Study On Mechanism Of The Flavonoid Accumulation In The Leaves Of Ginkgo Biloba At High Altitude(Nyingchi)

Ginkgo biloba is an important economic tree species in China.Ginkgo biloba extract(GbE)is widely used in the treatment of cardiovascular and cerebrovascular diseases,Alzheimer’s disease and other diseases due to its rich bioactive substances such as flavonoids and terpene lactones.Ginkgo biloba is naturally distributed in more than 20 provinces and cities in China.There are differences in flavonoid content in ginkgo leaves at different altitudes,but its regulatory mechanism is still unclear.In this study,5-year-old ginkgo seedlings grown in Nyingchi,Tibet(high altitude)and its introduction area in Pizhou,Jiangsu(low altitude)were used as materials to analyze the differentially expressed genes and metabolites in ginkgo leaves,and identify key structural genes involved in flavonoid synthesis.By treating ginkgo seedlings with different environmental factors such as UV,light intensity,and temperature,the main environmental factors and key genes that promote the accumulation of flavonoids in ginkgo were screened.The main results are as follows:(1)According to the measurement and analysis of physiological indicators of ginkgo seedlings in two different regions,we found that compared with the Pizhou region,the seedlings in Nyingchi region have the characteristics of being shorter,with yellow leaf color,fewer leaf cracks,smaller leaf area,and lighter fresh leaf weight.For example,in the Pizhou area,the plant height reaches about 270 cm,the diameter at breast height is about 9.8 mm,and there are 3-4 leaf cracks.However,the plant height in the Nyingchi region of Tibet is about 190 cm,the diameter at breast height is about 7.8 mm,and there are 1-2 leaf cracks.However,the flavonoid content in ginkgo leaves in the Nyingchi area is as high as 35 mg/g,which is 1.59 times higher than that in low altitude areas.The above results indicate that high altitude limits the growth of ginkgo plants but significantly increases the accumulation of flavonoids in their leaves.(2)Through extensive targeted metabolomics analysis of leaves grown in two regions,a total of 310 differential metabolites were identified in 9 major categories,including flavonoids,lipids,organic acids,amino acids,and phenolic acids.The content of primary metabolites such as amino acids,lipids and phenolic acids in ginkgo leaves in high altitude areas is generally lower than that in low altitude areas,while the content of secondary metabolites is higher than that in low altitude areas.In particular,there are differences in the content of eight kinds of flavonoids identified,which are significantly increased biflavone,flavonoid glycoside,flavanol,flavonol,dihydroflavonol,flavanone,dihydroflavone,isoflavone.(3)A total of 2745 differentially expressed genes were identified by transcriptome sequencing of ginkgo leaves from two altitudes.Among them,32 genes were enriched in phenylpropanoid biosynthesis,and except for 6 genes that showed an upward trend in expression in high-altitude areas,the expression of all other genes was significantly downregulated.A total of 33 genes were enriched in the flavonoid synthesis pathway,most of which were upregulated in leaves of high-altitude areas,especially structural genes closely related to the synthesis of chalcone,flavonols,and other substances,such as C4H and CHS,FLS,and F3 ’H,which were significantly upregulated.The combined analysis of metabolome and transcriptome showed that the main reason for the high flavonoid content in ginkgo leaves at high altitude might be due to the increased expression of flavonoid synthesis structure genes.(4)To explore the influencing factors of high flavonoid synthesis in ginkgo leaves in high-altitude areas,annual ginkgo seedlings were treated with different environmental factors indoors,including different intensities of UV-B radiation(0 KJ/m 2 7.14 KJ/m 2、21.42 KJ/m 2)、different temperatures(25℃,15℃,5℃)and different light intensities(300 μ mol m-2 s-1、500 μ mol m-2 s-1、800 μMol m-2 s-1)treatment and determination of flavonoid content and gene expression of key synthase GbFLS in ginkgo leaves.The results showed that compared with the control,UV-B radiation,low temperature,and high light intensity all promoted the accumulation of flavonoids in ginkgo leaves.After UV-B radiation treatment,the highest flavonoid content increased by 2.2 times,the low temperature treatment increased by 1.5 times,and the high light intensity treatment increased by 2.8 times.Among the environmental factors tested,the high light intensity(500 μmol m’2 s-1)is the main environmental factor that promotes flavonoid synthesis.(5)Analysis of the expression level of the flavonoid synthesis structure gene GbFLS by environmental factor treatment revealed that the expression level of GbFLS was upregulated up to 4 times,2.2 times,and 3.75 times after UV-B,low temperature,and high light intensity treatments,respectively.We cloned GbFLS,transiently transformed ginkgo callus,and stably transformed Arabidopsis.Compared with the control,the flavonoid content in GbFLS overexpressed callus was 3.5 times higher than that of the wild-type,indicate that GbFLS can response several environmental factors and positively regulate flavonoid synthesis.