| Eucalypt is an important fast-growing economic tree species,which is a general name of Angophora,Corymbia and Eucalyptus species of Myrtaceae.By 2018,the planting area of Eucalyptus trees in China reached 5.46 million hectares,and the annual output of Eucalyptus leaves was about 5,500 tons.However,most Eucalyptus leaves are discarded or burned,which lead to the waste of resources.Eucalyptus leaves contain a variety of secondary metabolites,among which polyphenols are the most abundant.Oenothein B(OeB)is a representative substance of Eucalyptus polyphenols.Oenothein B(OeB)is a representative substance of Eucalyptus polyphenols,which has antioxidant activity,bacteriostatic,immunological and anti-tumor effects,and can be used in feed,food,medicine and cosmetics,etc.,with great potential for development.In this study,the extraction methods of OeB from Eucalyptus urophylla×E.grandis(DH32-29)were firstly studied and explored.On this basis,the OeB content in Eucalyptus leaves of different growth stages was extracted and analyzed by using the optimized extraction method.Three different growth stages of Eucalyptus leaves with significant differences in OeB content were further selected for metabolome and transcriptome correlation analysis,and key genes involved in the regulation of OeB synthesis were identified and screened.It lays a foundation for further elucidating the molecular mechanism of OeB biosynthesis.The main results of this study are as follows:1.The effects of ethanol concentration,extraction temperature,extraction time and solid-liquid ratio on the extraction yield of OeB were investigated.The optimal extraction conditions were determined by orthogonal experiment,that is,ethanol concentration was 10%,extraction temperature was 87℃,solid-liquid ratio was 1:50(g/mL).Under these conditions,the extraction yield was 12.41%.In addition,the scavenging ability of OeB on DPPH and ABTS radical was 74.25%and 83.60%,respectively.2.Based on the optimal extraction method of OeB from Eucalyptus leaves,the OeB content of Eucalyptus leaves in 8 different growth periods was determined.It was found that the OeB content gradually increased in young Eucalyptus leaves,gradually leveled off in mature Eucalyptus leaves,and decreased in aged Eucalyptus leaves.3.A total of 284 metabolites were identified in leaves of Eucalyptus urophylla×E.grandis at different growth stages,which were divided into 13 categories.With the increase of eucalyptus leaf age,the accumulation of metabolites such as caffeic acid,apigenin and L-phenylalanine showed significant differences,mainly enriched in metabolic pathways,phenylpropane biosynthesis,amino acid biosynthesis and other biosynthetic pathways.GO functional enrichment results showed that the different expressed genes(DEGs)were mainly enriched in organic acid biosynthesis,glucan metabolism and secondary metabolic pathways.KEGG enrichment analysis showed that DEGs mainly distributed in phenylpropane biosynthesis pathway,flavonoid biosynthesis pathway,phenylalanine,tyrosine and tryptophan biosynthesis pathway,etc.Four key genes,EuCOMT,EuPAL,EuCSE and EuAOC3,which may be involved in OeB synthesis in Eucalyptus leaves were identified by transcriptional sequencing and metabolic analysis.4.The structural characteristics and expression patterns of CSE gene family members in Eucalyptus urophylla×E.grandis(EuCSE)were analyzed.It was found that EuCSE genes generally contained light responsive elements,especially EuCSE4 contained eight Box 4 light responsive elements,indicating that EuCSE genes was regulated by light signals.In addition,the protein domain of EuCSE were found to belong to c121494 superfamily(α/β-hydrolase superfamily)by constructing the genetic relationship and predicting the motif structure,indicating that the function of EuCSE genes was related to hydrolase. |
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