Cloning and molecular characterization of a functional flavonoid 3'-hydroxylase gene from Brassica napus.

A flavonoid 3'-hydroxylase (F3'H) gene, denoted BnF3'H-1, was cloned from oilseed rape (Brassica napus). The gene of 3038 base pairs (bp) contains 3 introns. The complementary DNA (cDNA) consists of 1820bp and has an open reading frame of 1536bp encoding a polypeptide of 511 amino acids with a molecular weight of 56.62kDa and an isoelectric point of 7.08. BnF3'H-1 shows high homology to known F3'H genes, especially F3'H from Arabidopsis thaliana. Untranslated regions (UTRs) may play important roles in regulating the expression of BnF3'H-1. Besides containing a Kozak sequence, the first 77-bp region is C-rich but G-poor, and the 26-bp 5'-UTR contains 3 sites of ACCACT-like sequences. Alternative polyadenylation in the 3'-UTR is adopted by this gene to generate heterogeneous transcripts. Conserved domain search and motif characterization identified BnF3'H-1 as a cytochrome P450. All F3'H-featured motifs, VVVAAS, GGEK and VDVKG, are unchanged in BnF3'H-1. The N-terminal signal peptide/anchor and 3 transmembrane helices were predicted in BnF3'H-1, and its subcellular localization is most probably at the endoplasmic reticulum. Since 16 phosphorylation sites could be predicted, phosphorylation may be a necessary post-translational modification of BnF3'H-1. The secondary structure is dominated by alpha-helices and random coils. Most helices are located in the middle region, while extended strands mainly intersperse in terminal regions. DNA gel blot analysis indicated that 2 different F3'H genes might exist in B. napus. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and RNA gel blot analysis showed that flowers have the highest F3'H expression, followed by pericarp and seed, and lower levels in some other organs. This species-featured expression pattern is in obedience to multiple functional roles that F3'H gene(s) play(s) in various organs of B. napus. The BnF3'H-1 coding region was expressed in Escherichia coli, and enzyme activity of the His-tagged protein was demonstrated by monitoring the conversion of the substrate naringenin using high-performance liquid chromatography (HPLC), suggesting that BnF3'H-1 is catalytically functional. RT-PCR analysis suggests that transcription level of the F3'H gene(s) is not the reason for the different seed colorations found in near-isogenic lines (black-seeded L1 and yellow-seeded L2) of B. napus.