Molecular Cloning Of The Chalcone Synthase Gene From Abelmoschus Manihot (L.) Medicus

Flavonoids, derivatives of chromone or chromane, are built upon a C6-C3-C6 flavone skeleton in which the three-carbon bridge between the phenyl groups is commonly cyclized with oxygen. There are diverse group of plants containing flavone, isoflavone, flavonol, chalcone, ect. Flavonoids form an important class of secondary metabolites which are abundant in all higher plants. They play important roles in flower pigmentation, protection against UV light, defence against phytopathogens, signal transduction between plants and microorgnism interactions, pollen fertilization and growing regulation. Flavonoids are the mostly active components in the officinal plants, and exhibit a wide range of human health-promoting activities that are currently the focus of intense study. Flavonoids have been found to own potent antioxidant and free radical scavenging activities in vitro. There is growing evidence from human consumption studies supporting the protective roles of flavonoids in cardiovascular diseases and cancer. Many flavonoids have been found to possess antiphlogistic, estrogenic or anti-allergenic properties.Chalcone synthase (CHS) is the first committed enzyme in the flavonoid biosynthesis catalyzing the condensation of 3 moleculars of malonyl-CoA and one molecular of hydroxycinnamoyl-CoA ester to form a naringenin-chalcone intermediate. Further isomerization and substitution of this central intermediate leads to the synthesis of other flavonoids. CHSs has been isolated from different plant species, including both monocots and dicots, some of their genomic DNAs and/or cDNAs have also been isolated. CHS is encoded by a multigene family in many plants, such as Petunia, Pea, Soybean, and so on. The amino acid identity among CHSs from different species is high, ranging from 79% to 91 %. The three-dimensional structure of CHS elucidates the chemical basis of plant polyketide biosynthesis and provides a framework for engineering CHS-like enzymes to produce new products. Enhancing flavonoid biosynthesis in chosen crops may provide new raw materials that have the potential to be used in foods designed for specific benefits to human health.In this study, a novel cDNA encoding chalcone synthase was cloned from the flower of Abelmoschus manihot (L.) Medicus by RT-PCR and RACE. The open read frame of the gene has 1173bp and encodes a polypeptide of 390 amino acids. We named this gene AmCHS. We presume this new cDNA is the CHS full-length cDNA by BLAST and alignment of nucleotide and amino acid. A plant constitutive expression vector under the control of CaMV35S promoter was constructed by inserting the AmCHS cDNA into pCAMBIA2301-pT Q 4A. The AmCHS gene was introduced to tobacco by Agrobacterium-mediated transfer method, and 15 individual kanamycin resistant plants have been obtained. Results of PCR analysis showed that the foreign AmCHS gene has been integrated into the tobacco genome in two transformants.