Cloning of gene encoding chalcone isomerase (CHI) from P. candollei and expression of genes involved isoflavonoid biosynthesis pathway in seedling plants

Abstract

Pueraria candollei (Fabaceae), a Thai medicinal plant used for rejuvenation in elderly, is major source of isoflavonoids [1]. Like other flavonoids, isoflavones are synthesized from phenylpropanoid pathway [2]. The aims of this investigation were to clone the gene encoding chalcone isomerase (CHI) from P. candollei using PCR-base cloning and to study the expression of genes involved isoflavonoid biosynthesis pathway. Total RNA from leaf, stem and root of twenty-day-old seedling plants were extracted then the first-strand cDNA was synthesized to be DNA templates. For cloning of CHI, forward and reverse primers were designed according to chalcone-flavanone isomerase cDNA from P. lobata (GenBank no. Q43056). The plasmids contain 672 bp of open reading frame CHIs, code for 223 deduced amino acids, were sequenced and then were aligned to compare the similarity. Multiple alignment of CHI from P. candollei with other leguminous plants showed highly identity (>80%). In addition, the CHI encoding genes showed the active sites residues including conserve regions as same as CHI from other plants [3]. For expression study, semi-quantitative RT-PCR analyses were employed using primers designed based on existing sequences of 4 genes from P. lobata and 2 genes from Glycine max. The optimal PCR conditions for amplification of each target gene are summarized. The highest expressions of chalcone reductase, chalcone synthase, chalcone isomerase, isoflavone synthase and hydroxy-isoflavone dehydratase were found in roots whereas isoflavone-O-glucosyltransferase was found highly in leaves. The results are useful for further study in the isoflavonoid biosynthesis pathway in Pueraria candollei.