Study On MaUGT79 Involving In Scopolin Biosynthesis And Drought Stress In Melilotus Albus

Drought is one of the major environmental factors that restrict global agricultural production.To cope with drought stress,plants have evolved the capacity to produce an impressive arsenal of stress-protective secondary metabolites.As a glycosylated coumarin,scopolin is a common secondary metabolite in plants and the formation is mediated by UDP-glucosyltransferases（UGTs）in plants.However,the relationship between scopolin content and drought tolerance remains unclear.Melilotus albus is a diploid legume herb and excellent rotation crop that can be used as forage,green manure and nectar source,and it is drought tolerate.However,the scopolin biosynthesis gene and the molecular mechanism of whether it enhances the drought tolerance by regulating the accumulation of scopolin is still unknown.In this study,the identification,drought tolerance of MaUGT79 and the transcriptional regulation by MaMYB4 and MabHLH11 related to scopolin biosynthesis in M.albus were studied by BSA,overexpression,RNA interference and other technics.It is of great significance to identify the related UGT gene in M.albus for understanding the biosynthesis of scopolin and the molecular mechanism of the strong drought tolerance of M.albus.The main results are as follows:1.A total of 189 MaUGT genes were identified from the M.albus genome,which were distributed differentially among its 8 chromosomes.According to the phylogenetic relationship,MaUGTs could be classified into 13 major groups,of which group E is the largest.A total of 76 MaUGT genes contained intron sequences,of which I-4（intron 4）was highly conservative.MaUGT gene family members had tissue-specific expression patterns.Sixteen MaUGT genes were differentially expressed between genotypes of JiMa46 and JiMa49.About 73.55%and 66.67%of the MaUGT genes were differentially expressed under abiotic stress in the shoots and roots,respectively.These results indicated that most MaUGT genes may be involved in the growth and development,biosynthesis of secondary metabolites and response to abiotic stress of M.albus.2.MaUGT79 was mapped as a key enzyme gene of scopolin biosynthesis.Based on the bulk segregant analysis sequencing（BSA-seq）,the scopolin biosynthesis gene was mapped to 67,539,223 bp to 67,540,565 bp region on chromosome 5,which was an UDP-glucosyltransferase enzyme coding gene MaUGT79.And 6 SNP and a neighbourinig In Del of 10 bp in the CDS of MaUGT79 can cause frameshift mutations that lead to premature termination of protein translation.MaUGT79 gene was differentially expressed in roots of JiMa46 and JiMa49.The tobacco subcellular location analysis showed that MaUGT79 protein was localized in the cytoplasm and nucleus.The recombinant MaUGT79 protein can catalyze the formation of scopolin using UDP-glucose as a sugar donor,and scopoletin as the possible substrate in vitro.We found that the scopolin accumulation in MaUGT79-overexpressed（OE-MaUGT79）transgenic hairy roots is much higher than that in control,and the scopolin content showed a significant reduction in MaUGT79-RNAi transgenic hairy roots.Overexpression of MaUGT79 significantly decreased MDA,H₂O₂ and O₂^-contents,increased scopolin content,up-regulated the expression levels of dought related marker genes in transgenic hairy roots under drought stress,and the drought tolerance of M.albus plants with transgenic roots was improved.It has been confirmed that MaUGT79mediates scopolin biosynthesis of M.albus and improves plant drought tolerance by removing reactive oxygen species under drought stress.3.MaMYB4 transcriptionally up-regulated MaUGT79 was involved in modulating scopolin biosynthesis and improving plant drought tolerance.A large number of MYB binding sites in the promoter region were found.The MaMYB4transcription factor of M.albus was identified by bioinformatics analysis.The tobacco subcellular location analysis showed that MaMYB4 was localized in the nucleus.The expression of MaMYB4 was induced by drought treatment.Yeast one-hybrid assay verified that transcription factor MaMYB4 could directly bind to the-1609 to-1201 bp of MaUGT79 promoter,and dual-luciferase reporter assay further verified that MaMYB4 could activate the expression of MaUGT79.The expression level of MaUGT79 and scopolin content significantly increased in MaMYB4-overexpressed（OE-MaMYB4）transgenic hairy roots in comparison to the control.Overexpression of MaMYB4 significantly decreased MDA,H₂O₂ and O₂^-contents,increased the expression of MaUGT79 under drought stress,and the drought tolerance of M.albus plants with transgenic roots was improved.While the MaMYB4-RNAi transgenic hairy roots showed the opposite trend.Herefrom,we preliminarily analyzed the mechanism of MaMYB4 binding to the promoter of MaUGT79 to up-regulate the transcription level of MaUGT79,and then positively regulate scopolin biosynthesis and plants drought tolerance.4.A MabHLH11 transcription factor interacted with MaMYB4 involved in socopolin accumulation.The tobacco subcellular location analysis showed that MabHLH11 was localized in the nucleus.The expression of MabHLH11 was also induced by drought treatment.The yeast two-hybrid and bimolecular fluorescence complementation assays verified that MaMYB4 could interact with MabHLH11.The dual-luciferase reporter assay and co-overexpression of MaMYB4 and MabHLH11 in M.albus showed that both MabHLH11 and MaMYB4 could activate the expression of MaUGT79,and the interaction between MabHLH11 and MaMYB4 additively increased the expression of MaUGT79 and induced scopolin accumulation compared with that under the expression of MabHLH11 or MaMYB4 alone.In summary,this study identified a MaUGT79 gene,and analyzed the molecular mechanism of MaUGT79 involved in scopolin biosynthesis and response to drought stress.The transcription of MaUGT79 was demonstrated to be directly and positively regulated by MaMYB4,which is a key integrator of both scopolin biosynthesis and drought tolerance.In addition,it was found that MabHLH11 interacts with MaMYB4and regulate the scopolin biosynthesis.This study provides new insights into the regulatory mechanism for plant growth adaption to drought stress through accumulation of scopolin and important theoretical basis and genetic resources for molecular improvement and germplasm innovation of M.albus.