Molecular Cloning And Analysis Of Flavones Synthases Ⅱ Gene From Scutellaria Baicalensis

Flavonoids, one kind of plant secondary metabolites which widely exist in plant kingdom, have a wide range of pharmacological activity. In flavonoid biosynthesis pathway need many enzymes’ involvement, and flavone synthase(FNS), a key enzyme in flavonoids biosynthesis pathway, controls the conversion from flavanones to flavonoids. In this paper, one FNS gene sequence was amplified from Scutellaria baicalensis leaves RNA, and then sequenced and analysed by bioinformatics tools. The main results were as follows:1. Genomic DNA was extracted from Scutellaria baicalensis by kit method. Two pairs of primers were designed by Primer Premier 5.0 for the amplification of FNS. Through optimizing PCR amplification system, one pair of optimal primers FNS001 was selected and amplification condition was optimized.2. One part of FNS gene was amplified by RT-PCR from wild Scutellaria baicalensis leaves RNA. The amplified fragment is about 700 base pairs as we expected. It then was recycled and cloned into p MD18-T vector and transformed into JM109 component cell. A recombinant plasmid was selected and identified by PCR, restriction enzyme digestion and sequencing.3. The results of bioinformation analysis showed that the amplified DNA fragment was 640 bp and inserted into p MD18-T in reversed direction. Campaired with the FNSⅡ gene complete sequcece of Perilla frutesanshoucens the homology is 46.6%, with FNSⅡgene complete sequcece of Medicago truncatula is 46.2%. It contained an ORF which has a size of 639 bp(open reading frame), could encode a 213 amino acid polypeptide that belongs to the cytochrome P450 superfamily. It contained a commom proline-rich region of FNSⅡ. So the cloned DNA fragment is one part of FNSⅡ gene.The sequence was registed to Gen Bank, the accession number is KU756482. The recombinant plasmid was named p MD18-T-FNS and kept in our lab for further research.