Biological Function Analysis Of Gibberellin Synthesis Gene MtGA3ox1 In Medicago Truncatula

With the completion of genome sequencing,Medicago truncatula has become a model plant for studying the gene function of legumes.In the current study,three mutant lines of MtGA3ox1 was identified by forward genetics screening and reverse genetics screening,and the molecular identification and functional analysis of the mutant materials were performed.Proteomics and metabolomics were carried out to analyze the potential molecular regulation mechanism of gibberellin and MtGA3xo1,and analyze the downstream pathway of gibberellin regulation.To provide a new idea for exploring the role of the gibberellin in legumes.The main results are as follows:The flank sequence analysis of mutant lines showed that Tnt1 insertion were located in the first exon of the MTR₂g102570 gene at 149 bp,and 416 bp,downstream of the ATG start code in NF13294 and NF 18131,as well as located at 16 bp downstream of the first exon in NF12434 in the dwarf mutant lines in Medicago truncatula.The MTR₂g102570 gene belongs to the GA3 oxs family and is responsible for encoding MtGA3ox1,which is mainly involved in the synthesis of GA1 and GA4.HPLC-MS was performed and the contents of GA3 and GA4 the ga3ox1 mutant were significantly lower than those of R108.The ga3ox1 mutant was lower than R108 in root length,petiole length,aboveground biomass and leaf and petiole cell size,while chlorophyll content?chlorophyll a,chlorophyll b and carotenoid?was higher than R108.MtGA3ox1 is mainly expressed in flowers and pods,and the relative expression levels are from high to low: fruit pod > flower > root > leaf > stem.Mt GA3ox1 expression was affected by other plant hormones and the external environment.Low temperature,ABA,IAA and salicylic acid treatment inhibited MtGA3ox1 expression,while dark treatment induced MtGA3ox1 expression.Histochemical staining showed that the length of the promoter determines GUS activity,and it has strong GUS activity in shoot tip and hypocotyl.In addition,exogenous gibberellins treatment?GA1,GA3,GA4,and GA7?restored the ga3ox1 mutant dwarf phenotype.Similarly,the dwarf phenotype and seed coat color were restored in overexpressing transgenic lines and the complementary transgenic lines in ga3ox1 mutant.The fresh weight,root length and number of lateral roots of over-expression MtGA3ox1 lines in Arabidopsis thaliana were significantly higher than that in Col-0.Through proteomics and metabolomics analysis,456 differentially enriched proteins?including 238 up-regulated and 218 down-regulated?and 131 differential metabolites?including 53 up-regulated and 78 down-regulated?were identified.The up-regulated proteins were mainly participated in flavonoid synthesis,isoflavone synthesis and lignin synthesis.The down-regulated proteins were mainly participated in photosynthesis,oxidative phosphorylation and nitrogen metabolism.While the differential metabolites were mainly involved in phenylpropanoid synthesis and isoflavone synthesis.A combination of proteomics and metabolomics showed that 36 metabolic pathways were co-enriched,including phenylpropanoid synthesis,flavonoid synthesis and isoflavone synthesis.Correlation analysis between differential enriched proteins and differential metabolites showed that a positive correlation was exerted between proteins and metabolites,which mainly concentrated on the interaction of flavonoids and isoflavone metabolites with proteins.In this study,the protein level and transcription level of anthocyanin synthesis pathway genes and nitrogen metabolism and transport genes were analyzed,which provided a reasonable explanation for the involvement of gibberellin in negative regulation of the anthocyanin synthesis pathway and positive regulation of nitrogen metabolism and nitrogen transport by mediated ubiquitination of DELLA protein.Furthermore,gibberellin may participate in the negative regulation of isoflavone synthesis and flavone and flavonol biosynthesis.