Characterization And Function Study Of Key Genes Involved In Flavonoid Biosynthetic Pathway In Carthamus Tinctorius L.

Safflower(Carthamin tictorius L.)is valued as a kind of Traditional Chinese Herbal by dry tubular flower with the effects of activating blood circulation and stimulating meridians.Modern pharmacological research demonstrates that it has a broad range of effects,for example,anti-thrombus,antioxidant,anti-inflammatory,anti-allergic.The main active components are kaempferol and its glucosides,quercetin and its glucosides,HSYA,carthamin.Up to date,flavonoid biosynthetic pathway has been well-documented in model plant,which originated from phenylalanine pathway and went through chalcone synthase into flavonoid biosynthesis.However,in safflower,its biosynthetic pathway has rare been reported,especially for unique chalcone pathway.In this study,basing on differential analysis of transcriptome in Y(HSYA-present)and W line(HSYA-absent),we screened out some significant regulation genes involved in flavonoid biosynthesis,including CHS,CHI,UGT,MYB.At first,by the method of RACE technology,5' end and 3' end of CtCHS1,CtCHI1,some UGTs and MYBs were cloned.Through SeqMan tool,the full-length were assembled.Multiple sequence alignment and phylogenic analysis predicted Ct CHS1 own 87% similarity with other CHI proteins from other species,and CtCHI1 share 92% similarity with CHI from Saussurea medusathese,wich can enhance apigenin content in hair root.In vitro,by the construction of recombination plasmid,CtCHI1 was successfully isolated and expressed and proved to own enzymatic activity that catalyzed 2,,4,,4,6,-tetrahydroxychalcone into naringenin,active CtCHS1 can converts p-coumaroyl-CoA and 3 malonyl-CoA into naringenin.In vivo,we constructed the transient expression plasmids.Through the transient expression in model plant mediated by GV3101,we concluded that CtCHS1 mainly was located in cytoplasm and CtCHI1 in nucleus.In vivo,we succeed to transform safflower Y line by agrobacterium-mediated pollen-tube method and obtained transgenic plants.In overexpression of Ct CHS1 transgenic plants,upstream genes PAL2,PAL3,4CL2,CHS4,CHS6 and Ct CHS1 were up-regulated.On the contrary,4CL1,4CL3,4CL5,DFR1 and CHI2 were inhibited.For quinochalcone cumpounds,HSYA and carthamin obviously increased by 19.83% and 29.48%,respectively.The rest of compounds decreased,especially for quercetin,quercetin-3-O-?-D-glucoside and luteolin by 50-60%,however,kaempferol and rutin decreased by 14.41% and 24.89%,kaempferol-3-O-?-D-glucoside by 17.06% and D-Phenylalanine by 39.51%.From this,we believed that CtCHS1 can positively regulate chalcone accumulation and inhibit flavonol branch production.In transgenic tobacco plant,overexpression of CtCHI1 can induce the expression of upstream genes,NtC4 H and Nt4 CL.HPLC results displayed that anthocyadin and quercetin contents dramatically decreased,while kaempferol increased by 10%~20%.Therefore,overexpression of CtCHI1 can direct to different metabolites branches.In transgenic safflower,overexpression of CtCHI1 can strength the expression of upstream genes,in parallel,inhibit the expression of CtF3 H and CtDFR2.To evaluate the whole metabolic database,the pattern recognition methods such as PCA and PLS-DA were performed.The unsupervised PCA was utilized as an unbiased statistical method to investigate general interrelation between groups and got a separation.This suggests a significant alteration in the metabolic profile induced by CtCHI1-overexpression.In parallel,to discover variation of metabolic profiles between control and ovx groups,a supervised PLS-DA method was used to promote the metabolites detection.And the PLS-DA model from negative mode generated better differentiation between groups.Metabolites analysis by UPLC-QTOF/MS demonstrated that CtCHI1-overexpression can enhance HSYA,rutin,kaempferol-3-O-rutinoside and dihydrokaepferol accumulation.Through differential expression in two lines,we figured out that CtUGT3,CtUGT16 and CtUGT25 own different character.Phylogenic analysis showed that CtUGT3 and CtUGT16 were classified under the UGT71 subfamily involved in metabolite process,whereas CtUGT25 has high identities with PoUGT both catalyzing the glycosylation of flavonoids and belonging to the UGT90 subfamily.Given the high identities of the reported flavonoid GTs in multiple sequence alignment,three CtUGT genes can be tentatively assigned as a flavonoid glycosyltransferase in C.tinctorius.Therefore,to functionally characterize UGT in safflower,CtUGT3,CtUGT16,and CtUGT25 were cloned and analyzedIn WoLF PSORT,protein subcellular localization predicted CtUGT3 may be located in cytoplasm,CtUGT25 in chloplast.Signal P4.1 Server predicted three UGTs have no signal peptide(SP).These results indicated that the three UGTs may not take part in protein transport and act in the catalytic activity of the enzyme in the cell cytoplasm and chloroplast.Further,we constructed “gene-compounds” coefficient network under MeJA-induced condition.This study indicated that CtUGT3 and CtUGT25 can enhance kaempferol-3-O-?-D-glucoside accumulation in Y line while promote the production of quercetin-3-O-?-D-glucoside in W line.CtUGT16 can positively regulate quercetin-3-O-?-D-glucoside content in both lines.In addition,we screened out 21 R2R3-MYB transcription factors from transcriptome database and firstly performed systematic bioinformatic analysis of MYB gene family in safflower.Utilizing multiple em for motif elicitation(MEME)and Simple Modular Architecture Research Tool(SMART),we analyzed 10 motifs location and function,which motif 3 and motif 6 were conserved DNA binding domain.By “gene–compound” correlation network,we figured out myb13 and myb14 were positively related to kaempferol-3-O-?-D-glucoside accumulation.In order to study MYB transcription factors regulation mechanism,safflower secondary yeast library was built firstly by our team.By the method of Y2 H co-transformation,we are trying to screen important interaction proteins with myb13(CtSDGL,CtEPGL,CtPHOX1 L,CtB2L,CtASC1,CtMPL1,CtMPL2),further study is ongoing.In briefly,overexpression of Ct CHS1 and CtCHI1 can positively regulate PAL and CHS expression and inhibit downstream genes expression,and powerfully promote quinochalcones production.CtUGT3,CtUGT16 and CtUGT25 may wildly participate in glycosylation of flavonoid in safflower.MYB13 transcription factor may positively regulate HSYA and kaempferol-3-O-?-D-glucoside production.This study provides important scientific basis for illustrating active compounds biosynthesis in safflower.