above the sea level),to investigate the level of DNA methylation.By using MSAP technology we studied the DNA methylation level and change mode of the tested tobacco in the five developmental stages.The research results are summarized as follow.The MSAP result showed that DNA methylation level was dynamically changed in seedling resuscitation period, rosette stage,strengtrly growing stage,flower budding stage and mature stage.It also showed that CCGG locus is mostly double-strand methylated in internal cytosines (CmCGG) in every growth stage.Among the five stages,the DNA methylation level of the tenth leave was the lowest in flower budding stage.It might be related to the change of growth mode in this stage.According to the PCA analysis,in the vegetative growth period (seedling resuscitation period,rosette stage and strengtrly growing stage),DNA methylation difference is relatively small or even there is no difference in the same growth stage of the tobacco planted in the same area.However,during the flower budding stage,when vegetative growth changed to reproduction growth,methylation levels were different between the fourth and the tenth leaves.The most different of methylation in the maturity stage of the tobacco leaf development indicated the relationship of methylation and growth.In the mean time,the methylation level changed dromatically between two planting area,which indicated that environmental factors cannot be ignored as for the pattern of gene expression for tobacco leaves.It coincided with the fact that the tobacco products from different areas have various style characteristics.During developmental stages,in addition to the difference among DNA methylation level,DNA methylation patterns differences occurred in "CCGG" locus between two adjacent stages,including monomorphism (2kinds) and polymorphism(12kinds).It also showed that polymorphism was significantly higher than other stage from strengtrly growing stage to the flower budding stage and the flower budding stage to mature stage,which may be related to the development changed during these two stages.The results indicated the polymorphic loci may be involved in developmental and physiological changes.Methylation pattern were more complicated.During the overall development,there were methylation and demethylation, which may related to some repeat change factors.The research of methylation pattern of tobacco leaves showed47%methylation sites may involved in the normal development and functional representation. In the mean time,53%may related to environmental factors and Endosymbiont bacteria metabolism.The results revealed the pattern of DNA methylation and the complexity of methylation regulation,which can provides evidence for the epigenetics and the regulation mechanism of specific gene expression in Nicotiana tabacum L. var. Honghuadajinyuan for further study. |
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