| The DNA methylation phenomenon, which plays an important role in the regulation of gene expression is widely existed in eukaryotes. The DNA methylation status can be affected by many factors, such as temperature, salt stress, drought and waterlogging, light conditions, et al. When one of these conditions changes, or even plant disease and insect pests can make contributions to the convertion of plants' methylation status. In this study, we employed the MSAP (Methylation sensitive amplification polymorphism) technology to make an investigation on the methylation extents and patterns of Nicotiana tobacum L.cv. Honghuadajinyuan, which were planted in five different areas (Nanjian, YunLong, Weishan, Binchuan, Midu, respectively) of Dali county, YunNan province. All samples were collected in vegetative growth and reproductvie growth stage. After that, we also have recycled and then cloned some of the MSAP fragments which have showed significant polymorphism in the MSAP analysis into the pMD-19T simple vector. All of these clones were then sequenced in order to investigate the effects of multiple environment factors contributing to the methylation status of HongDa. According to ten pairs of different primers based on MSAP technology, the results have showed that:(1) This study employed the MSAP technology to investigate the methylation extents and patterns of two group samples (The4th piece and the10th piece of leaf from the same individual plant, separately) which were collected from five different areas in vegetative growth and reproductive growth stage, and424loci were created totally. We classified all of the bands we have generated by MSAP technology into three basic types---Type Ⅰ:non-methylation (E-H/E-M,+/+), Type Ⅱ: hemi-methylation (E-H/E-M,+/-), Type Ⅲ:full-methylation (E-H/E-M,-/+). In these three type band, type Ⅰ takes the predominant status (range from27.59%-58.25%), and type Ⅲ next to type Ⅰ(range from19.34-42.45%), type Ⅱ takes the minimum proportion(range from3.54%-14.86%).(2) In this study, the PCA and UPGMA analysis method were applied to investigate HongDa samples, the results showed us that the samples which were collected from different areas in vegetative growth stage have certain distinction: The samples come from NanJian and YunLong have a much similar methylation pattern, thus, they were much closer to each other in the PCA coordinate graphs, and they were also classified into one cluster in the UPGMA cluster figure. Analysis on the samples collected from WeiShan, BingChuan and MiDu in the same development stage have showed a similar result. These results implied that, in vegetative growth stage, the geogen has certain affects on the HongDa's methylation status, and these results have showed a high confidence level in this experiment.(3) After the reproductive growth stage, most of the samples collected from different areas have a methylation situation which spreading in the PCA coordinate graphs in a mess, this meant the samples'methylation difference in transition from reproductive growth stage to senescence stage have a trend to become more and more significant. The UPGMA analysis also showed a parallel result, therefore, these results have showed a high confidence level because both PCA and UPGMA analysis have reached to a same conclution. From these results, it can be infered that, the combination of environment difference, different development stages and long-term accumulated methylation difference may take responsibility for the great methylation difference between samples collected in the reproductive growth stage.(4) In this experiment, the leaf samples, which were collected in vegetative growth stage, have showed a regional-related methylation difference feature, while samples collected in reproductive growth stage have showed a much higher methylation difference level. The accumulation of methylation difference caused by mutiple factors, and the metabolic process engaged by different endophytes in different areas, may contribute to different chemical propotion and special quality of sense organ in tobaccos which were planted in different areas.(5) According to sequencing and blastn analysis, we have found that most of these bands which have showed significant polymorphism are highly homologous to some other methylation-sensitive related sequence in tobacco and other plants, and many of them have a close relationship to the environment factors, such as the temperature, metal iron stress and diease, et al. Another part of fragment sequences are homologous to some plant organelles, such as chloroplast and mitochondria.These results have proved our suspects:The methylation status of tobacco were affected by different area environments through some environment stimulus. The different development may also have a close relationship to the methylation status. and we believe that these results can provide some valuable assistance and reference to other related future research. |
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