白菜和甘蓝类黄酮3‘-羟化酶基因的克隆和序列分析

Flavonoid 3’-hydroxylase (F3‘H) is a key enzyme involved in biosynthesis of flavonoids such as anthocyanins, flavonols and proanthocyanidins for determination of important plant traits such as flower colors, seed coat pigments and plant surface pigmentation. In this research, the 3067-bp BrF3‘H and 3128-bp BoF3’H full-length genes were cloned from Brassica rapa ssp. oleifera and B. oleracea L. var. acephala DC respectively. They both have 3 introns with standard splicing. Their leader sequence contains a predicted Kozak sequence and is C-rich but G-poor, and more than one potential poly(A) signals exist in their 3‘UTR. Full-length cDNAs of BrF3’H and BoF3‘H are 1871 bp and 1874 bp respectively, both encoding 511 amino acids in their open reading frames (ORFs). BrF3‘H has a molecular weight of 56.636 kD and an isoelectric point of 7.10, whereas 56.567 kD and 7.08 for BoF3‘H. BrF3‘H and BoF3‘H are typical cytochromes P450, containing all the F3‘H-featured motifs. Based on predictions, BrF3‘H and BoF3‘H contain a signal peptide and at least 2 transmembrane domains, being located at the endoplasmic reticulum and probably modified by phosphorylation. α-helices and random coils are their major secondary structures, and most helices are located in the middle region. BrF3‘H and BoF3‘H are orthologous genes of Brassica napus BnF3‘H-1 and Arabidopsis thaliana AtF3‘H with high homologies, and they also show wide homologies to other F3‘H genes. GenBank sequence tags and this research both point to only 1, 1, and 2 F3‘H gene(s) in B. rapa, B. oleracea and B. napus respectively, implying that loss of paralogous gene(s) possibly occurred after the triplication of F3‘H in Brassiceae ancestor.