| Flowering which is the process of transformation of higher plants from vegetative growth to reproductive growth is a key developmental stage of plant development and future generations of breeding. This developmental process of change is decided by development condition of the plant itself and external environmental factors. Temperature is one of the important environmental factors affecting the flowering of plants.90%oilseed rape cultivated in China is winter oilseed rape. Winter oilseed rape at the seedling stage needs to undergo a period of low temperature period to flowering and fruiting, this developmental period which determined Brassica napus L the change of vegetative growth to reproductive growth called vernalization. Vernalization is very important in Brassica breeding and seed breeding production. Existing research results show that DNA demethylation has played an important role in plant vernalization. The research on DNA methylation and plant gene expression and regulation of the relationship is currently most focused on the model plant Arabidopsis. wheat, cabbage and other plant have also been reported in recent years. Plant DNA methylation was reduced and flowering was promoted in vernalization process.In this study, B. napus L Jinyu oil No.1used as experimental materials. Early spring B. napus L was sowed in different stages on the experimental fields to simulate the time of the low-temperature vernalization on rapeseed plant. Used flowering for the plant budding switched to reproductive growth and by vernalization standard to survey the ratio of plant vernalization. Detected the extent of genome-wide methylation by the methylation-sensitive amplified fragment polymorphism. Excel2010and Origin7.5of software were uesd to analysis the effects of the various periods by vernalization on plant genomic DNA methylation levels and patterns. At the same time, methylation inhibitor5-azacytidine treated B. napus L to assisted verification vernalization reduce the plant DNA methylation level. This study provided a theoretical basis for the promotion of flowering and artificial vernalization. The main results are reported as follows:(1) Different sowing of B. napus L whose length of vernalization time were different plant genomic DNA methylation levels were all lower, but the levels of DNA methylation reduced were different. The lower plant genome DNA methylation level, the higher the ratio of the plant by vernalization. Plant in the same sowing which passed the Vernalization genome DNA methylation levels(12.50%) were higher than plants which did not pass the Vernalization(13.38%).(2) B. napus L in the vernalization process demethylation was a major change of pattern, at the same time, small part of the DNA was methylated. The ratio of genomic DNA hemimethylated (7.76%to10.49%) were all higher than the ratio of complete methylation(2.47%to4.89%).(3) Different concentrations (5、10、50、100、150、250、300、500、1000μmol·L-1) of methylation inhibitor5-azacytidine treated B. napus L found that the high concentration of5-azacytidine severely inhibited plant height and root length. All treated cotyledon genomic DNA methylation level had varying degrees of reduction, but between each treated plant genome DNA methylation levels changed little and the difference of DNA methylation levels were not significant. Genomic DNA methylation level of the control group cotyledon was15.62%, genomic DNA methylation levels of the treated groups cotyledon were reduced35.3%、35.65%、35.65%、33.93%、34.63%、32.26%、31.95%、36.75%and40.59%respectively. Plant demethylation was a major change of pattern in each treatment group, the ratio of demethylation(10.48%to7.46%) were higher than the ratio of methylation(1.61%to4.48%).B. napus L in the vernalization process demethylation was a major change of pattern, at the same time, small part of the DNA was methylated. Different sowing of B. napus L, plant genomic DNA methylation levels were all reduced. The lower of plant genome DNA methylation levels, the higher the ratio of plant through vernalization. In the same sowing period, the plant which genomic DNA methylation level was12.50%could through the vernalization and which genomic DNA methylation level was13.38%could not through the vernalization. Deduce that B. napus L by reducing the level of genomic DNA methylation regulated the plant vernalization related gene expression in the vernalization process. When the plant genome DNA methylation level decreased to a certain level, such as12.50%, the plant can through the vernalization, on the contrary, the plant can not by vernalization. This study provided a theoretical basis and reference in promote early flowering technology of B. napus L and artificial vernalization. |
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