안개초(Gypsophila paniculata)로부터 Flavanone 3β-Hydroxylase 유전자의 분리 및 분석

Abstract

Flavanone 3β-hydroxylase (FHT) is an enzyme acting in the central part of the flavonoid biosynthesis pathway. FHT catalyses the hydroxylation of flavanone to dihydroflavonols in the anthocyanin pathway. In this paper we describe the cloning and expression of the genes encoding the flavonoid-biosynthetic enzyme FHT in Gypsophila paniculata L. A heterologous cDNA probe from Dianthus caryophyllus was used to isolate FHT-encoding cDNA clones from Gypsophila paniculata L.. Inspection of the 1471 bp long sequence revealed an open reading frame 1047 bp, including a 190 bp 5' leader region and 288 bp 3' untranslated region. Comparison of the coding region of this FHT cDNA sequence including the sequences of Arabidopsis thaliana, Citrus sinensis, Dianthus caryophyllus, Ipomoea batatas, Matthiola incana, Nierembergia sp, Petunia hybrida, Solanum tuberosum, Vitis vinifera reveals a identity higher than 69% at the nucleotide level. The function of this nucleotide sequences were verified by comparison with amino acid sequences of the amino-terminus and tryptic peptides from purified plant enzyme, by northern blotting with mRNA from wild type and mutant plants, by in vitro expression yielding and enzymatically active hydroxylase, as indicated by the small dihydrokaempferol peak. Genomic southern blot analysis showed the presence of only one gene for FHT in Gypsophila paniculata.