| Ferns are a class of vascular plants which propagating by spores.The evolutionary status of ferns is between mosses and seed plants.It is reported that ferns are rich in flavonoids,and the average content of total flavonoids is higher than that of mosses and seed plants.However,there are few studies on anthocyanidins or anthocyanins,important species of flavonoids,in ferns.Anthocyanins are not only important plant pigments,but also have the function of protecting plants from a variety of biotic and abiotic factors.They can scavenge free radicals,reduce photo damage and protect plant photosynthesis.The leaves of some ferns appear dark red,and whether these pigments are anthocyanidins have not been reported before.Due to the large number of chromosomes in most ferns and the lack of reference genomes,there are few studies on functional genes of ferns.Although there are certain foundations for the biosynthesis of anthocyanidins in flowering plants,the biosynthesis of anthocyanidins in ferns are still have many unclear pathway.In particular,dihydroflavonol 4-reductase(DFR),a key enzyme in the biosynthesis of anthocyanidins and proanthocyanidins,were not reported in the spore plants beforeIn order to investigate the composition and the biosynthesis of anthocyanidins in ferns,we firstly detected the content and types of anthocyanidins in 44 species of ferns and analyzed the distribution of anthocyanidins in ferns.Then,we screened 36 genes involved in anthocyanidin biosynthesis from Dryopteris erythrosora by transcriptome sequencing.Finally,the functions of five DeDFR genes were comprehensively analyzed from protein structure prediction,gene expression analysis,enzyme activity detection and transgenic Arabidopsis.The main results are as follows1.The contents of anthocyanidins,proanthocyanidins and flavonoids in 7 orders,14 families and 44 species of ferns collected in Jinggangshan national nature reserve were analyzed.The results showed that all of these ferns contained anthocyanidins Blechnaceae ferns contain large amounts of cyanidin,and Dryopteridaceae ferns not only contain cyanidin but also delphinidin.The types of anthocyanidins show a certain correlation with the family of ferns.In addition,higher levels of proanthocyanidins and flavonoids were detected in most samples of the families Blechnaceae and Dryopteridaceae.In general,the contents of anthocyanidins,proanthocyanidins and flavonoids were dominant in the families Blechnaceae and Dryopteridaceae,and the main anthocyanidins in Dryopteridaceae were more2.The transcriptome of the D.erythrosora was sequenced by the Illumina Hiseq 2000.The annotation results show that homologous genes related to anthocyanidin biosynthesis mainly include chalcone synthase(CHS),chalcone isomerase(CHI),and flavanone 3-hydroxylase(F3H),DFR,flavonoid-3'-hydroxylase(F3'H),flavonoid 3',5'-hydroxylase(F3'5'H),anthocyanidin synthase(ANS)and flavonoid 3-0-glucosyltransferase(UFGT).The results of sequence alignment and phylogenetic tree showed that these genes have low similarity and belong to different clades to the genes of seed plants,which may be related to the special evolutionary status of ferns3.The functions of the five DeDFR genes obtained from the transcriptome data of D.erythrosora.The results showed that the ORF regions of tiese DeDFRs were 972-1026 bp,and the similarity of encoded amino acids to Arabidopsis were 39.02-46.34%DeDFRl and DeDFR2 proteins have DFR catalytic function,which can catalyze DHK and DHQ to the target product,but have no effect on DHM.The Arabidopsis DFR mutant complementation experiment showed that the DeDFR2 gene can restore the phenotype and deepen the color of seed coat.The DeDFR3 protein does not have the function to catalytic substrate in vitro,and its transgenic Arabidopsis mutants do not restore phenotype.However,its overexpression in Arabidopsis wild type will promote development,and the seed size of the overexpressing strain is about 1.33 times that of the wild type.The DeDFR4 and DeDFR5 proteins also do not have the activity of catalyzed dihydroflavonol in vitro.In our results these two genes were mainly expressed under low light conditions in D.erythrosora.And the transgenic Arabidopsis with these two genes grow better then wild type under low light.In total,five DeDFRs genes have different regulatory functions on plant growth,and DeDFRl and DeDFR2 are involved in the biosynthesis of anthocyanidins4.Found a new type of DFR function,namely Arg-type DFR.In addition to DeDFRl and DeDFR2,which have been identified with DFR functions,Azolla filiculoides AfDFRl-6 and Salvinia cucullata ScDFR1-4 are also belong toArg-type DFR.Previously,Arg-type DFR had never been reported in seed plants,and it may be unique in ferns.Site-directed mutagenesis techniques and in vitro experiments were used to verify the effect of the DFR substrate binding critical site on DFR activities The results show that when the critical site was changed to Asp,the amount of product will be significantly reduced with DHQ as a substrate;when the critical site was changed to Asp,the ability of catalyze DHK decline,and the catalytic ability of DHM is lost.Therefore,the difference in the side chain of the amino acid residues at the key sites is an important factor affecting the catalytic ability of the substrate.The characteristics of Arg-type DFR can catalyze DHK and DHQ,and do not catalyze DHMIn summary,this study first analyzes the distribution of anthocyanidins in ferns,then explored the genes involved in the biosynthesis of anthocyanidins from the fern D erythrosora by transcriptome sequencing.By studying the function of DeDFRs gene,we investigated the effect of DFR genes on the biosynthesis of anthocyanidins in ferns These work not only have important value in studying the functional evolution of plant DFR,developing and utilizing fern anthocyanins,exploring the synthesis of anthocyanin compounds in ferns,but also providing information and methods for the study of other functional genes in ferns. |
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