| Peony(Paeonia lactiflora Pall.)is a kind of important perennial flowers,which belongs Paeonia of Paeoniaceae and forms the characteristics of the bud dormancy in winter in the long-term process of evolution.Therefore,cloning and studying the target genes is an important content to analyze the molecular mechanism of endo-dormancy release.Gibberellin is a kind of important plant hormones,which involved in regulating various developmental and physiological processes of plants,such as the germination of seeds and buds,the elongation of stems,the expansion of leaves,the growth of roots and development of flowers and fruits and so on.Previous studies have shown that GAs can replace low temperature to break dormancy completely or partly.GA20 ox is a key rate-limiting enzyme in gibberellin biosynthesis,which can regulate the biosynthesis of GAs.Therefore,it has great theoretical and practical significance to elucidate the relationship between GA20 ox and dormancy,which may provide a new strategy for breaking dormancy ahead.So far,the GA20 ox gene has not been isolated from P.Lactiflora.Therefore,the full-length cDNA sequence of PlGA20 ox gene was cloned based on transcriptome data,and emphasised on its expression pattern in the process of dormancy and identify function simply,in order to further reveal the biological function and lay foundations for clarifying the molecular mechanism of dormancy release.The main conclusions of this study are as follows:(1)The full-length cDNA sequence of PlGA20 ox gene(GenBank accession number:KU886552)in gibberellin biosynthesis pathway was cloned on the first time.It is 1351 bp in length,containing 1146 bp open reading frame(ORF)encoding 381 amino acids.(2)PlGA20ox is a kind of stable protein with a predicted molecular weight of 43209.1Da and belongs to C19-GAoxs without transmembrane domain or signal peptide.Amino acid sequence analysis indicated that PlGA20 ox possesses conserved 20G-Fe(II)-Oxy protein domain,highly conserved Fe2+ and 2-oxoglutaric acid binding sites.The homology between PlGA20 ox and GA20 ox from P.suffruticosa is as high as 96%,indicating the closest genetic relationship and the results indicated that GA20 ox gene is more conservative and consistent with the law of genetic evolution.(3)PlGA20ox was expressed in each organ of P.lactiflora but differentially.It was extremely high in buds,followed by petals,and weakly present in the leaves,stems,sepalsand roots.(4)The expression level of Pl GA20 ox showed an overall downward trend with an initial rise,which is positively correlated to endogenous GA3 content during bud endodormancy release induced by chilling.The exogenous GA3 could increase the endogenous GA3 content and inhibit the expression of PlGA20 ox significantly,indicating that PlGA20 ox can regulate the synthesis of GA3 meanwhile receive negative feedback regulation from active GA3 in plants.(5)Constructed an expression vector of pROKII-PlGA20ox-GFP and expressed in the leaves of Nicotiana benthamiana transiently.Subcellular localization showed that the PlGA20 ox protein was localized in the cytoplasm.(6)PlGA20ox transformed into Arabidopsis thaliana,and observed phenotype and determined the morphological indexes of transgenic plants.The transgenic plants exhibited an early bolting,increased height and improved vegetative growth.But it could not promote the germination of seeds obviously. |
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