Flavonoids Biosynthesis Pathway And Key Genes Function Analysis In Meconopsis Horridula At Different Altitudes

Meconopsis horridula Hook.f.& Thoms.is a poppy species and an annual herbaceous plant in the Meconopsis genus.As a traditional Tibetan medicine plant in the Qinghai-Tibet Plateau region,it grows in highaltitude areas of 3600-5100 meters and is a commonly used medicinal material in Tibetan medicine,playing an important role.Currently,research on Meconopsis horridula focuses more on resource surveys,molecular identification,chemical composition analysis,and pharmacological research,while research on its environmental adaptability mechanism,and secondary metabolite synthesis are relatively scarce.Flavonoids are important secondary metabolites in Meconopsis horridula.They play a crucial role in the growth,development,and response to environmental changes of Meconopsis horridula.Flavonoids are also one of the main active components in Meconopsis horridula used in traditional medicine.In the face of the depletion of wild resources,the need to protect resources and utilize them as drugs,it is urgent to integrate modern genomics and molecular biology techniques to conduct comprehensive basic research on Meconopsis horridula.This will promote the sustainable utilization of Meconopsis horridula and provide basic data for the study of the biosynthesis mechanism and adaptation to extreme environments of flavonoids in Meconopsis horridula.This study,conducted in 2020,focused on the Meconopsis horridula at different altitudes(DDC: 4872 m,DQC: 4925 m and DDG: 3980 m),as the research object.The researchers used transcriptomics and metabolomics methods to analyze the differences in genes and metabolites between different groups,and screened out key structural genes,chalcone synthase(Mh CHS),and chalcone isomerase(Mh CHI)in the flavonoids metabolic pathway of Meconopsis horridula.The Mh CHS and Mh CHI also conducted preliminary explorations of their physical and chemical properties,protein structures,and molecular biological functions.The main results and conclusions of the study are as follows:(1)Metabolomics analysis was conducted on three different populations of Meconopsis horridula at different altitudes(DDC: 4872 m,DQC: 4925 m and DDG: 3980 m),and the results showed that a total of771 metabolites were identified among the three populations,including406 differentiated metabolites.In the annotation of plant secondary metabolites,the number of flavonoids,alkaloids and terpenoids was higher,with 22,11 and 9,respectively..Enrichment analysis showed that flavonoid biosynthesis,flavone and flavonol biosynthesis isoflavone biosynthesis,and anthocyanin biosynthesis pathway were significantly enriched.(2)Transcriptomics analysis was conducted on the Meconopsis horridula at different altitudes,and a total of 60.15 Gb of sequencing data were obtained.After assembly,75,793 Unigene sequences were obtained,with an average N50 length of 1637 bp.Differential analysis,annotation,and enrichment analysis showed that differentially expressed genes were mainly enriched in primary metabolic pathways related to energy metabolism and secondary metabolic pathways related to stress resistance.The flavonoids biosynthesis pathway and the upstream phenylpropanoid metabolic pathway were also significantly enriched.(3)Key structural genes in the flavonoids metabolic pathway were screened through co-occurrence network analysis,and the sequences of the CHS and CHI genes were obtained by cloning and sequencing of the Meconopsis horridula.The physical and chemical properties and protein structures of the CHS and CHI genes were predicted and analyzed.The Mh CHS gene encodes 390 amino acids,with a relative molecular weight of 42,620.18,an isoelectric point of 5.8,an instability coefficient of 36.85,and an average hydrophobicity of-0.078,making it a hydrophilic stable protein.The Mh CHI gene encodes 222 amino acids,with a relative molecular weight of 24,763.2,an isoelectric point of 4.67,an instability coefficient of 47.10,and an average hydrophobicity of-0.101,making it a hydrophilic unstable protein.(4)The p ET-28a-Mh CHI and p ET-28a-Mh CHS prokaryotic expression vectors were successfully constructed and transformed into Escherichia coli BL21 to obtain expression strains.The target proteins of24.76 KD and 42.62 KD were successfully induced by IPTG as an exogenous inducer at 37°C and 28°C in E.coli BL21 cells.(5)The p FGC5941-Mh CHS and p FGC5941-Mh CHI plant overexpression vectors were successfully constructed,and the plant overexpression vectors were transformed into wild-type Arabidopsis by Agrobacterium-mediated flower dip method.Through vector resistance screening and molecular verification,stable overexpression lines were obtained in the T3 generation.The total flavonoid content of the T3 generation transgenic plants was measured,and the results showed a significant increase in total flavonoid content in both p FGC5941-Mh CHS and p FGC5941-Mh CHI transgenic plantsThis study is the first to use transcriptomics and metabolomics to analyze the flavonoids biosynthesis pathway in Meconopsis horridula.Key structural genes in the flavonoids biosynthesis pathway were screened,and their biological functions were preliminarily analyzed,providing fundamental data for the molecular regulatory mechanism,development and utilization of flavonoids compounds in Meconopsis horridula,and the study of its high-altitude adaptive evolution.