Analysis of elicitor inducible cytochrome P450 induction in Astragalus chrysochlorus cells

Abstract

In this study, elicitor-inducible cytochrome P450 biosynthesis in Astragalus chrysochlorus was investigated for further analysis on phenylpropanoid metabolism. In order to analyse cytochrome P450s under yeast extract elicited conditions, we used non-radioactive P450 targeted differential display method. The P450 targeted differential display of mRNA technique was performed with upstream primers based on the conserved heme-binding region [PFG] of P450s, as a result 56 clearly differential bands were revealed; 37 of the bands were correctly analysed, and one of the PCR products was contained the P450 fingerprint. This sequence has been confirmed to be up-regulated and subsequently cloned and sequenced. Homology analysis of the 400 bp long sequence revealed that 81 % similarity with cinnamate 4-hydroxylase in the manner of amino acid. Quantitative real-time-PCR analysis showed that putative C4H gene was up-regulated 13,24-fold by 6h yeast extract treatment unlike untreated control. 1338 bp long cDNA fragment (Accession no: GQ844863) of A. chrysochlorus C4H (AcC4H) has been obtained by PCR with degenerate primers. Bioinformatics analyses revealed that putative AcC4H (1338 bp) was highly similar (95 %) to trans-cinnamate 4-monooxygenase (EC 1.14.13.11). As a result, we have isolated a putative C4H fragment from A. chrysochlorus suspension cells under yeast extract elicited conditions. This knowledge will use for obtaining whole C4H sequence, and to manupulate phenylpropanoid metabolic pathway of this medicinal plant.