Molecular cloning of trans-cinnamate 4-hydroxylase and purification of cytochrome P450 proteins from aspen.

摘要

A complementary DNA (PtC4H) encoding trans-cinnamate 4-hydroxylase has been cloned from an aspen developing xylem cDNA library. To our knowledge, this is the first trans-cinnamate 4-hydroxylase (C4H) cDNA that has been isolated from a forest tree species. Later this clone was designated as CYP73A13. During growing seasons, aspen xylem C4H showed a bi-phasic gene expression pattern, which is in the agreement with the expression pattern of a lignin bi-specific caffeic acid/5-hydroxyferulic acid O-methyltransferase (OMT). At the same time, the changes of the C4H, phenylalanine ammonia-lyase (PAL), 4-courmarate: coenzyme A ligase (4CL), and OMT enzyme activity in xylem also fit into a similar pattern. Furthermore, since RT-PCR Southern analysis revealed CYP73A13 had stronger expression level in xylem than in bark and in leaf, and genomic Southern analysis also revealed that C4H exits as an small gene family in aspen, it is possible that CYP73A13 represents one of the gene which mainly expressed in developing secondary xylem and involved in the monolignol biosynthesis.;A simple two-step purification procedure including detergent solubilization and a novel Ferulate-