Flavonoids Biosynthesis Pathway Chalcone Synthase Genes Function Research In Carthamus Tinctorius L.

Flavonoids are an important class of secondary metabolites with a wide range of pharmacological activity and determine the quality of a lot of Chinese herbal medicines.Thus,studying the gene function in flavonoid biosynthetic pathway is crucial to elucidate the molecular mechanism of the quality formation in traditional Chinese medicine.Safflower(Carthamus tinctorius L.)is a annual herb of Asteraceae family,which its floret is commonly used for blood circulation and stasis analgesic in traditional Chinese medicine.Modern pharmacological studies confirm that it has a variety of pharmacological effects,including anti-clotting,anti-oxidation,anti-inflammatory,analgesic and prevention of thrombosis.Quinochalcones and flavonols are considered as the characteristic and active constituents of safflower,such as safflor yellow A and hydroxysafflor yellow A,quercetin and its glycosides,kaempferol and glycosides.However,progress on safflower flavonoid biosynthetic pathway were rather slow because plant secondary metabolites biosynthetic pathway is extremely complex,and HSYA,as a quality ingredients in safflower,is only specific accumulation in flower,combining with the difficult of safflower tissue regeneration and rooting,which collectively restricting the pace of enhancing safflower quality through plant genetic engineering.Numerous studies showed that Chalcone synthase was positioned at the entrance of flavonoid metabolism pathway,regulating the synthesis of flavonoid product.Therefore further research on chalcone synthase gene function in safflower is of significance important to explain the molecular mechanisms of quality formation and thereby oriented regulate quality of safflower.On the basis of our previous studies on constructing cDNA library of safflower Corolla,gene sequencing and annotation as well as comparison among gene expression in different flowering time on the Corolla safflower via situ synthesis microarray,the gene expression of HSYA timing differences were obtained.Also,significant difference gene in flavonoid metabolic pathway in KEGG database were obtained through KOBAS analysis,such as cinnamic acid 4-hydroxylase(C4H),chalcone synthase(CHS),chalcone isomers enzyme(CHI),2-hydroxy flavanones enzyme(F2H),AT4G33360 genes.And CHS(chalcone synthase,CHS)gene CtCHS533 and other genes in flavonoids metabolic pathways were cloned.On the basis of investigation above,two chalcone synthase,namely CtCHS3720 and CtCHS1515,were first cloned from safflower on the basis of the pre-building corolla cDNA library and sequencing.Bioinformatics analysis showed that Ct CHS3720 amino acid sequence had the highest homology with chrysanthemum(Chryscanthemumboreale)CHS amino acid sequence(AGU91424.1),showing 94% of coverage and 91% of matching degree.The encoding protein molecular weight and isoelectric point of CtCHS3720 separately were 43667.25 Da and 5.72 according to prediction on protparam.While the highest homology of amino acid sequence of CtCHS1515 displayed 93% of coverage and 82% of matching degree with amino acid sequence of Comospore Yang(Populustrichocarpa)CHS(XP006375238.1).The encoding protein molecular weight and isoelectric point of CtCHS1515 separately were 43016.36 Da and 5.67 according to prediction on protparam.Based on the phylogenetic tree analysis,The distance among CtCHS3720,CtCHS533 and CtCHS1515 were far in clustering branches,proving a big difference in catalytic function among three CHS genes.To further study the three CHS gene in safflower,two species of safflower(HSYA type with main component of HSYA(Yunnan Weishan species)and KAEG type with main component of kaempferol and their glucoside(Xinhonghua No.7)grown in the greenhouse were sprayed with methyl jasmonate(MeJA,100?M)to head of ball Corolla on the first flowering day.After 0.1 h,3 h,6 h,12 h,the flowers were collected and RNA were abstracted.Then the relative expression of CtCHS533,CtCHS3720 and CtCHS1515 were analysed using qPCR method.The results showed that three genes in response to MeJA induced patterns varied in different varieties.CtCHS3720 gene expression at 0.1 h,3 h and 6 h in Weishan species were significantly increased,especially the expression level at the induction of 6 h was increased by 2.3-fold.The CtCHS3720 expression induced 12 h did not change significantly.In Xinhonghua No.7 species,CtCHS3720 gene expression under MeJA induce was somewhat lower,which reduced most significantly after 6 h and 12 h induction.In Weishan species,the CtCHS1515 gene expression after 0.1 h ?3 h? 6 h and 12 h inducion were significantly decreased.The CtCHS1515 expression in different time points in Xinhonghua No.7 species were lower in response to MeJA induction.The CtCHS533 gene expression were lower in response to MeJA induction after 3 h?6 h and 12 h,and the Xinhonghua No.7 species in MeJA induced 0.1 h the expression was significantly increased,while its expression were not significantly changed after 3 h 6 h and 12 h inducion.These results indiacted that CtCHS3720,CtCHS1515 and CtCHS533 genes may be involved in the formation of different chemical type strains in flavonoid biosynthetic pathway of safflower.12 flavonoids in safflower corolla with MeJA treatment at different time points(0.1 h,3 h,6 h,12 h),containing D-Phenylalanine,Hydroxysafflor yellow A,Rutin,Kaempferol-3-O-?-D-glucoside,Kaempferol-3-O-?-rutinoside,Kaempferol,Apigenin,Dihydrokaepferol,Quercetin-3-?-D-glucoside,Scutellarin,Carthamin,Luteolin were detected using UPLC-QTOF / MS.It was found that 12 flavonoids in different strains are of significant different accumulation patterns induced by MeJA.Hydroxysafflor yellow A,Carthamin,Luteolin,Kaempferol-3-O-?-D-glucoside only in Weishan species has accumulated,in the induction of 3 h,6 h.these compounds in MeJA-treated corolla was higher than those in control group.While the accumulation of Kaempferol,Kaempferol-3-O-?-D-glucoside,Luteolin,Quercetin 3-?-D-glucoside is subject to different degrees of inhibition.Dihydrokaepferol,Apigenin,Scutellarin had accumulated only in Xinhonghua No.7.After 0.1 h,3 h,6 h induction,D-Phenylalanine,dihydrokaepferol and Kaempferol Quercetin-3-?-D-glucoside were suppressed,while Kaempferol-3-O-?-rutinoside,and Rutin persistently rised after the induction,Scutellarin were suppressed in all the time.suggesting that different accumulation patterns and different types of safflower strains of flavonoids may be an important cause of the formation of chemical type.The associated analysis between the gene expression of CtCHS3720,CtCHS1515 and CtCHS533 the amount of flavonoid compound in response to MeJA were carried out.The results showed that Hydroxysafflor yellow A,D-Phenylalanine and Carthamin in Yunananweishan species were positively correlated with CtCHS3720,CtCHS1515 and CtCHS533expression(r =0.92,0.88,0.76);CtCHS3720 and CtCHS533 may be hey genes of Hydroxysafflor yellow A and Carthamin.The gene expression of CtCHS1515 displayed positive correlation(r =0.64)with Scutellarin in Xinhonghua No.7 species,indicating CtCHS1515 may be a key gene of cutellarin flavonoids formation of Xinhonghua No.7 species.Plasmid of CtCHS3720 with eukaryotic expression vector(pMT39)was constructed by seamless cloning and then transformed to Agrobacterium tumefaciens LBA4404.The result of subcellular localization of onion epidermal displayed that CtCHS3720 was located in the nucleus and the cell membrane.Enzymatic reaction system was build and further demonstrated that the encoding enzymes of CtCHS3720 could catalyze p-coumaryl-COA and malonyl-COA to generated naringenin in vitro,confirming the function enzyme activity of CtCHS3720 in the safflower.