Effects Of Exogenous ABA On Flavonoid Synthesis In Ginkgo Biloba Leaves And Mining Of Functional Genes

As the main medicinal active ingredient in Ginkgo biloba extract,flavonoids are widely used in medicine,food,health products and other fields.ABA is an important plant hormone,which can increase the content of flavonoids in plants in production practice.As a MYB family gene,R2R3-MYB transcription factor has been confirmed to play an important role in the regulation of Ginkgo biloba flavonoid metabolism.To explore the effects of exogenous application of ABA on the content of flavonoids in Ginkgo biloba leaves,the activity of key enzymes for synthesis and the expression of related R2R3-MYB transcription factors,screen key genes and identify their functions,which is of great significance to reveal the metabolic regulation mechanism of flavonoids in Ginkgo biloba leaves and molecular breeding.This study was carried out in the artificial climate culture room.Taking one-year-old Ginkgo biloba seedlings as test materials,five concentration gradients were set for the foliar spraying treatment of exogenous ABA.The changes of the content of total flavonoids and components,the activity of key enzymes in the flavone synthesis pathway and the expression trend of nine key enzyme structure genes in Ginkgo biloba leaves were measured.On this basis,the RNA expression of Ginkgo biloba leaves treated with exogenous ABA was analyzed by transcriptome sequencing technology.Combined with transcriptome data and the previous research group’s research on Ginkgo biloba related MYB genes,two key genes GbMYB4 and GbMYB61,which are significantly responsive to ABA induction and most likely related to Ginkgo biloba flavonoid synthesis,were screened for cloning,transformation and functional analysis.The main results are as follows:（1）Exogenous ABA treatment had a significant effect on the contents of total flavonoids and its components quercetin,kaempferol and isorhamnetin in Ginkgo biloba leaves.With the increase of ABA treatment concentration,the contents of total flavonoids and its components in Ginkgo biloba leaves increased first and then decreased.The contents of total flavonoids,quercetin and isorhamnetin in treatment D(150μmol·L^-1)were the highest,while the contents of kaempferol in treatment C(100μmol·L^-1)were the highest.Exogenous ABA can improve the synthesis and accumulation of flavonoids in Ginkgo biloba as a whole,and the effect of ABA concentration of150μmol·L^-1 is the best.（2）Exogenous ABA treatment had a significant effect on the activities of key enzymes（PAL,C4H,4CL）in the synthesis of flavonoids in Ginkgo biloba,but there were certain differences in the changing trends of different enzymes.The enzyme activity of PAL increased gradually with the increase of ABA concentration,The enzyme activity of C4H increased and decreased synchronously with the content of total flavonoids,The enzyme activity of 4CL in treatment C(100μmol·L^-1)and D(150μmol·L^-1)was significantly higher than that in the control,while that in treatment B(50μmol·L^-1)and E(200μmol·L^-1)was lower than that in the control.（3）Under ABA treatment,2190 genes were significantly up-regulated and 2430 genes were significantly down-regulated in Ginkgo biloba leaves.The differential expression analysis of 48structural genes of Ginkgo biloba flavonoid synthesis pathway combined with Ginkgo biloba transcriptome and genome data showed that the expression levels of most genes were significantly up-regulated,which was consistent with the results of real-time fluorescence quantitative PCR at the metabolic level.It was further proved that ABA treatment could significantly increase the expression levels of several key enzyme structural genes in Ginkgo biloba flavonoid synthesis pathway,such as GbPAL,GbC4H,Gb4CL,GbCHS and GbCHI,thereby promoting the synthesis and accumulation of flavonoids.（4）The differential expression of 69 GbR2R3-MYB genes screened by the research group in the transcriptome data was analyzed,and 26 GbR2R3-MYB genes were found to be significantly different under ABA treatment,among which Gb＿19333 had the highest expression and the change was the most significant,followed by Gb＿26833 gene,its expression was basically consistent with the change trend of quercetin,isorhamnetin and total flavonoids.Based on the responses of the structural and regulatory genes related to flavonoid synthesis in Ginkgo biloba to ABA treatment,Gb＿19333（GbMYB4）and Gb＿26833（GbMYB61）genes were selected as candidate genes for functional identification.（5）The complete sequences of GbMYB4 and GbMYB61 were obtained by cloning and transformation,and the two genes were located in the nucleus by subcellular localization.The overexpression vector was constructed for the genetic transformation of Arabidopsis thaliana.The verification results at the DNA and transcription levels showed that GbMYB4 and GbMYB61 was successfully expressed heterologously in Arabidopsis thaliana.The possibility of false positives was excluded by resistance screening.The T₃ generation Arabidopsis thaliana homozygote was obtained for the determination of total flavonoids.Compared with the wild type,the transgenic plants of GbMYB4 and GbMYB61 were significantly improved.It is preliminary speculated that these two genes act as positive regulators to regulate the synthesis of Ginkgo biloba flavonoids.