Functional Study Of Glycosyltransferase Genes CtUGTs In The Flavone Biosynthesis Pathway Of Carthamus Tinctorius L.

Carthami Flos,a traditional Chinese medicine which is widely used in clinic for promoting blood circulation and removing blood stasis.Modern pharmacological studies have shown that its main pharmacodynamic substances are chalcone compounds represented by Hydroxysafflower yellow A（HSYA）and flavonols represented by nicotinosin,which have good protective activities against cardiovascular and cerebrovascular injury.The biosynthesis pathway of flavonoids has been widely studied in a variety of plants and crops.However,the synthesis pathway of flavonoids in safflower especially the post modification enzymes related to the biosynthesis of flavonoids glycosides in safflower such as HSYA has not been fully clarified.UDP glycosyltransferase（UGT）is a terminal modifying enzyme for the formation of flavonoid glycosides,and the completion of flavonoid glycosylation is closely related to the activity of the compound.Therefore,in-depth screening and research on safflower flavone glycosyltransferase is of great significance to explain the molecular mechanism and directionally regulation of safflower quality.In this paper,58 UGTs genes were screened based on existing safflower corolla transcriptome database.Pearson correlation analysis was carried out based on the expression of corolla genes at different flowering time points in the gene chip and the content of active components in the metabolome database.The research showed that CtUGT18 was closely related to the accumulation of HSYA,naringin,kaempferol-3-O-rutoside,phenylalanine and carthamin（r>0.5）.And CtUGT25 was closely related to the accumulation of nine active components including HSYA,quercetin,quercetin-3-O-glucoside,kaempferol-3-O-glucoside,luteolin,rutin,scutellarin,kaempferol-3-O-rutoside and phenylalanine（r>0.5）.The total length of CtUGT18 gene was 1738 bp,including 1458 bp ORF region,encoding 485 amino acids.The molecular formula of the protein was C₂₄₄₄H₃₇₉₇N₆₄₅O₇₁₄S₃₃,the molecular weight of the protein was 54697.91 Da,and the theoretical isoelectric point（PI）was 5.73;the total length of CtUGT25 gene is 1746 bp,including 1473 bp in ORF region,encoding 490 amino acids.The molecular formula of the protein was C₂₄₉₅H₃₉₂₉N₆₆₉O₇₁₀S₂₀,the molecular weight of the protein was 55298.90 Da,and the theoretical isoelectric point（PI）was 5.92.Wolf PSORT subcellular localization predicted that CtUGT18 was located in the cytoplasm and CtUGT25 was in the nucleus.Both CtUGT18 and CtUGT25 were unstable hydrophilic proteins without transmembrane region,which were non-transmembrane proteins.The analysis of CtUGT18 and CtUGT25 expression characteristics in different parts of safflower showed that CtUGT18 mainly accumulated in the root,CtUGT25 mainly accumulated in the corolla and its expression was highestduring full flowering stage which was in accord with the flavonoids accumulation mode in the corolla of safflower.So it was speculated that CtUGT25was involved in the biosynthesis of flavones in the corolla.In order to further study the function of CtUGT18 and CtUGT25 in safflower,a constructed eukaryotic expression vectors CtUGT18-pmt39 and CtUGT25-pmt39 were transferred into Agrobacterium GV3101,and the genes were transformed by pollen tube channel method to obtain 7 strains of CtUGT18 overexpressed safflowers and 8 strains of CtUGT25 overexpressed safflowers.The target genes expression of T₂transgenic safflower were analyzed by q RT-PCR.The expression of CtUGT18 gene in CtUGT18-ovx plants was increased by 2.78 times and that of CtUGT25 gene in CtUGT25-ovx plants was increased by2.17 times.The content of flavonoids was determined by UPLC-Q-TOF/MS.The results showed that kaempferol-3-O-β-D-glucoside and Orientin increased significantly after overexpression of CtUGT18 while the content of kaempferol-3-O-β-D-rutoside decreased.It was speculated that CtUGT18 mainly responsible for the participation of kaempferol-3-O-β-D-glucoside and Orientin;the contents of kaempferol-3-O-β-D-glucoside and HSYA increased significantly（p<0.05）after the overexpression of CtUGT25 indicating that CtUGT25 may have substrate heterogeneity and participate in the synthesis of chalcone glycosides HSYA and other unique compounds in safflower.By constructing the prokaryotic expression vectors of CtUGT18-pet28a（+）and CtUGT25-pet28a（+）,the proteins of CtUGT18 and CtUGT25 were expressed in E.coli Rosetta（DE3）,and their glycosyltransferase functions were verified in vitro.The results showed that CtUGT25 could catalyze the glycosylation of quercetin,naringin,apigenin,kaempferol,luteolin and 2-hydroxynaringin;CtUGT18 has catalytic activity for2-hydroxynaringin.and their glycosyltransferase functions were verified in vitro based on the prediction results of Autodock Vina docking protein substrate.The results showed that CtUGT25 which had two active pockets could catalyze the glycosylation of quercetin,naringin,apigenin,kaempferol,luteolin and 2-hydroxynaringin;CtUGT18 has catalytic activity for 2-hydroxynaringin.In conclusion,two glycosyltransferase genes,CtUGT18 and CtUGT25,which are closely related to the synthesis of safflower flavonoids,were screened in this paper.Their molecular characteristics were analyzed,and their functions were verified in safflower by transgenic means.It was proved that CtUGT18 was a glycosyl modifying enzyme for kaempferol-3-O-β-D-glucoside accumulation;CtUGT25 promoted the accumulation of kaempferol-3-O-β-D-glucoside and HSYA.Therefore CtUGT25 might be the glycosyl modifying enzyme for the biosynthesis of HSYA,which was the unique active component in safflower.What’s more,CtUGT25 protein showed catalytic activities for a variety of flavone substrates.It offered an important reference to further regulate the quality of safflower through molecular biotechnology,especially the industrialized production of the unique component HSYA in safflower,and also provided data for the research of related genes of other plants.