关键词: 牡丹, DNA甲基转移酶, PsROS1, 赤霉素, DNA甲基化, 休眠解除

Abstract:

【Objective】 Application of exogenous gibberellin (GA) is a commonly method to break floral bud dormancy in the anti-season production of tree peony. However, the mechanism of how gibberellin plays a role in breaking dormancy of tree peony is an unsolved problem. The objective of this study is to clarify whether GA participates in dormancy regulation through epigenetic mode. 【Method】 In this study, 4-year-old ‘Luhehong’ was used as material, and 500 mg·L^-1 exogenous GA₃ was applied. Terminal buds were harvested and DNA was extracted at 0.5, 1, 3 and 5 d after GA₃ applied. The DNA of peony floral buds was extracted using CTAB method and DNA methylation level of floral buds was analyzed by HPLC technology. Three types of DNA methyltransferase genes, PsCMT, PsMET and PsDRM, and one DNA demethylase gene PsROS1 were obtained using RACE amplification based on transcriptome analysis. Bioinformatics method was performed to analyze gene sequences and possible domains, and MEGA 5.0 was used to construct phylogenetic tree. Using Actin as reference gene, the tissue expression characteristics and response to GA₃ of these genes were analyzed by qPCR method. 【Result】The open reading frame (ORF) of the three DNA methyltransferases (Dnmts) varies in length. The ORF length of PsCMT, PsMET and PsDRM is 1 118, 1 056 and 2 175 bp, respectively. The number of amino acids encoded is 372, 351 and 724, respectively. All three DNA methyltransferases have methyltransferase domains. The DNA demethylase gene PsROS1 has a long sequence with an ORF of 6 636 bp and encodes 2 211 amino acids, and there is a conserved DNA glycosylation domain in PsROS1. Phylogenetic analysis indicated that the PsCMT and PsDRM proteins in peony were closely related to the VvCMT2 protein of grape, PsMET and PsROS1 were grouped with most woody plants and located far away from tobacco. PsCMT, PsMET and PsDRM were expressed in all tissues (roots, stems, leaves, bracts, sepals, petals, stamens, carpels) at the early flowering stage of peony, and the expression level of PsCMT, PsMET and PsDRM was higher in the root of peony than that of other tissues, while the expression level of PsROS1 was the highest in carpels. Application of GA₃ dramatically accelerated buds sprouting and flowering. Bud sprouting could be observed 5 d after GA₃ application, and the germinating rate of bud was 97.5% with a 92.5% flowering rate after 60 d of GA₃ application. But in the control group, only a few buds germinated at 20 d, and the germinating rate was only 23.1% at 60 d. GA₃ significantly decreased the DNA methylation level of peony bud (P<0.01), from 38.9% before treatment to 28.7% at 5 d after treatment. GA₃ treatment increased the expression of PsCMT and PsROS1, decreased the expression level of PsDRM, but there was no significant difference in PsMET expression. 【Conclusion】GA₃ treatment induces PsROS1 expression and inhibits PsDRM expression, which leads to DNA hypomethylation and thus promotes dormancy release of peony, which may be an important way for gibberellin to play its role.

Key words: Paeonia suffruticosa, DNA methyltransferase, PsROS1, gibberellin, DNA methylation, dormancy release