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

Summary 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 1820 bp and has an open reading frame of 1536 bp encoding a polypeptide of 511 amino acids with a molecular weight of 56.62 kDa 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 α -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 .