Cloning And Functional Verification Of Anthocyanin-related Genes In Begonia Semperflorens

Begonia semperflorens,perennial evergreen herbaceous flowers.Because it blooms in four seasons.Red flower green leaves and long ornamental period,convenient cultivation and management of soil and climate adaptability is very strong,used for street and landscaping.Flower color is one of the most important quality traits of ornamental plants,and about 80 % of angiosperms flower color is determined by anthocyanin;in this study,two anthocyanin structural genes BsDFR and BsUFGT were cloned and analyzed using Begonia semperflorens as materials,and the mechanism of these two genes regulating flower color formation of Begonia semperflorens was studied.The main research results are as follows :1.An anthocyanin biosynthesis pathway gene,named BsDFR(OL329827),was cloned from B.semperflorens.The length of BsDFR was 1020 bp,encoding 339 amino acids,the relative molecular mass was 38122.76,the theoretical isoelectric point p I was 5.91,and the total atomic number was 5331,indicating that it was a stable protein.The total average hydrophobic index is-0.228,belonging to hydrophilic protein.In the secondary structure,α-helix and random coil are more.BsDFR encodes a non-transmembrane protein without signal peptide characteristics,suggesting that it is a non-secretory protein.Domain analysis showed that DFR protein encoded by BsDFR gene contained dihydroflavonol 4-reductase domain belonging to PLN02650 supergene family,and NADB-Rossmann super family conserved domains,namely NADPH binding domain and substrate specific binding domain.Phylogenetic tree analysis showed that BsDFR had the closest relationship with Vv DFR.The subcellular localization results showed that BsDFR was localized to the cytoplasm.2.An anthocyanin biosynthesis pathway gene,named BsUFGT(OL329828),was cloned from B.semperflorens.The length of BsUFGT was 1536 bp,encoding511 amino acids.The molecular weight was 56385.34,the theoretical isoelectric point was 5.25,and the total number of atoms was 7920,which belonged to unstable protein.The total average hydrophobic index is-0.122,belonging to hydrophilic protein.In the secondary structure,α-helix and random coil are more.The protein encoded by BsUFGT is non-transmembrane protein and has no signal peptide,so it is speculated to be non-secretory protein.Domain analysis showed that the UFGT protein encoded by BsUFGT gene had a UDPGT domain located in 275-441,which was a member of the UDPGT superfamily and had a UDP-flavonoid glycosyltransferase Yji C characteristic region.Phylogenetic tree analysis showed that BsUFGT was closely related to Syzygium oleosum.Subcellular localization results showed that BsUFGT was mainly localized in cell membrane and nucleus.3.The anthocyanin content in green leaves,red stems,bracts and full blooms of B.semperflorens in the four seasons increased gradually,and the anthocyanin content in full blooms was the highest.The two anthocyanin structural genes BsDFR and BsUFGT have different transcriptional expression levels in the above tissues,showing a gradual upward trend.The transcription expression of BsDFR and BsUFGT was positively correlated with anthocyanin content in these four tissues,especially in full bloom.4.BsDFR and BsUFGT of B.semperflorens were transformed into Nicotiana tabacum ‘ NC89 ’ by Agrobacterium-mediated leaf disc method for functional verification.With tobacco Nt Tub A1 reference gene as the control,semi-quantitative results showed that tobacco Nt Tub A1 reference gene was normally expressed in both wild-type and transgenic lines.BsDFR and BsUFGT genes could be normally expressed in transgenic tobacco lines,but not expressed in wild-type lines,and the band brightness of transgenic tobacco was stronger than that of wild-type,indicating that BsDFR and BsUFGT genes were successfully transcribed and expressed in tobacco lines.Compared with wild type,corolla color of BsDFR and BsUFGT transgenic tobacco turned dark red,and corolla color of BsDFR transgenic tobacco and BsUFGT transgenic tobacco turned red.Royal Horticultural Society of the UK compared the color of wild-type tobacco corolla with RED-PURPLE 64D;BsDFR tobacco corolla color OE1-RED-PURPLE 63 B,OE2-RED-PURPLE 63 A,OE3-RED-PURPLE 63A;BsUFGT tobacco corolla color OE1-RED-PURPLE 63 B,OE2-RED-PURPLE 67 C,OE3-RED-PURPLE 68 A.The anthocyanin components were determined by high performance liquid chromatography(HPLC).The main anthocyanin components of BsDFR and BsUFGT transgenic tobacco corolla and wild type tobacco were cyanidin-3-O-rutinoside.Compared with wild type,BsDFR and BsUFGT transgenic tobacco had significant differences in the content of cyanidin-3-O-rutinoside.With wild type as control,real-time quantitative qPCR analysis showed that in BsDFR and BsUFGT transgenic tobacco corollas,except for the low expression of flavonoid 3 ’ 5’hydroxylase(F3 ’ 5’H)gene,the gene expression of other anthocyanin biosynthesis pathways was up-regulated in varying degrees.