Cloning, Expression Analysis And Functional Analyses Of Transcription Factor Genes CtMYB1 Related With Safflower Yellow Pigment Biosynthesis

MYB transcription factor is one of the larger members in the family of plant transcription factors, participates cell differentiation and cell cycle regulation, and plays an important role in biosynthesis of plant flavonoid metabolism. The red flower petals in this study was acted as experimental material in different periods. According to safflower(Carthamus tinctorius L) transcriptome sequencing, the high expression Unigene123933 sequence was gained. A length of the safflower MYB transcription factor cDNA sequence was cloned from safflower petals using RT-PCR and RACE, named as CtMYB1.Using quantitative PCR and HPLC technique for different flowering period expression and pigment accumulation of CtMYB1 gene and hydroxy safflower yellow A. Construct yeast expression vector and study its transcription activation, construction of plant expression vector and transform in Arabidopsisplants, construct prokaryotic expression vector for prokaryotic expression analysis.The main research results are as follows:1. Using RT-PCR and RACE, CtMYB1 gene first was cloned from safflower petals, its total length is 893 bp, and its ORF is 750 bp, and encoding 249 amino acids. R2 and R3 MYB domain of the family was a typical R2R3-MYB discovered by amino acid sequence alignment. There was higher homology between an important regulatory factor CtMYB1 protein sequence and other plants in anthocyanin synthesis with multiple alignment analysis.2. Using quantitative PCR, the expression of CtMYB1 gene of safflower in different flowering period was: the seventh day> the fifth day> Bud> the seventh day of flowering.3. Determinating by HPLC chromatography, the expression of hydroxy yellow pigment A of safflower in different flowering period was: the seventh day> the fifth day> Bud> the seventh day of flowering.4. The constructing yeast effect plasmid pGBKT7-CtMYB1 was transferred into yeast competent cells, which did not grow in SD/-Trp-His-Ade + 3-AT-deficient medium. It is speculated that CtMYB1 was transcriptional activation suppressor factor.5. The constructing fusion expression vector pBASTA-CtMYB1 was successfully transferred 12 into Arabidopsis.6. The constructing CtMYB1 prokaryotic expression vector was transformed into E. coli BL21(DE3). Then, a higher purity of the target protein can be get 4 h after induction with IPTG 1.0 mmol/L at 28oC.We can get a higher purity of the target protein for the experiments of interaction with subsequent DNA.