关键词: 桂花, 肉桂酸4–羟基化酶, 黄酮类化合物, 原核表达, 基因表达

Abstract: According to the expressed sequence tags from transcriptome assembly and RACE method，two cinnamate 4-hydroxylase genes，which are the second key enzymes involving in phenylpropanoid biosynthesis，were obtained from petals of Osmanthus fragrans. The ORF of these two C4H genes were separately 1 518 bp and 1 611 bp，encoded 505 and 536 amino acids and named OfC4H1 （GenBank No. KR861466）and OfC4H2（GenBank No. KF254842）. Phylogenetic analysis showed that OfC4H1 protein was clustered together with Petunia hybrida C4H1 and C4H2，and belonged to ClassⅠ type，whereas OfC4H2 protein was clustered with Lonicera japonica C4H in ClassⅡ type. Multi-alignment of OfC4Hs proteins of several plants found that OfC4H1 and OfC4H2 had the characteristic conservative domains of the C4H family，and special conserved domains which were different in ClassⅠand ClassⅡ C4H. The results of time and space expression patterns by real-time PCRshowed that OfC4H1 had the highest amount of expression in the petals. While OfC4H2 displayed higher expression in green tissues including peduncles，pistils and young leaves，comparing to petals. By constructing prokaryotic expression vector pET6xHN-C4H1 and pET6xHN-C4H2，and transforming into Escherichia coli Transetta（DE3），the result showed that the target proteins can be well expressed in the prokaryotic expression system，and consistent with the expected size，but unable to enzyme activity analysis due to the low solubility of the recombinant proteins.

Key words: Osmanthus fragrans, cinnamate 4-hydroxylase, flavonoid compound, prokaryotic expression, gene expression