Expression Analysis Of Key Enzymes PbCCOMT、PbCOMT Of Lignin Synthesis In Pyrus Bretschneideri Cv. Dangshan Su

Pyrus bretschneideri cv.Dangshan Su is initially planted in the Dangsahn county of Anhui province.It is also famous for an kind of output pear species in China.At recent years,because of the environmental variation and careless management,the quality of the fruit has a few depression,the researches on fruit quality improvement had observed that the stone cells are close related to the delicate tasty of pears.The studies indicate that the stone cells are came from lignified parenchymal tissue as a result of the secondary wall thickening,since the lignin monomer synthesized,polymerized as well as the lignin finally deposited at the cell wall.The increased stone cell is one of the significant factors which influence the flesh quality and taste as well.It has been confirmed that Caffeic acid O-methyltransferase(COMT)and Caffeoyl Co A3-O-methyltransferase(CCOMT)are both key enzymes of lignin synthesis pathway.This research uses the bioinformatics to analyze and identify the total OMT gene family in the whole genome.We have already cloned PbCOMT1 and PbCCOMT1 sequences as well as two gene’s promotor sequences to attempt to analyze the expression pattern of PbCOMT1 and PbCCOMT1 in lignin synthesis.It is expected to be helpful for the researches of lignin regulation in Pyrus.The main results as follow:1.Through the whole genome analysis we totally find 30 COMT and 9 CCOMT amino acid sequence candidates preliminarily,then 26 members identified belong to the OMT family,among them there are 19 COMTs and 7 CCOMTs,and we named them as PbCOMT1-19,PbCCOMT1-7 in turn.They divided into two classes,and the eight subclasses,the results indicate that PbCOMT1 and PbCCOMT1/3 might be involved in the lignin synthesis.2.Chromosomal location analysis showed that members from the OMT family are more likely in the form of a gene cluster distributing in the pear genome,combining with the phylogenetic tree,OMT family members were more probably expansion through tandem duplication in the genome.3.Analysis of the key elements of PbCOMT1、PbCCOMT1/3 promoters sequences,not only AC element but also a lot of light response elements as well as Me JA response element have been found.Generally AC element is considered a close relation with lignin synthesis,and Me JA and light might influence the expression of two genes to impact the stone cell development.4.QRT-PCR results show that both PbCOMT1 and PbCCOMT1 genes relative expression level present the rising first then decreasing,and expression quantity of PbCOMT1 reaches the peak at 63 d,but PbCCOMT1 reaches the peak at 55 d,In combination with the results of the researches before,different development period of pear fruit lignin content change express the same trend and peaked at 71 d,suggesting that two genes’ expression level is associated with lignin contents.5.PbCOMT1 and PbCCOMT1 promoters sequences have been cloned,and getting various DEL1-6 fragments for lacking of 5’,construction of eukaryotic expression vector.