Molecular Cloning And Functional Studies Of Three New GbDFR Family Genes In The Pathway Of Flavonoid Biosynthesis From Ginkgo Biloba L.

Dihydroflavonol 4-reductase(DFR)is a key enzyme involved in the downstream synthesis of anthocyanins in the flavonoid synthesis pathway.Its not only affects the color of plant flowers and fruits,but has a certain regulatory effect on the distribution of different flavonoid components in plants.As the oldest relict plant,ginkgo has high nutritional value and medicinal value,which has a significant effect on the prevention and treatment of cardiovascular and cerebrovascular diseases.Flavonoids is one of the main active components of ginkgo extract,it is a great practical significance to study the flavonoid synthesis pathway of Ginkgo biloba.In this study,the Arabidopsis thaliana AtDFR gene was used as a reference to compare the genome sequences of Ginkgo biloba,and finally found three new genes in the GbDFR family of Ginkgo biloba.It explored the influence of external environment on the synthesis of ginkgo flavonoids and the three gene expression,and preliminarily analyzed the functional differences of the three gene in response to external stimuli.Then,the expression vectors of these three genes were constructed respectively,and the functions of these three genes were preliminarily verified by in vitro enzyme activity experiments after transient injection of tobacco and Arabidopsis thaliana tt3 mutants.Finally,the functions of these three genes were further verified by transforming wild type tobacco and Arabidopsis thaliana tt3 mutants.The main research contents and results are as follows:1 ? According to the AtDFR gene sequence of Arabidopsis thaliana,bioinformatics technology was used to search the genome sequence of Ginkgo biloba,and the sequences of three genes of Ginkgo biloba were obtained.Through sequence alignment analysis,we found that these three genes are different from the previously reported three GbDFR genes,so they were named GbDFR4,GbDFR5 and GbDFR6.2?The effects of exogenous application of salicylic acid(SA)and jasmonic acid(JA)on the flavonoid content of Ginkgo biloba and the expression of three genes were investigated.The results showed that both SA and JA treatments could affect the content of flavonoids in Ginkgo biloba,but their regulation of the three GbDFR genes was complicated.In addition,the anthocyanin content of Ginkgo biloba has increased month by month since April,but the expression levels of the three GbDFR genes have decreased month by month.3?These three GbDFR genes were cloned and their 35 S overexpression vectors were constructed.4?GbDFR protein was transiently expressed by injection of leaves from tobacco and Arabidopsis thaliana tt3 mutants.We performed in vitro enzyme activity analysis on the extracted protein.The results showed that the proteins encoded by the three genes have the enzymatic activity of DFR in vitro.5?The expression vectors containing GbDFR4,GbDFR5 and GbDFR6 were transferred into wild type tobacco by disc method to investigate the effect of exogenous gene expression on tobacco flower color and anthocyanin content.The results showed that the anthocyanin content of transgenic tobacco is higher than that of wild type,and the color of flower is deeper.6?Three GbDFR genes were transformed into Arabidopsis thaliana tt3 mutants by inflorescence infection method for functional recovery experiments,and multiple growth points were observed in the transgenic positive seedlings just obtained,presumably associated with auxin transport.In summary,the experimental results of this paper preliminarily determined that these three genes can function as DFR genes and have dihydroflavonol 4-reductase activity.