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园艺学报 ›› 2015, Vol. 42 ›› Issue (10): 2015-2022.doi: 10.16420/j.issn.0513-353x.2015-0167

• 观赏植物 • 上一篇    下一篇

矮生观赏杉木DNA甲基化的水平与模式分析

刘琼瑶,黄华宏,冯惠平,楼雄珍*,童再康   

  1. 浙江农林大学林业与生物技术学院,亚热带森林培育国家重点实验室培育基地,浙江临安 311300
  • 出版日期:2015-10-25 发布日期:2015-10-25
  • 基金资助:

    国家‘863’计划项目(2011AA100203);国家自然科学基金项目(31300565);浙江省农业科技重点项目(2012C12908-11);
    浙江农林大学亚热带森林资源培育中心项目(CCSFR2013002)

Analysis of DNA Methylation Levels and Patterns in Dwarf Ornamental Cunninghamia lanceolata

LIU Qiong-yao,HUANG Hua-hong,FENG Hui-ping,LOU Xiong-zhen*,and TONG Zai-kang   

  1. College of Forestry and Biotechnology,Zhejiang Agriculture and Forestry University,Nurturing Station for State Key Laboratory of Subtropical Silviculture,Lin’an,Zhejiang 311300,China
  • Online:2015-10-25 Published:2015-10-25

摘要:

为探讨杉木矮化变异与DNA甲基化的关系,以矮生观赏杉木与野生杉木为试验材料,采用基于DNA甲基化敏感扩增多态性分析(Methylation-sensitive amplification polymorphism,MSAP)方法,研究其DNA 序列CCGG 位点的甲基化水平及模式变化特征。应用20个引物组合,在矮生观赏杉木和野生杉木的叶片DNA中均检测出745个CCGG位点,其中甲基化位点数分别为508个和505个,分别占总扩增位点数的68.17%和67.83%;在矮生观赏杉木与野生杉木木质部DNA中分别检测到742个和737个CCGG位点,其中甲基化位点数分别为471个与498个,分别占总扩增位点数的63.52%和67.51%,差异达极显著水平(P < 0.01)。与野生型相比,矮生观赏杉木叶片和木质部DNA甲基化模式发生了一定变化,在叶片DNA中,去甲基化率为17.81%,明显高于超甲基化率15.44%;在木质部DNA中,去甲基化率17.25%,也明显高于超甲基化率14.65%。通过甲基化序列的初步克隆及比对分析发现,矮生观赏杉木中参与MAPK级联途径的蛋白磷酸酶IBR5基因启动子区域的甲基化水平上升。因此推测,植物激素信号转导及其调控基因的甲基化变化可能是矮生观赏杉木形成的原因之一。

关键词: 杉木, 矮生变异, DNA甲基化, 甲基化敏感扩增多态性分析

Abstract:

In this study,methylation sensitive amplification polymorphism(MSAP)analysis was performed in dwarf ornamental Cunninghamia lanceolata(dwarf-type)and wild-type,to characterize the DNA methylation levels and patterns of CCGG sites in leaves and xylem. A total of 745 CCGG loci were both amplified in leaf of dwarf-type and wild-type using 20 primer combinations. And among them,508(68.17%)and 505(67.83%)loci were methylated respectively. While the primer combinations totally amplified 742 and 737 CCGG loci,respectively in the xylem of dwarf-type and wild-type. A 63.52% of loci were methylated in the xylem of dwarf-type,which was significantly less than that of wild-type(67.51%). It was found that the DNA methylation patterns changed in dwarf-type compared with wild-type. The hypermethylation rate was higher than hypermethylation rate both in leaves and xylem.Subsequent cloning and analysis of methylation DNA sequences showed that methylation level of IBR5 promoter sequence increased in dwarf-type. IBR5 is a phosphatase that modulates phytohormone signal transduction. Therefore,it was inferable that methylation of genes involved in phytohormone signal transduction could be a cause of dwarf-type formation.

Key words: Cunninghamia lanceolata, Chinese fir, dwarf variation, DNA methylation, methylation-sensitive amplification polymorphism(MSAP)

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