Analysis Of Transcription,Protein And Metabolic Regulation Mechanisms Of Flavonoids Synthesis In Ginkgo Biloba L. Leaves

Ginkgo biloba is a precious tree species in China.It has typical warmth and sunshine characteristics,wide adaptability,strong resistance,long life,less pests and diseases,and high economic value.Ginkgo biloba has great leaf value,nut value,ornamental value,ecological value and scientific research value because of its morphological characteristics and evolutionary status.At present,the medicinal value of Ginkgo biloba has been widely concerned worldwide.The completion of the whole genome sequencing work provides a useful reference genome for Ginkgo biloba high-throughput sequencing technology,but the sequencing of the transcriptome has not been widely used to explain the biological phenomenon of Ginkgo biloba;Proteome,metabolome,especially multi-group Joint analysis technology is an effective tool to explain biological phenomena,but it has not yet been applied in Ginkgo biloba.Although many genes related to the synthesis of flavonoids in Ginkgo biloba have been cloned so far,the depth of their research is relatively shallow,and the biosynthesis pathway of flavonoids in Ginkgo biloba has not been well understood.In this study,the Ginkgo biloba cultivar ’Taishan（TS10） family’ was used as the research object.We conducted annual dynamic observations on the flavonoids in this family,and found out the annual variation of flavonoids.In addition,the quantitative and qualitative analysis of the class of compounds was carried out in Ginkgo biloba leaves.For the first time,transcriptome,metabolome and proteomics were analyzed in three key developmental stages of Ginkgo biloba leaves.The mechanism of flavonoid biosynthesis in Ginkgo biloba leaves was systematically studied.Different genes,proteins and metabolites were obtained through different periods.Through the relationship between the expression of differential genes,differential proteins and differential metabolites in different periods,the key regulatory genes,proteins and metabolites in the biosynthesis of flavonoids in Ginkgo biloba leaves were screened out.The aim of this study is to explore the key regulation mechanism of flavonoids biosynthesis,and to provide molecular and biological basis for improving the content of flavonoids in Ginkgo biloba leaves,and to promote the development and utilization of medicinal value of Ginkgo biloba.The main results of this study are as follows:（1）Seasonal changes of flavonoids in Ginkgo biloba leaves and different tissuesThe annual content of flavonoids in Ginkgo biloba leaves has a very significant difference with the seasons.In the early stage of leaf development,the content of flavonoids was higher.The content of flavonoids decreased gradually with the growth of leaves in May.Since June,the content of flavonoids gradually increased,reaching the highest value in September.After the yellowing of Ginkgo biloba leaves in autumn,the flavonoids decreased sharply and reached a very low value before and after defoliation.The difference of flavonoids content in different tissues of Ginkgo biloba was extremely significant.In April,July and October,the difference of flavonoids conten: leaves > roots > stems was present.There are also significant differences in the content of flavonoids among different individuals in the same family in the same period.（2）Quantitative and qualitative analysis of metabolites and flavonoids in Ginkgo biloba leavesA total of 615 metabolites were detected in Ginkgo biloba leaves,which were divided into 32 categories.Among them,the most common species were organic acids and their derivatives,with 64 species,accounting for 10.41% of the total.The most abundant metabolites in Ginkgo biloba leaves were lipid-fatty acids,followed by sugars and flavonoids.A total of 165 flavonoids were detected in Ginkgo biloba leaves,which were divided into 8 categories.The most abundant of which was astragalin;while 3-hydroxyflavone is the lowest content.Metabolites such as amino acid derivatives,flavonoids,and flavonols are positively correlated with the total amount of flavonoids,while nucleotides and derivatives,hydroxycinnamoyl derivatives,lipid-glycerol phospholipids and other metabolites are negatively correlated with the total amount of flavonoids.（3）Differential regulation analysis of flavonoid biosynthesis in Ginkgo biloba leaves in different periods based on multi-omics analysisIn this study,a high-quality cDNA library was obtained by sequencing the transcriptome,and the average comparison rate with the reference genome of Ginkgo biloba was 89.42%.Therefore,it is feasible to use the transcriptome sequencing technology to explain the biological phenomenon of Ginkgo biloba.During the three periods（August/H1,July/H2,June/H3）,the leaves were subjected to a combination of transcriptome,metabolome and proteomic analysis.2835 differential genes,64 differential proteins,and 166 differential metabolites were detected in the H1 vs H2 combination.7421 differential genes,197 differential proteins,and 164 differential metabolites were detected in the H1 vs H3 combination;3278 differential genes,23 differential proteins,and 51 differential metabolites were detected in the H2 vs H3 combination.Pathway analysis showed that the flavonoid biosynthesis pathway in H1 VS H2 combination enriched 15 differential genes,5 differential proteins and 15 differential metabolites.The biosynthetic pathway of phenylpropane inpre-reaction pathway of flavonoid biosynthesis pathway was enriched 26 differential genes,4differential proteins,14 differential metabolites;In H1 VS H3 combination,flavonoid biosynthesis pathway enriched 20 differential genes,5 differential proteins,13 differential metabolites,and its pre-reaction pathway-biosynthesis pathway of propane was enriched with40 differential genes,6 differential proteins,and 10 differential metabolites.19 metabolites were differentially expressed in the three combinations,including 5 flavonoids,one anthocyanins and two kinds of hydroxycinnamoyl derivatives.In addition,a large number of bHLH and MYB-related transcription factors are enriched in each sample.Flavonoids are an important secondary metabolite and play an important role in the resistance and adaptability of Ginkgo biloba.（4）The key mechanism of the regulation of flavonoids in Ginkgo biloba leavesThe genes,proteins and metabolites related to the flavonoid biosynthesis in different combinations of Ginkgo biloba leaves were screened accurately.Eight key enzymes,such as FLS,LAR and DFR,were chosen during the synthesis of flavonoids from Ginkgo biloba,and the 16 key genes such as Gb₁4029（FLS）,Gb₁4026（FLS）,Gb₀8047（LAR） were also selected.The expression of key genes was verified in different tissues of Ginkgo biloba.The expression levels of Gb₃3746（LAR）,Gb₁4024（FLS）,Gb₀7705（CCR） and Gb₀9086（DFR） were positively correlated with the content of flavonoids,While the expression levels of Gb₀6448（LDOX）,Gb₁3074（Flavonoid 3’-Monooxygenase） and Gb₁0488（HCT） were negatively correlated with the content of flavonoids.The biosynthesis of flavonoids in Ginkgo biloba leaves has strong correlation with phenylalanine,tyrosine and tryptophan biosynthesis,fatty acid metabolism,fatty acid biosynthesis and other pathways,which are regulated by a series of genes,proteins and metabolic processes.Through the annotation of GO and KEGG functions of differential genes in different periods,the key regulation pathways of flavonoids in Ginkgo biloba were sorted out.CCR（cinnamoyl-CoA reductase） and FLS（flavonol synthase） are key rate-limiting enzymes in the synthesis of flavonoids from Ginkgo biloba.