Effects Of Exogenous Salicylic Acid On Flavonoid Metabolism Of Ginkgo Biloba And Functional Verification Of GbMYB33

Ginkgo biloba flavonoids,as secondary metabolites with great medicinal value,also require many simple and efficient techniques to increase their content in actual production.Foliar application of salicylic acid is a simple,non-toxic,residue-free and low-cost operation technique.MYB transcription factor,as one of the largest family of plant transcription factors,is involved in the regulation of ginkgo flavonoid metabolism.Therefore,by studying the effects of foliar application of salicylic acid on the metabolic pathways of flavonoids in Ginkgo biloba leaves,it is of great significance to explore the differential expression of MYB transcription factors related to flavonoid metabolism under different concentrations of salicylic acid.In this study,different concentrations of salicylic acid were sprayed on the leaves of two-year-old seedlings of Ginkgo biloba half-sib families,and the changes of some physiological characteristics and total flavonoid content of Ginkgo biloba leaves under different concentrations of salicylic acid were analyzed.At the transcription level,the changes in the expression levels of key enzyme structural genes in the flavonoid metabolism pathway of Ginkgo biloba under different treatments were studied.Analyze the effects of different concentrations of salicylic acid on Ginkgo-related MYB transcription factors.One Gb MYB33（Gb＿18153）whose expression change trend was the same as that of total flavonoid glycosides was screened out,and cloned and transformed for verification.The main research conclusions are as follows:1.With the increase of the spraying salicylic acid concentration,the content of chlorophyll a,chlorophyll b and chlorophyll a+b first decreased,then increased and then decreased,and both increased by more than 10%when 2 mmol/L was applied externally;With the increase of the sprayed salicylic acid concentration,the intercellular CO₂ concentration decreased first,then increased,the transpiration rate,stomatal conductance,and net photosynthetic rate first decreased,then increased and then decreased,the non-photochemical quenching coefficient showed a significant downward trend.The maximum photochemical efficiency of photosystem II first decreased,then increased and then decreased,while the actual light energy conversion efficiency and photochemical quenching coefficient did not change significantly;With the gradual increase of the treatment concentration,the content of abscisic acid（ABA）decreased significantly,the content of gibberellin（GA₃）first decreased and then increased;the content of zeatin（ZR）first increased and then decreased;The content of auxin（IAA）first decreased,then increased and then decreased,reaching the highest when 2 mmol/L salicylic acid was applied.2.When 2 mmol/L salicylic acid was applied externally,the total flavonol glycosides increased significantly by 39.90%（P<0.001）.The content of the three components quercetin,kaempferol and isorhamnetin was also significantly increased by more than 17%when 2 mmol/L salicylic acid was applied externally.When 1,3 mmol/L salicylic acid was applied externally,the contents of total flavonol glycosides,quercetin,kaempferol and isorhamnetin were lower than those in the control group.3.Transcriptome sequencing of ginkgo leaves was performed,and Clean Reads of each sample with a comparison rate of more than 94%were obtained.After the samples are filtered,the Q30 in the total sequence is above 96%.The correlation coefficients between samples are all above0.93,which can be considered as having good biological reproducibility.By comparing the differential genes between the three different treatments and the control,1391 differential genes were significantly up-regulated and 1522 genes were down-regulated between the control group and externally administered 1 mmol/L salicylic acid;Between the control group and externally administered 2 mmol/L salicylic acid,735 differential genes were significantly up-regulated and912 genes were down-regulated;between the control group and externally administered 3 mmol/L salicylic acid,there were 1163 differential genes that were significantly up-regulated,and 442genes were down-regulated.At the same time,there are 2012 genes with significant differences.4.Through GO function annotation analysis,most genes are annotated as"metabolic process","celiular process","membrane part","cell part","organelle","binding"and"catalylic activity";In the analysis of the gene KEGG enrichment pathway,the pathways that are enriched in the secondary metabolic pathway include Flavonoid biosynthesis,Phenylpropanoid biosynthesis,Diterpenoid biosynthesis,Monoterpenoid biosynthesis,Sesquiterpenoid and triterpenoid biosynthesis,etc.When analyzing the expression changes of key enzyme genes in the flavonoid anabolic pathway,it was found that Gb DFR（Gb＿26256）was up-regulated after 1 mmol/L salicylic acid treatment,and Gb FLS（Gb＿14029）,Gb F3’H（Gb＿29563）and Gb ANS（Gb＿10028）were up-regulated after 2 mmol/L salicylic acid treatment.Gb DFR（Gb＿26256）and Gb F3’H（Gb＿29563）were up-regulated after treatment with 3 mmol/L salicylic acid.5.In the MYB gene family analysis under different concentrations of salicylic acid,18 genes were significantly up-regulated and 36 genes were significantly down-regulated.Combining the differential analysis of the expression levels of the above genes in leaves treated with different concentrations of salicylic acid,the R2R3-MYB transcription factor Gb MYB33 was successfully cloned,and the gene was determined to be located in the nucleus.The Arabidopsis was genetically transformed and verified to obtain T1 generation transgenic Arabidopsis seedlings.The total flavonoid content of the T1 generation was determined,and the total flavonoid content was significantly increased by 26.36%compared with the wild-type Arabidopsis.It is preliminarily speculated that Gb MYB33 can positively regulate the synthesis of ginkgo flavonoids.