Cloning And Functional Identification Of Salix Viminalis Gibberellin Related Genes SvGID1s And SvIDD1

Willow is the collective name of all 500 kinds of willow plants,with wide ecological width and strong adaptability,it is an important native species in China and is widely used in landscaping,soil conservation,water pollution control and handicraft preparation.However,willow’s flocculation and dispersing powder problems seriously affect people’s production life.Studies have shown that gibberellin（GA）is involved in the flowering process of plants,but it has been less studied in woody plants.GID1 is the receptor of gibberellin,and the active GA first forms a GA-GID1 dimer with the receptor GID1,and then binds to DELLA protein to form a ternary complex after which DELLA is ubiquitinated and degraded by SCF E3 ubiquitin ligase to produce gibberellin effect in plants.IDD is a unique class of transcription factors in plants,and it has been reported that IDD can interact with DELLA protein to participate in gibberellin signaling to regulate plant flowering.Salix viminalis is an ideal material for studying floral bud differentiation in woody plants because of its fast growth rate,short growth period and small genome.Based on the results of transcriptome sequencing,we cloned SvGID1b,SvGID1c and SvIDD1 genes from S.viminalis and transformed Arabidopsis thaliana,and analyzed their functions as follows.1.A bioinformatic approach was used to construct a GID1 phylogenetic tree,and it was found that S.viminalis SvGID1s was most closely related to Salix suchowensis.SvGID1s had a tertiary structure consisting mainly ofαhelix,irregular coiling,a small amount ofβfolding and corner turning,and did not contain transmembrane structural domains.A study of the expression of SvGID1s at different developmental periods revealed that their expression levels were significantly higher at 16 and 24 weeks after bloom（WAB）than at other periods.The Phenotypic analysis showed that the phenotypes of SvGID1s overexpressing Arabidopsis differed from those of the wild type.The transgenic plants showed earlier shoot extraction time and flowering time than the wild type,smaller rosette leaf number and rosette leaf diameter than the wild type,and significantly longer hypocotyl length than the wild type,but no significant differences in fruit pod size and fruit pod spacing.The endogenous gibberellin content and the transcription level of GA3_OX1 gene related to gibberellin synthesis in transgenic Arabidopsis showed no significant changes.Compared with the wild-type,the expression levels of genes that promote flowering in transgenic Arabidopsis,such as FUL,SOC1,and FT,were upregulated,while the expression levels of genes that inhibit flowering,such as FLC and GAI,were downregulated.2.The phylogenetic tree of SvIDD1 was constructed by bioinformatics,and it was found that SvIDD1 was most closely related to Salix brachista,and the tertiary structure of SvIDD1mainly consisted ofα-helix,irregular curl,a small amount ofβ-fold and corner,and did not contain transmembrane structural domains.The expression of SvIDD1 at different developmental stages was found to be gradually increasing in S.viminalis leaves after flowering.Subcellular localization experiments revealed that SvIDD1 protein was located in the nucleus.Yeast two-hybrid assay demonstrated that SvIDD1 protein has transcriptional self-activating activity.Phenotypic analysis revealed that Arabidopsis overexpressing SvIDD1 gene had later flowering time than wild-type,shorter plant height than wild-type,elongated pistil,decreased number and diameter of rosette leaves,curled rosette leaves,decreased lignin content,and significantly lower expression of flowering-related genes FUL,KS,and FLY than wild-type.In summary,DELLA protein is a key factor in the negative regulation of flower formation in S.viminalis by the gibberellin pathway.The active gibberellin binds to the receptor GID1 to form a dimer,which in turn binds to DELLA to form a trimeric protein complex,the formation of which leads to the ubiquitination and degradation of DELLA protein,resulting in gibberellin effect in plants.The dimerization of SvIDD1 and SCL3 protein leads to the conformational change of SvIDD1 and the exposure of the C-terminus of the peptide chain,followed by the trimerization of DELLA protein with the C-terminus of SvIDD1,which inhibits the expression of gibberellin precursor synthesis genes and flowering-related genes by means of the transcriptional binding region of IDD1.