Cloning and characterisation of chs-specific DNA and cDNA sequences from hop ( Humulus lupulus L.)

Abstract

A complete sequence of chalcone synthase (CHS) gene from hop was cloned. The gene designated chs_H1 consists of two exons and one 187 bp intron. CHS protein predicted from chs_H1 cDNA has 42.5 kDa and retains conserved domains and residues including 26 amino acids at positions identical to those identified by crystallography as characteristic for catalytic domains of alfalfa CHS (EC 2.3.1.74). Cloned CHS_H1 protein shows specific CHS activity with 4-coumaroyl-CoA. Structure modelling revealed clear differences between CHS_H1 and phlorisovalerophenone synthase, the only published CHS-like homologue from hop. Conserved motifs like H, and G boxes characteristic for the light regulated and stress inducible genes were identified within promoter region of chs_H1 gene. Highly specific expression of chs_H1 mRNA was detected by quantitative RT PCR in glandular trichomes during cone maturation. Much lower, but significant levels of chs_H1 mRNA were detected at the stage of hop flowering in petioles (100%), developed flowers (96%), and in stem apexes (78%), while the lowest levels of mRNA were found in the roots (31%) and leaf blades (9%). Southern blot analyses predicted at least five additional chs-like genes related to chs_H1. A genomic arrangement different from phlorisovalerophenone synthase sequences was found for these genes. RFLP analyses using DNA from 15 genotypes revealed several distinct dendrogram clusters, suggesting specific re-arrangements of hop chs-like genes during evolution and/or during the breeding and selection processes.