Cloning Of MsGID1 Homologous Gene From Magnolia Sieboldii K.Koch And Transformation Into Arabidopsis Thaliana

The seed of Magnolia sieboldii K.Koch has the characteristic of deep physiological dormancy,which causes troubles for sowing and growing seeding.Based on the previous study of laboratory,exogenous gibberellin treatment can effectively relieve dormancy of M.sieboldii seed,in this study,the transcriptome of Magnolia sieboldii obtained by Illumina high-throughput sequencing as a reference,and starting from receptor genes?GIBBERELLIN INSENSITIVE DWARF1,GID1?of gibberellin signaling pathway,Ms GID1 homologous genes were obtain by cloning.On the basis,through real-time quantitative PCR,the effect of different temperatures?4 ?and 22??on the expression patterns of Ms GID1 homologous gene during seed imbibition were explored;and the expression patterns of Ms GID1 homologous gene after the seeds were soaked with GA₃ and PAC solution of different concentrations at 4 ? cold stratification.Simultaneously,the recombinant overexpression vector was successfully constructed by using double enzyme digestion method,and transformation of Ms GID1 homologous gene into Arabidopsis thaliana is accomplished by Agrobacterium-mediated inflorescence dip method.The paper provided a foundation for the gene function identification and a theoretical basis for removing seed dormancy by gibberellin treatment.The main results are as follows:1.Screening differential gene showed GID1 was a candidate gene.And The full-length sequences of gibberellin receptor genes,Ms GID1 b and Ms GID1 c on M.sieboldii are respectively 996 bp and 1 032 bp.They encode respectively 331 and 343 amino acids,and the sequence similarity is up to 84.88%.They are a unstable hydrophilic protein without signal peptide with 37.021 k D and 38.363 k D molecular weight respectively and belong to the ?/?-hydrolase fold superfamily by CDD Searcher in NCBI.Through multiple comparative analysis,the amino acid sequences of Ms GID1 b and Ms GID1 c contain 17 key sites,of which 15 are conserved.Among the 13 functional domains,only the TWVLIS domain in Ms GID1 b had amino acid substitution.These key sites and conserved sequences are helpful for GA recognition,ubiquitination of DELLA proteins,and GA signaling transmission.2.4? cold imbibition was more favorable for the gene expression of Ms GID1 b and Ms GID1 c in seeds of M.sieboldii,among which the relative expression level of Ms GID1 c was significantly higher than Ms GID1 b.It suggests that Ms GID1 c plays a major regulatory role in seed dormancy-breaking process.3.During 4? cold stratification,exogenous GA₃ solution can increase the content of endogenous GA₃.It is known that Ms GID1 b and Ms GID1 c was positively regulated with increase of GA₃ solution concentration;after 49 d at 4 ? cold stratification,the seed germination rate increased by 10%,22% and 44% compared with that of water treament.Furthermore,with the increase of PAC solution concentrations,Ms GA20OX6?GA 20-oxidase 6?,Ms GID1 b and Ms GID1 c were significantly regulated,while the relative expression of Ms RGL2?RGA-LIKE 2?was not remarkably changed.It is worth nothing that the seeds treated by PAC did not germinate after 49 d stratification.In conclusion,It is proved that exogenous gibberellin hormone treatment increases the content of endogenous gibberellin hormone in seeds at cold stratification process,positively regulates the expression of GA receptor GID1 s,promotes GA-GID1 complex to combine DELLA proteins,even destructed the GID1-GA-DELLA complex through the ubiquitin-proteasome pathway,accelerates the GA hormone signal transmission process,finally breaks dormancy and promotes M.sieboldii seed germination.4.In the study,Ms GID1 b and Ms GID1 c were respectively constructed into p RI–GUS?Ca MV35S?and named them p RI-GUS-Ms GID1 b and p RI-GUS-Ms GID1 c.Subsequently,to lay the foundation for subsequent verification of gene function,they were respectively transferred into wild Arabidopsis thaliana?WT?and T0 generation seeds were obtained.