Molecular Cloning And Characterization Of Isoflavone Aynthase Gene From Astragalus Membranaceus(Fisch.) Bge.var. Mongholicus(Bge.) Hsiao

Isoflavone, which is one category of natural secondary metabolites, posseses special functions of health-care and prevention of many diseases. With the development of molecular biology and secondary metabolite engineering, the synthesis metabolite pathway of isoflavone was elucidated clearer , and some key enzymes' genes of this pathway were cloned from some plants, such as Glycin max, Cicer arietinum and Pisum sativum etc.Isoflavone synthase is the key enzyme of isoflavone synthesis pathway-phyenlalanine metabolism branch pathway. For further isoflavone synthase gene research, a novel isoflavone sythase gene was firstly isolated from roots of Astragalus membranaceus (Fisch) Bge. var. Mongholicus (Bge.) Hsiao via RACE technique. Characterization and functional prediction of this new gene were studied. The analytical results of the gene were very helpful to elucidate its function, molecular evolution and secondary metabolic engineering. The major experiment results from this work were followings:Astragalus membranaceus (Fisch.) Bge. var. Mongholicus (Bge.) Hsiao roots were treated by reduced glutathione for isoflavone synthase gene induction expression. Then total RNA was extracted. Using homology gene clone method, with the technique of RT-PCR, 673bp sequence was obtained. By application of RACE, the full length cDNA was l,995bp, including an open reading frame (l,578bp ) which encoded 525 amino acids. The gene was named as mhifs. The molecular weight of its deduced polypeptid was about 60kDa. Bioinformation analysis suggested that full length cDNA sequence of mhifs showed 82%, 84%, 82%, 83% similarity to Glycin max, Cicer arietinum, Pisum sativum and Glycyrrhiza echinata respectively, and isoflavone synthase amino acid sequence of Astragalus membranaceus (Fisch.) Bge. var. Mongholicus (Bge.) Hsiao had 80%, 83%, 79%, 83% identity and 88%, 89%, 87%, 88% similarity with Glycin max, Cicer arietinum, Pisum sativum and Glycyrrhiza echinata respectively. After analyzing amino acid conserved domains, it was found that Astragalus membranaceus (Fisch.) Bge. var. Mongholicus (Bge.) Hsiao isoflaovne synthase was similar to other leguminosae plants isoflavone synthase to have three conserved domains. Based on the above results, it could be inferred that mhifsmight have isoflavone synthase gene function.