Cloning And Expression Analysis Of Cinnamate-4-hydroxylase Gene From Pyrus Bretschneideri Cv. Danshan Su

The phenylpropanoid pathway,one of three secondary metabolism pathways in plants,is a plant specific metabolism pathway.The phenylpropanoid's final product is used to synthesize lignin monomer and flavonoid.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.C4 H gene belongs to CYP73 A subfamily of cytochrome P450-dependent monooxygenase superfamily,and specifically converts the trans-cinnamic acid into p-coumaric acid.However,it has not been elucidated that how C4 H gene affects the polymerization of lignin and the formation of stone cell so far.In this study,we cloned the C4 H gene from Pyrus bretschneideri cv.Dangshan Su.Subsequently,bioinformatics anaysis,together with in vivo transcription and bacterial expression,was carried out on the C4 H gene.In addition,we constructed eukaryotic expression vector to identify its subcellular localization and to be used for genetic transformation.These data could facilitate further studies and applications to improve the quality of Pyrus bretschneideri cv.Dangshan Su fruit by genetic engineering technology.The major results were listed as follows:(1)The cinnamate 4-hydroxylase gene(designated as PbC4H)was cloned from Pyrus bretschneideri cv.Danshan Su pear.The full-length PbC4 H cDNA(Gene Bank No.KF663548.)comprises 1764 bp,with an ORF of 1515 bp encoding a putative protein with504 amino acid residues.(2)According to the bioinformatic analysis,the deduced molecular mass of PbC4 H is57.8 kD and the isoelectric point is 9.05.Homology comparison indicated that the amino acid sequence of PbC4 H was highly homologous to the sequences of amino acids enccoded by other C4 H genes,indicating C4 H gene conservation during evolution.PbC4 H protein consists of a transmembrane domain,with some typical characteristics of P450 s.(3)qRT-PCR analysis showed that PbC4 H relative expression peaked at 31 days after flowering and 55 days after flowering during the fruit development of Pyrus bretschneideri cv.Dangshan Su.PbC4 H relative expression are rise to peaked before the stone cells and lignin content.The expression of PbC4 H was changed by cross-pollination,PbC4 H relative expression peaked at 31 days after flowering and 55 days after flowering in the Dangshan su(?)�Wonhwang(?)and at 31 days after flowering and 79 days after flowering in the Dangshan su(?)�Yali(?)?(4)PbC4H gene was constructed into prokaryotic expression vector of pET-32 a,and the recombinant PbC4 H protein was expressed in E.coli BL21 strain.(5)We constructed the plant expression vector pCAMBIA1304-PbC4 H and antisense RNA expression vector pBI121-antiPbC4 H.PbC4H protein was observed to be localized on vacuolar membrane by subcellular localization analysis.After the recombinant plasmid pCAMBIA1304-PbC4 H was transformed into Arabidopsis c4 h mutant,and the recombinant plasmid pBI121-anti PbC4 H was transformed into Wild type Arabidopsis Col-0,the corresponding transgenic plants were screened out,respectively.