Establish Safflower (Carthamus Tinctorius L.) In Vitro Regeneration System, Clone And Identify Genes Of Key Enzymes On Flavones Biosynthesis Pathway

Safflower (Carthamus tinctorius L.) is an important annual or biennial plant, belonging to the family Compositae. Its florets are harvested as a traditional Chinese medicine with pharmacological effects of dilatating coronary artery, lowering blood pressure, anti-coagulation, anti-inflammatory and analgesic. Flavones are the main active components in safflower and their contents decide the quality of this drug. So, clone and identify genes of key enzymes on safflower flavones biosynthesis pathway have great significance to clarify the molecular mechanism of safflower quality formation. It will also set basement for the regulation of safflower flavones biosynthesis through transgene. An efficient and reproducible plant regeneration system is a prerequisite for successful plant trangenes. This study aimed to establish an in vitro safflower regeneration system, clone and identify genes of key enzymes on safflower flavones biosynthesis pathway.As results, cotyledons cut from6-8days old seedlings were the most responsive explants, the culture temperature set at24/16℃rhythmic condition, illuminance at9000lux and humidity at60%were the most favorable culture condition. Medium (14) MSo+TDZ (12.0mg/L)+IBA (2.5mg/L)+2-ip (1.5mg/L) had a highest regeneration frequency of79.1%. The regenerated buds at this condition was healthy and reproducible, it demonsteted medium (14) was a desirable inducing medium for safflower in vitro regeneration. The Regenerated buds elongated successfully and grew well on MS basal medium.Contig897、Contig1988、Contig2129、Contig3482、Contig3855were candidate genes of safflower F3H (flavanone3-hydroxylase) screened by our laboratory. In this study, we took these5Contigs as research objects and cloned5’and3’fragments of the5Contigs by RACE. We had got the full-length gene sequences through sequencing and linking the sequences of5’and3’fragments of each Contig and cloned these5full-length genes by PCR. Bioformatic analysis showed that, Contig897was highly homologous with F3H of other plants for more than72%identities and the anino acid sequence coded by Contig897had80.16%consistency with F3H of other plants in SWISS-PROT enzyme database. Contig1988and Contig3482had64%and45%homologous with predicted Solanum lycopersicum FLS (flavonol synthase) or F3H-like partial sequence separately. The anino acid sequence coded by Contig1988and Contig3482had74.83%and75.44%consistency with F3H of other plants in SWISS-PROT enzyme database separately. Contig2129had60%homologous with putative Ricinus communis FLS or F3H sequence and the anino acid sequence coded by Contig2129had75.73%consistency with F3H of other plants in SWISS-PROT enzyme database. Contig3855had64%homologous with predicted Glycine max FLS or F3H-like sequence and the anino acid sequence coded by Contig3855had74.36%consistency with F3H of other plants in SWISS-PROT enzyme database.Subsequently, we tried to validate functions of these5genes through prokaryotic expression study. We obtained the specific fragements P897and P2129of Contig897and Contig2129by PCR. Analysis sequences of P897and P2129showed P897was highly homologous with F3H of other plants and it could be applied to prokaryotic expression experiment. P897and pET-32a (+) vector were cut with designed endonuclease and linked to constitute the recombinant plasmid. Recombinant plasmid was successfully tranferred to BL21(DE3)pLysS host cell. The positive strain was induced at different concentration and for different inducing time. Result of induction suggested bacteria OD600at0.6induced for3-5h and bacteria OD600at0.8induced for3h were suitable inducing conditions.This study had established an efficient safflower regeneration system, cloned5genes of key enzymes on safflower flavones biosynthesis pathway and initially identified functions of these5genes. Results of this study had set basement for clarifing the molecular mechanism of safflower quality formation and regulating safflower flavones biosynthesis through transgene.