Molecular Mechanism Of GbHY5 Transcription Factor Regulating Flavonoid Synthesis In Ginkgo Biloba In Response To UV-B

Ginkgo biloba L.,known as "living fossil",is an important economic forest specie in China.Ginkgo biloba extract（GBE）is widely used in the treatment of cardiovascular and cerebrovascular diseases because it is rich in flavonoids,terpene lactones and other medicinal components.The biosynthesis of flavonoids is complex and affected by many internal and external factors,among which UV-B radiation can significantly affect the synthesis of plant flavonoids.With the deepening of flavonoid research,it has been found that HY5 transcription factor is induced by UV-B and plays an important role in flavonoid transcription regulation.However,the role and molecular mechanism of UV-B and HY5 in ginkgo flavonoid synthesis are not clear.Therefore,this study screened out the GbHY5 transcription factor of Ginkgo biloba whose expression significantly upregulated under UV-B radiation based on the whole genome data of Ginkgo biloba and our early transcriptome data of UV-B-treated Ginkgo biloba leaves.The function and regulation of GbHY5 in flavonoid synthesis were studied by means of genetic transformation,transcriptomic analysis,yeast two hybrid and double luciferase assay.The molecular mechanism of GbHY5 in regulating ginkgo flavonoid biosynthesis was preliminarily revealed.The main research contents are as follows:（1）Through UV-B treatment of Ginkgo biloba seedlings,the H2O2 content of Ginkgo biloba leaves had an largest increase on the 7th day.The Malondialdehyde（MDA）content increased first and then decreased with the extension of treatment time,reaching the peak on the 7th day.The activity of Superoxide dismutase（SOD）and Catalase（CAT）in Ginkgo biloba leaves treated with UV-B increased significantly and continued enhancing with the extension of treatment time.The activity of CAT also showed the same trend as that of SOD.The content of flavonoids in leaves also increased significantly after UV-B treatment.These results showed that UV-B radiation could lead to the accumulation of Reactive oxygen species（ROS）in Ginkgo biloba leaves,enhance the activity of ROS scavenger enzymes,and increase the content of flavonoids in Ginkgo biloba leaves.（2）Our previous transcriptome sequencing data showed that GbHY5 was significantly up-regulated in Ginkgo biloba leaves treated with UV-B for 14 days.In order to further clarify the expression pattern of GbHY5 under UV-B treatment,we detected the expression level of GbHY5 in Ginkgo biloba leaves treated for 0-7 days.The results showed that the expression of GbHY5 increased significantly under UV-B treatment at different times,and its expression increased with the extension of UV-B treatment time.By constructing the overexpression vector of GbHY5 and transferring it into Ginkgo callus and Arabidopsis thaliana,it was found that the content of flavonoids in GbHY5 transgenic materials increased significantly.These results showed that GbHY5 was induced by UV-B,which promoted the synthesis of flavonoids.（3）In order to explore the potential downstream genes of transcription factor GbHY5,we sequenced the transcriptome of GbHY5 overexpressed Ginkgo callus and found that the differential genes were mainly enriched in flavonoid biosynthesis and other related pathways.Among them,FLS and other key structural genes of flavonoid synthesis in flavonoid synthesis pathway were significantly up-regulated.In addition,22 MYB transcription factors were differentially expressed,of which 11 members were significantly increased.Transcriptome sequencing showed that GbHY5 transcription factor may mediate the flavonoid synthesis through regulating structural genes（e.g.FLS）in flavonoids synthesic pathway and transcription factors（e.g.MYB）.（4）qRT-PCR results showed that the expression levels of GbMYBl（gb₂7027）and GbFLS（Gb₀1811）in GbHY5 overexpressed callus were significantly higher than those in the control group,suggesting that these two genes may be regulated by GbHY5.In order to explore the role of GbMYBl and GbFLS in regulating flavonoid synthesis,we constructed 35S::GbMYBl and 35S::GbFLS overexpression vectors and transferred them into Ginkgo callus and Arabidopsis.It was found that GbMYBl and GbFLS could significantly increase the content of flavonoids in Ginkgo transgenic callus and Arabidopsis,indicating that both GbMYB1 and GbFLS could promote the synthesis of flavonoids.（5）In order to clarify the regulatory relationship between GbHY5 and GbMYB1,GbHY5 and GbFLS,yeast two hybrid and double luciferase experiments were carried out.Firstly,it was found that GbHY5 protein did not have autonomous transcriptional activation activity after transforming pGBKT7-GbHY5 vector into yeast cells.Then,yeast two hybrid experiment was carried out between pGADT7-GbMYB1 and pGBKT7-GbHY5.It was found that GbHY5 and GbMYB1 protein could interact.GbFLS promoter was connected to pGreen Ⅱ 0800 Luc vector.Through double luciferase experiment,it was found that GbHY5 induced GbFLS promoter activity to increase by about 1.5 times and GbMYB1 induced GbFLS promoter activity to increase by about 3 times compared with the control group.When GbHY5 and GbMYB1 were co expressed,GbFLS promoter activity increased by more than 4-folds.These results confirmed that the transcription factors GbHY5 and GbMYB1 could bind to the promoter of GbFLS alone and promote its transcription.Furthermore,the binding of GbHY5 and GbMYB1 could strengthen the transcription of GbFLS.In conclusion,this study found a pathway of regulating ginkgo flavonoid synthesis,namely UV-B-GbHY5-GbMYB1-GbFLS pathway.The discovery of the UV-B pathway of Ginkgo biloba not only reveals the mechanism of promoting flavonoid synthesis in other plants,but also provides a certain reference for the study of other UV related molecules.