| Diosgenin is the main active compound in Dioscorea zingiberensis.It can be used to synthesize a variety of steroid hormone drugs,and the demand is second only to antibiotics in the world.Extracting and separating from plants is the only way to obtain diosgenin.It inevitably leads to the waste of plant resources and environmental pollution.The use of microbial heterologous expression of plant secondary metabolism synthesis pathway can produce a large number of products in a short time,which has become an effective means to replace traditional extraction and chemical synthesis.However,this technology is based on the full understanding of the metabolite synthesis pathway.Diosgenin is mainly synthesized by the cytoplasmic mevalonate pathway(MVA),possibly with the participation of the plastid methylerythritol phosphate pathway(MEP),but its downstream synthesis process is still unclear.At present,the genome of Dioscorea zingiberensis has not yet been obtained.In this paper,the key enzyme genes squalene epoxidase(SE),cycloartenol synthase(CAS)and 1-deoxy-D-xylulose-5-phosphate reductoisomerase(DXR)of MVA and MEP pathways were selected,and the gene functions were studied by gene cloning,subcellular localization,prokaryotic expression,enzyme activity analysis and genetic transformation,providing a theoretical basis for the downstream pathway analysis and metabolic regulation of diosgenin synthesis.The DzSE gene was cloned from Dioscorea zingiberensis using RACE technology,and the bioinformatics analysis was performed with known DzCAS and DzDXR.DzSE,DzCAS and DzDXR encode 514,759 and 469 amino acids,respectively.The secondary structure predicts that the three proteins have fewer flexible regions,almost consisting of ?-helix and ?-sheet.Through the comparative analysis of tertiary structures,the catalytic and binding sites coincide with the positions of homologous proteins,indicating that these three proteins are highly conserved in the evolutionary process.Subcellular localization results showed that the fluorescence signal of MVA pathway 3-hydroxy-3-methylglutaryl coenzyme A reductase(DzHMGR),squalene synthase(DzSS),DzSE,DzCAS fused with GFP were overlapped with the endoplasmic reticulum.Farnesyl diphosphate synthase(DzFPPS)was distributed in the cytoplasm,while DzDXR and 2-C-Methy-D-erythritol2,4-cyclodiphosphate synthase(DzMDS)in MEP pathway were localized in the chloroplast.This result indicates that the endoplasmic reticulum is critical for the diosgenin synthesis and may provide a venue for multi-step oxidation modification reactions.The tissue expression pattern of the gene was analyzed by real-time quantitative PCR.The results showed that three genes had the highest expression in the leaves,followed by the stem,and DzDXR was hardly expressed in the rhizomes.The content of diosgenin and gene expression in different tissues at different growth stages were combined for comprehensive analysis.The result showed that the expression of DzSE and DzCAS was positively correlated with the change in saponin content.We speculate that diosgenin is mainly synthesized in leaves,and then transported to rhizomes via stems after connecting glycosyl groups,and the rhizomes also have the ability to synthesize diosgenin.MBP-fused protein DzSE,DzCAS and DzDXR were successfully expressed by prokaryotic vector p Mal-c2 x,with sizes of 98.1 KDa,129.3 KDa and 93.3 KDa,respectively.The enzyme activity analysis showed that DzSE was able to catalyze the synthesis of 2,3-oxidosqualene from squalene in vitro.This reaction also requires the auxiliary participation of NADPH and P450 cytochrome reductase(DzCPR).The DzCAS protein was added in the reaction subsequently,but the formation of cycloatenol was not detected.This indicates that prokaryotic expression is unable to maintain the biological activity of certain eukaryotic genes.In addition,diosgenin polyclonal antibodies were prepared to study the distribution and content differences in the tissues of Dioscorea zingiberensis.The titer of the antibody reached 1:16000 and the sensitivity was 63.43 ng/m L.The content of diosgenin in leaves was significantly higher than rhizomes,which proved that leaves were the main organ for synthesizing diosgenin.At the cellular level,the content of furostanol saponins were significantly higher than spirosteroidal saponins,and spirosteroidal saponins mainly existed in vacuoles and vesicles.It is speculated that furostanol saponins may be converted into spirosteroidal saponins in the cytoplasm and transported to vacuoles for storage by intracellular vesicles.The DzSE,DzCAS and DzDXR genes were transformed into fenugreek mediated by Agrobacterium,and the corresponding over-expressed plants were obtained.The metabolite differences caused by gene transformation were detected through mass spectrometry.Among the three gene overexpressed strains,the content of sterols such as cholesterol,campesterol,sitosterol,stigmast-5-ene and diosgenin,were all increased compared with wild type.DzCAS had the most significant effect on the synthesis of sterols.This indicates that both MVA and MEP pathways are involved in the biosynthesis of diosgenin,while the MVA pathway plays a dominant role.DzCAS can directionally regulate the metabolic flux to sterol synthesis.The overexpression of DzDXR promoted the synthesis of the metabolites in the plasmid pathway,and the content of phytol,neophytadiene and tocopherol were significantly increased,while DzSE and DzCAS had no effect on plastid pathway metabolism.Due to the complex genetic background of Dioscorea zingiberensis,the transformation efficiency depends on the appropriate genotype.After the improvement of the regeneration system and transformation conditions of Dioscorea zingiberensis,a DzDXR knockdonwn strain was obtained by using CRISPR/Cas9 editing technology.The comprehensive study on the function of key enzyme genes contributes to analyze the source and formation mechanism of diosgenin,and also provides a theoretical basis for the molecular breeding of Dioscorea zingiberensis. |
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