Cloning And Expression Analysis Of Ferulate 5-hydroxylase Gene From Pyrus Bretschneideri Cv. Dangshan Su

Pyrus bretschneideri cv.Dangshan Su,is originated in Dangshan in Anhui Province,China.With a long cultivation history and a largest cultivation area in China,it has been a main export product for the local economy.However,the local economy associated with this fruit is under threat.Recently,stone cell content significantly increased in the pear fruit resulting from natural environment changes,extensive field management,and other issues.Stone cell is a type of sclerenchyma cell,formed by the secondary deposition of lignin on the primary walls of parenchama cells.Development of stone cells is closely related to synthesis,transfer and deposition of lignin.Ferulate 5-hydroxylase?F5H?is a key enzyme involved in lignin biosynthesis pathway that mainly regulates syringyl lignin.In this study,we cloned the F5H gene?designated as PbF5H?from Pyrus bretschneideri cv.Dangshan Su fruit.Subsequently,bioinformatics anaysis,together with in vivo transcription and bacterial expression,was carried out on the PbF5H gene.In addition,we constructed eukaryotic expression vector to identify its subcellular localization and transfer into Arabidopsis f5 h mutant.Thess data laid the foundation for the research on modification of lignin content via genetic engineering.The major results were listed as follows:?1?A F5H gene was cloned from Pyrus bretschneideri cv.Dangshan Su fruit using homology-based cloning methods,named PbF5H?GenBank accession No.KC852907?.The full-length PbF5H cDNA comprised 1978 bp with an Open Reading Frame of 1551 bp,which encoded a putative protein containing 516 amino acid residues with a typical P450 conservative domain.PbF5H had a molecular weight of 58.4 kDa and a PI of 6.49,with molecular formula C₂₆₁₄H₄₁₂₈N₇₀₄O₇₄₇S₂₅.?2?Phylogenetic analysis indicated that PbF5H protein shared a high homology with other plant F5Hs.The highest homology?90%?was found between PbF5H and Prunus persica.Combined with chromosome localization,these analysis showed that,PbF5H maybe the gene with the function of Ferulate 5-hydroxylase in Pyrus bretschneideri cv.Dangshan Su fruit.?3?The stone cell development of Pyrus bretschneideri cv.Dangshan Su showed distinct phases: 15 to 39 d,the early stone cell development,the lignification degree of stone cell was relatively low,and the PbF5H gene appeared first expression peak.In this period,S-lignin was the majority component in lignin matrix;39 to 79 d,the peak of stone cell development,the stone cell and lignin content showed a significantincrease in Dangshan Su fruit,with a significantly positive correlation between two factors.At this stage,the PbF5H gene transcription reached the second peak;79 to 145 d,the end stage of the stone cell development.At this stage,the PbF5H expression level was stable,leading to the lignin containing G-lignin as the majority component.?4?The PbF5H gene was cloned into the prokaryotic expression vector pET-32a?+?.The recombinant protein PbF5H was expressed in E.coli BL21?DE3?with a 80 kDa,consistent with molecular mass of the deduced protein.?5?The eukaryotic expression vector plasmid pCambia1304-PbF5H-GFP was constructed to be used for subcellular localization and genetic transformation.It was showed that the fusion protein PbF5H-GFP was localized on plasma membrane of epidermis cells.And transgenic Arabidopsis fah1 mutant with PbF5H was screened for future investiogation.