Cloning And Functional Identification Of The Genes Encoding The Key Enzymes In The Biosynthetic Pathway Of Flavonoids In Safflower

Safflower (Carthamus tinctorius L.) is an important oil crop and medicinal plant. Asthe secondary metabolites in safflower, the flavonoids are the major components of theextracts from the flowers with medicinal function. Hydroxysafflor yellow A (HSYA), oneof the most important flavonoids and unique presence in flower petals of safflower, playsa major role in the pharmacological effects of flavonoids. There are few studies about thebiosynthesis of flavonoids of safflower.Plant cell suspension cultures are widely used in plant biology as a convenient toolfor the investigation of a wide range of phenomena, bypassing the structural complexityof the plant organism in toto.The explants are placed on solid growth media (MS basalmedium supplemented with1mg/L TDZ and0.1mg/L NAA),callus tissue will appear onthe explants in the course of2–6weeks.The dense and green texture callus issubsequently cut from the explant and further subcultured in liquid medium (9.31mg/LNAA and0.563mg/L6-BA). The whole process,from plant to a stable cell suspensionculture, may take6–9months. We produce agrowth curve with the stable cell suspensionculture. Methyly asmonate and yeast elicitor were used to induce the flavonoidsbiosynthesis and growth condition.Gene expression analysis is increasingly important in the research of safflower.Quantitative PCR (qPCR) has become the powerful method for gene study. Referencegenes are the one of the major qualification requirements of qPCR because they canreduce the variability. Considering the similarity to reference genes,9candidate geneswere selected from the EST library of safflower: CtACT (actin), CtGAPDH(glyceraldehyde3-phosphate dehydrogenase), CtEF1(elongation factor1alpha), CtTUA(alpha-tubulin), CtTUB (beta-tubulin), CtPP2A (serine/threonine-protein phosphatase),CtE1F4A (eukaryotic initiation factor4A), CtBUI(Ubiquitin) and Ct60S (60S acidicribosomal protein). We assessed the expression stability of these candidate genesemploying four different algorithms. According to the analysis results, we used the CtUbIand Ct60S as reference genes and evaluated the expression of CtFAD2-10and CtKASIIin safflower. After analysis, we suggested the combinations of CtUbI and Ct60S assuitable reference genes for accurate normalisation of qPCR data for experimentsinvestigating the gene expression of safflower.The biosynthetic pathway of flavonoids is one of the most intensive study of plantmetabolic pathways.In this pathway, Chalcone synthase(CHS), Chalcone isomerase(CHI), Flavonol synthase(FLS), dihydromyricetin reductase(DFR), anthocyanidin synthase(ANS) and glycosyltransferase(GT) are involved in the formation of flavonoids.According to the analysis of EST libraries and microarray of safflower, we selected thegenes encoding the key enzymes in the biosynthetic pathway of flavonoids and thecircadian rhythmas of plant as the candidate genes. The full length of CtCHI1177,CtCHI3626, CtGT837, CtGT1097, CtA097C, CtGTD07C, CtFLS897, CtCKA2856,CtCKB33071and CtWNK3724were cloned successfully.Prokaryotic expression is the method using the bacterial strains to express thecorresponding protein in vivo. According to the sequences of the target genes, specificprimers were designed to get the target gene into the prokaryotic expression vector.Under appropriate culture conditions, IPTG was used to induce the expression of targetgenes in vitro. By double digestion and ligation, the target genes and pMAl-c5x wererecombinanted. The recombinant vector was transformed into the prokaryotic expressionhost strain BL21(DE3)Plys and producd recombinant proteins under0.3mM IPTGinduction. Using affinity chromatography and enzyme digestion, we got the purifiedprotein. In order to characterise the function of the enzyme, we use HPLC to detect thesubstrates and products after adding a certain amount of enzyme catalysis. The enzymeof CtFLS897was ientification.Agrobacterium-mediated transformation is a common tool for studies on genefunction of plants. By agrobacterium-mediated transformation, the recombinant vectorcontaining the target gene was transfered into plant cells and the gene of interest wasintegrated into the plant genomes. Seven key genes of flavonois biosynthesis and pMT39were reorganizated and transfected into Agrobacterium tumefaciens LBA4404byelectroporation method. Those containing the recombinant vector to agrobacterium wereselected for subsequent conversion operations. Floral dip method is the transformationusing to disseminate inflorescence plants. Arabidopsis inflorescence was disseminated byagrobacterium suspension and seeds were obtained by resistance screening to identifysuccessfully transformed transgenic Arabidopsis. The functionality of genes was verifiedby molecular biology and chemical composition tests of transgenic Arabidopsis. Thefluorescence detection, PCR validation, and chemical composition were used to test thefunction of the corresponding gene after the use of agrobacterium infection explantsinduced callus formation. We obtained the transgenic seedling of Arabidopsis by floraldip and also successfully obtained transgenic callus. The safflower suspension cell system provide a good platform for metabolic andmolecular biology of safflower; The identification of two stable reference genes willenable accurate and reliable gene expression studies of safflower;Agrobacterium-mediated transformation is an important method for gene function studiesin safflower; The enzymes can be obtained by prokaryotic expression in vitro which isthe essential research tool. These techniques and methods, which are used in geneticresearch of safflower, supplement and complement each other. The exploration ofmethods and establishment of subject system can establish the basis for genomics andmetabolomics research of safflower.