| Iris is a perennial herb in the Iridaceae family and is an important ornamental plant for garden applications.Species of Iris are rich and varied in form,with elegant blue-purple flowers,the coloring substance is mainly anthocyanins.There may be certain composition and content differences between different species of blue iris,thus forming different flowers,but the material basis and regulatory mechanism for the formation of such differences in flower color are still unclear,and the analysis of the mechanism of differential presentation of blue flowers is of great significance for the breeding of blue flowers.The DFR is an important enzyme downstream of the anthocyanin biosynthesis pathway,which often affects the expression of anthocyanins through specific selection of catalytic substrates or by regulating the expression of downstream genes.In this study,4 blue flowering iris species,Iris sanguinea,Iris typhifolia,Iris laevigata and Iris lactea var.chinensis,were used to analyze the material basis of different blue flowering iris coloration by anthocyanin metabolomics.The key gene of flower color regulation,DFR,were cloned and functionally verified.The mechanism of color presentation of blue iris was initially explored at the metabolite and molecular levels,laying the foundation for molecular breeding of blue flowers.The main findings were as follows:(1)The tepals of 4 species were measured using ‘Royal Horticultural Society Color Chart’and a compact colorimeter CR-10 Plus,all of which belonged to the purple-violet color family,but the CIELab spatial coordinate system constructed revealed large differences in the L*,a*,and b* parameters among the floral colors.(2)The precise quantification of anthocyanin glycosides in 4 species using liquid chromatography tandem mass spectrometry(LC-MS/MS),resulted in the detection of 61 anthocyanins,including 6 major groups of cornflower pigments,cyanidin,delphinidin,malvidin,pelnidin,peonidin,petunidin.Multiple linear regression equations were constructed and analyzed to determine that delphinidin was the main pigment for the blue color of the petals of 4 species.In addition,the accumulation of petunidin in Iris typhifolia and Iris laevigata caused their petals to shift from blue to purplish-red,while the accumulation of flavonoids gave a brighter color to Iris lactea var.chinensis.(3)The cDNAs obtained from the reverse transcription of total RNA of the tepals of the 4 blue iris species were used as templates to clone the different DFR genes of the four blue iris species,named IsDFR(GenBank MH091707),ItDFR(GenBank OP484852),IlDFR(GenBank OP484853),IlaDFR(GenBank KY907171.1)with gene lengths of 1071 bp,1075 bp,1068 bp,and 1074 bp,respectively,encoding 356,357,355,and 357 amino acids,respectively.Bioinformatics analysis revealed that all belonged to structurally stable hydrophilic proteins,and none of them had transmembrane regions or signal peptides.Among them,IsDFR protein and ItDFR protein have 7 glycosylation sites,IlDFR protein and IlaDFR protein have 8glycosylation sites,and all 4 DFR proteins have typical Epimerase structural domains.TheDFR protein sequences of 16 species with high similarity were selected to construct a phylogenetic tree,and were found to be the closest relatives to Iridaceae such as Iris x hollandica.Based on the Arabidopsis homologous protein interaction network,it was hypothesized that the four DFR proteins might affect the synthesis of phycocyanins,flavonols,catechins and proanthocyanidins,which might eventually influence the color formation mechanism of plants.(4)The pCAMBIA1300-GFP plasmid was linearized with two restriction endonucleases,BamHI and Sal I.The plasmid was ligated to the linearized vector by homologous recombination,and pCAMBIA1300-IsDFR-GFP,pCAMBIA1300-ItDFR-GFP,pCAMBIA1300-IlDFR-GFP,pCAMBIA1300-IlaDFR-GFP overexpression vectors,and the 4overexpression vectors were transferred into agrobacterium tumefaciens GV3101 using the freeze-transfer method,and were transferred into the model plant tobacco by agrobacteriummediated transfer,and were validated by antibiotic screening and PCR of resistant plants,demonstrating at the DNA molecular level that IsDFR,ItDFR,IlDFR and IlaDFR genes were successfully integrated into the tobacco genome,and three transgenic positive strains were obtained for each.However,the trans-IsDFR and trans-ItDFR strains were not retained due to the COVID-19.(5)The flower color of IlDFR transgenic tobacco was observed to be darker compared to wild-type tobacco(WT),while the flower color of IlaDFR transgenic tobacco was brighter and did not get significantly deeper.The anthocyanin content of perianth segments of transgenic and wild-type tobacco was measured by hydrochloric acid-methanol method,and it was found that the anthocyanin content of IlDFR transgenic tobacco was 65.54 μmol/g,which was108.20% higher compared with wild-type,while the content of IlDFR transgenic tobacco was28.90 μmol/g,which was 8.19% lower compared with wild-type tobacco.The expression of five key genes,including DFR,ANS,UFGT,ANR,and LAR,was detected in transgenic tobacco using real-time fluorescence quantification,and it was found thatDFR gene and ANS gene were significantly up-regulated in both IlDFR transgenic tobacco and IlaDFR transgenic tobacco,and UFGT gene was significantly up-regulated in IlDFR transgenic tobacco,while it was down-regulated in IlaDFR ANR and LAR genes were down-regulated in IlDFR and significantly up-regulated in IlaDFR transgenic tobacco,and the up-regulation of ANR and LAR genes in IlaDFR transgenic tobacco may directly or indirectly affect the synthesis of anthocyanins,thus not The up-regulation of ANR and LAR genes in IlaDFR transgenic tobacco may directly or indirectly affect the synthesis of anthocyanins,thus not deepening the flower color of tobacco.Both transgenic tobaccos showed faster growth,earlier flowering than wildtype tobacco,and it is possible that the DFR gene may have effects on other aspects of plant growth regulation. |
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