Construction Of Expression Vector、genetic Transformation And Functional Analysis Of Flavonol Synthase Gene In Carthamus Tinctorius L.

Carthamus tinctorius L. is a dry tubular floret annual herb. It is used mainly for invigorating the circulation of menstruation, promoting blood circulation, reducing pain and many kinds of pharmacological action. Flavonoid is one of the main effective components in Safflower, flavonoids metabolic pathway has been researched clearly in the genetic level. Flavonol synthase(FLS) is one of the key enzymes in the flavonoids metabolic pathways,which plays an important role in processes of plant development.On the basis of the full-length cDNA of FLS gene was obtained from petals of Carthamus tinctorius L, FLS gene of "jihongzaoshu" full-bloom stage petals was subcloned.Plant expression vector with safflower FLS gene was constructed and transferred into Arabidopsis thaliana,flavonoids content was determined in leafs.Meanwhile, prokaryotic expression vector with safflower FLS gene was constructed for over expression in e. coli. This study provided the foundation for studying the FLS biology function and flavonoids biosynthesis mechanism. The main results as follows:1) According to the full-length cDNA of FLS, specific primers were designed. Total RNA of "jihongzaoshu" full-bloom stage petals is extracted and reverse transcribed into cDNA as template. Safflower FLS gene was cloned. The cloned FLS gene sequence was identical to the obtained one by sequence analysis. The full-length is 1201 bp.2) Plant expression vector with safflower FLS gene was constructed by DNA recombination. Transferred into agrobacterium tumefaciens EHA105 and infected Arabidopsis thaliana and by Floral Dip.28 of the strains in resistant plants were detected by PCR,of them, 20 strains showed target bands..It means that the target gene has been transferred into Arabidopsis thaliana and successfully.3) Flavonoids content was determined in T3 generation of Arabidopsis thaliana, the results showed that transgenetic Arabidopsis was higher than wild type, the highest strain increased nearly 2.2 times. It means that FLS gene played an important role in flavonoids biosynthesis mechanism.4) FLS gene and pET28a(+)vector were ligased after restriction enzyme digestion. Constructed the pET28a-FLS prokaryotic expression vector and obtained 44 kD protein. It indicated that FLS gene can be successfully expressed in e. coli. Lay a foundation for subsequent research on the activity of the gene.