| Magnolia sieboldini is a small deciduous tree of the Magnoliaceae family,and it is a national key tertiary protected wild plant with extremely high ornamental and economic value.At present,Magnolia sieboldini is mainly propagated by seeding,and its propagation and promotion are limited due to the morphological and physiological dormancy characteristics of seeds.Group A PP2 C is an important negative regulator in the ABA signal transduction pathway,which is of great significance for revealing the mechanism of seed dormancy release.In this study,based on the transcriptome data of Magnolia sieboldini seeds in the group,three fulllength Magnolia sieboldini group A PP2 C genes(Ms AHG1,Ms AHG3,Ms HAB1)were obtained through gene cloning.The expression patterns of Ms AHG1,Ms AHG3 and Ms HAB1 genes in different tissues,during the process of seed imbibition and during the release of seed dormancy were explored by real-time quantitative PCR technology.The recombinant plant expression vector was constructed by single-enzyme cleavage homologous fusion method;The gene function was verified by subcellular localization and Arabidopsis genetic transformation,which provided a scientific basis for revealing the molecular mechanism of ABA signal transduction in the process of seed dormancy release in Magnolia sieboldini.The main results obtained are as follows:1.The genes of Ms AHG1,Ms AHG3 and Ms HAB1 were cloned successfully.The full length of the open reading frames were 1269 bp,1209 bp and 1695 bp,respectively,encoding 422,402 and 564 amino acids.Ms AHG1,Ms AHG3 and Ms HAB1 proteins are all nontransmembrane hydrophilic proteins,all have PP2 Cc domains,and belong to the PP2 Cc family.Homologous evolutionary tree showed that Ms AHG1,Ms AHG3 and Ms HAB1 of Magnolia sieboldini were closely related to Cm AHG1,Cm AHG3 and Cm HAB1 of Cinnamomum micranthum respectively.2.The expression pattern analysis of Ms AHG1,Ms AHG3 and Ms HAB1 genes showed that the expression of Ms AHG3 gene was the highest in dry seeds,which was significantly higher than that of Ms HAB1 and Ms AHG1 genes.During the imbibition process of Magnolia sieboldini seeds,Ms AHG1 and Ms AHG3 genes responded to the imbibition process.Compared with dry seeds,the relative expression of Ms AHG1 gene was higher than that of Ms AHG3 gene in imbibition 6-48 h,indicating that it plays a major regulatory role in the imbibition process.During the process of releasing the dormancy of Magnolia sieboldini,the gene expression levels of Ms AHG1,Ms AHG3 and Ms HAB1 all showed a trend of "first decreasing and then increasing",and all of them were the lowest at 16 d.The expression level of Ms AHG3 gene was higher than that of Ms AHG1 and Ms HAB1 in the whole process of dormancy release,indicating that it played a major regulatory role in the process of seed dormancy release.3.The p RI101-Ms AHG1-GFP,p RI101-Ms AHG3-GFP and p RI101-Ms HAB1-GFP plant expression vectors were successfully constructed.Nicotiana subcellular localization found that Ms AHG1,Ms AHG3 and Ms HAB1 were all localized in the nucleus,which was consistent with the predicted subcellular localization.4.By comparing the overexpression of Ms AHG1,restoration of Ms AHG1 function,and the germination rates of ahg1 mutants and Col-0,it was found that the transgenic seeds overexpressing Ms AHG1 were insensitive to ABA,while the ahg1 mutant was sensitive to ABA,while the transgenic seeds that complemented the function of Ms AHG1 could reduce the sensitivity to ABA.By comparing the germination rates of Ms AHG3 overexpression,ahg3 mutant and Col-0,it was found that Ms AHG3 overexpressed transgenic seeds were insensitive to ABA,while the ahg3 mutant was sensitive to ABA.By comparing the germination rates of Ms HAB1 overexpression,hab1 mutant and Col-0,it was found that Ms HAB1 overexpression transgenic seeds could slightly reduce the sensitivity to ABA,while the hab1 mutant was sensitive to ABA.Ms AHG1,Ms AHG3,Ms HAB1 genes all have the function of promoting the release of seed dormancy. |
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