Abstract：Lignin is a complex, noncrystalline polymer, which is one of the important components of the pear stone cells. Stone cells are a type of brachysclereid, contain abundant lignin and cellulose, which formed by the secondary deposition of lignin on the primary walls of parenchyma cells. The synthesis, transfer and deposition of lignin is closely related to the development of stone cells. Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) catalyzes the hydroxylation of trans-cinnamic acid to 4-hydroxycinnamate and is the second key enzyme of common phenylpropanoid pathway. C4H gene belongs to CYP73A subfamily of cytochrome P450-dependent monooxygenase superfamily, and specifically converts the trans-cinnamic acid into p-coumaric acid. In this study, the C4H (designated as PbC4H) from Pyrus bretschneideri cv. Dangshan Su fruit was cloned. The full-length PbC4H cDNA (GenBank No. KF663548) was 1 764 bp, 5'-UTR was 85 bp, 3'-UTR was 164 bp and contained 26 bp PolyA tail, which also with an ORF of 1 515 bp encoding a putative protein with 504 amino acid residues. Functional structural analysis showed that PbC4H had a transmembrane domain, and T binding groove motif A306AIETT311, E363-R366-R420 triplet, the necessary proline hinge structure P34PGPIPVP41 for the enzyme to correct orientation. In addition, PbC4H had 5 characteristic substrate binding site motifs (substrate recognition sites and SRSS): SRS1(S100RTRNVVFDIFTGEGQDMVFTVY),SRS2(L216AQSFDYNY), SRS4(I299VENINVAAIETTLWS), SRS5 (R368MAIPLLVP) and SRS6(K484GGQFSLHI). Homology comparison indicated that the amino acid sequence of PbC4H was highly homologous to the sequences of amino acids encoded by other C4H, indicating that C4H gene conservation during evolution. In order to further clarify the phylogenetic relations between PbC4H and other species C4H, a phylogenetic dendrogram was constructed using MEGA5.1 software and found that Rosaceae plant Pyrus bretschneideri, Malus and Prunus C4H clustered as a class. qRT-PCR analysis showed that PbC4H expression increased first and then decreased during the fruit development. The expression levels of 23~63 days after flowering was higher than that in other stages, expression peak of PbC4H was observed at 55 days after flowering. The change trends were consistent with the changes of lignin and stone cell content during fruit development, which suggested that there was a significant relationship between them. PbC4H was constructed into prokaryotic expression vector of pET-32a, and the recombinant PbC4H expressed in Escherichia coli strain BL21. The induced protein, which molecular weight is consistent with the predicted molecular weight. We constructed eukaryotic expression vector to identify its subcellular localization. PbC4H was observed to be localized on membrane by subcellular localization analysis. This study provides a theoretical basis for the regulation of the synthesis of lignin and the development of stone cells in pear by using molecular biology technology.
