The Effect Of UV-B Radiation On The Metabolism Of Flavonoids In Ginkgo Biloba Leaves And Its Regulatory Mechanism

The leaf extracts of Ginkgo biloba L.has a better curative effect on human cardiovascular and other diseases,among of which,quercetin,kaempferol and isorhamnetin are the main substances in the quality control of G.biloba leaves pharmaceutical preparations.Flavonoids can serve as a UV filter in plants due to their special absorption properties,thus appropriate UV-B radiation can promote the biosynthesis of flavonoids.In this study,the G.biloba seedlings were used as the main experimental materials,and they were treated with low-dose and high-dose UV-B radiation,respectively.We found that the contents of flavonols in G.biloba leaves increased significantly after UV-B treatment.Further studies were carried out on G.biloba exposure to UV-B radiation by means of physiology,molecular biology,bioinformatics and genetic transformation.The main results were as follows:（1）Morphological observation showed that during the stage of 7-14days of UV-B treatment,only the G.biloba treated with high-dose for 14days were slightly stressed,appearing leaf yellowing.After 21 days of UV-B treatment,G.biloba mainly appeared leaf yellowing,curling and even wilting and scorch under UV-B stress.After UV-B treatment,the changes of ROS metabolism in G.biloba leaves were as follows:H₂O₂content increased significantly after 7 days of UV-B treatment,while its content had the biggest increase after treatment for 14 days.MDA content and CAT activity both firstly increased and then decreased,and reached the maximum on 14 days.SOD activity increased significantly only at the late stage of high-dose treatment.In addition,GST activity increased rapidly in the early stage of high-dose treatment.（2）HPLC-MS was used to determine the content of flavonols in G.biloba leaves.Our results showed that the content of total flavonol glycosides in G.biloba leaves was 1.82 folds than that of the control after UV-B treatment.The content of isorhamnetin reached 16-42 folds of the control,and the content reached the maximum after 14 days of high-dose treatment.The content of quercetin in G.biloba leaves increased rapidly after 7 days of UV-B treatment,and the content appeared the peak after 14 days of high-dose treatment,which reached2.37 folds higher than that of the control.The content of kaempferol also increased slightly at the early stage of UV-B treatment.The results indicated that long-term UV-B treatment could significantly promote the content of flavonols（especially quercetin and isorhamnine）in G.biloba leaves.（3）We analyzed some genes related to the metabolism of antioxidant enzymes,a total of 28 genes encoding CAT,POD,APX and GST were detected to be differentially expressed.One gene Gb＿38467encoding CAT2 was up-regulated.There were 6 up-regulated genes encoding GST,of which Gb＿18136 had the highest up-regulated expression level.Most of the POD genes were down-regulated.All three APX genes were down-regulated.Combined with the physiological data,CAT and GST enzymes may play a major role in the removal of reactive oxygen species under UV-B treatment.（4）Four DEGs were identified to be involved in the UV-B signal pathway,including three genes Gb＿14008,Gb＿31523 and Gb＿40615encoding UVR8,and one gene Gb＿15627 encoding COP1.And q RT-PCR results showed that Gb＿14008（UVR8）was up-regulated and Gb＿15629（COP1）was down-regulated.Further conservative domain analysis revealed that they had similar domains with At UVR8 and At COP1 in Arabidopsis thaliana,respectively.A total of 21differentially expressed structural genes were identified in the flavonoids synthesis pathway.Among of them,FLS and F3’H showed up-regulated expression pattern after UV-B treatment.q RT-PCR results showed that Gb＿19792,Gb＿04545 and Gb＿11520（F3’H）,Gb＿22751（FLS）were consistent with trans-seq results,indicating that above genes may be involved in the biosynthesis of flavonols by responding to long-term UV-B radiation.In adition,MYB transcription factor is the largest family group of differentially expressed TFs.The q RT-PCR results verified the down-regulated expression pattern of 3 MYB genes（Gb＿18153,Gb＿40629 and Gb＿05115）,the up-regulated expression pattern of ODO1（Gb＿38090）,and the up-regulated expression pattern of 2 bZIP TFs（Gb＿00122 and Gb＿12012）.（5）Bioinformatics analysis of Gb＿12012 and Gb＿00122 showed that both of them had the characteristic structural domain（N-x7-R/K-x9-L-x6-L-x6-L）of bZIP,and Gb＿12012 had similar conservative structural domain with At HY5 in Arabidopsis thaliana,and both had the characteristic structural domain of HY5.Evolutionary tree analysis showed that Gb＿12012 was closely related to At HY5 and Pp HY5（Physcomitrella patens）.The binary vector of Gb＿12012 was constructed with p ENTR-L12-MCS and p K7WGF2-GFP,and the length of cloned sequence obtained was 774 bp.Furthermore,we carried out the transient transformation of tobacco leaves,and subcellular localization showed that the gene was mainly located in the nucleus and distributed in a small amount on the cell membrane.This indicated that the HY5 gene（Gb＿12012）may mainly play a regulatory role in the nucleus.