Identification Of Key Genes In Lignin Metabolism Pathway And PbBZR Function In Pear Fruit

The stone cell is an important factor affecting the quality of pear fruit.The stone cells are mainly formed by the thickening of lignin in the secondary wall.The lignin metabolism is closely related to the formation of stone cells in pear fruits.Therefore,the study of lignin metabolism pathway in pear fruit is also the focus of current researchers.Compared with the Pyrus communis,the content of the stone cells of Pyrus bretschneideri is higher,and the stone cell mass is larger.At present,there is no report about the causes of stone cell difference between two pear fruits.Based on comparative genome and transcriptome technology,this experiment analyzed the othologous gene pairs between P.communis and P.bretschneideri genome.Subsequently,we screened out these genes related to fruit lignin anabolism in pear fruits.The specific results are as follows:1.The othologous gene pairs between P.communis and P.bretschneideri genome were identified and their genomic characteristics,such as Ka,Ks,Ka/Ks and the content of GC were compared.The results indicated that positive selection genes(PSGs)and negative selection genes(NSGs)not only show differences in environmental selection pressure(Ka/Ks),but also differ in their genetic structure,function,and evolutionary characteristics.The expression patterns of orthologous genes during pear fruit development were compared.The function of most orthologous genes was sub-functionalized,which revealed that there were some differences between P.communis and P.bretschneideri in the genes related to lignin metabolism pathway.2.Eleven gene families(F5H,COMT,CCR,CAD,CSE,4CL,HCT,C3H,PAL,C4H and CoAOMT)of lignin metabolism pathways were identified in pear fruits.By volutionary analysis and expression analysis,these genes,including PbPAL2,PbC4H1,PbC4H3,Pb4CL12,PbHCT6,PbC3H1,PbCES9,PbCCoAMOT1,PbCCoAOMT2,PbF5H1,PbF5H3,PbCOMT3,PbCCR20,PbCADl and PbCAD2 were identified to involve in lignin metabolism pathway of P.bretschneideri.We also verified for the first time the interaction between PbC3H and PbC4H in P.bretschneideri.3.A total of 94 non-redundant PbPRXs were identified in P.bretschneideri.EST library revealed 41 of 94 PbPRXs expressed in fruits.The qRT-PCR showed that the expression of PbPRX2,PbPRX22,PbPRX34,PbPRX64 and PBPRX75 in P.bretschneideri during fruit development was basically consistent with the change trend of lignin content.These results inferred that these five genes might participate in the polymerization of lignin monomers.4.The genes of PbPRX2,PbPRX22,PbPRX34,PbPRX64 and PBPRX75 were cloned from P.bretschneideri fruit.Subcellular localization indicated that these genes are located on the cell membrane or cell wall.Enzymatic kinetics analysis showed that PbPRX2 exhibited high activity against sinapyl alcohol and soniferyl alcohol.It is speculated that PbPRX2 may play an important role in lignin polymerization of ligin in P.bretschneideri fruit.5.A total of 13 non-redundant PBBZRs were identified in P.bretschneideri genome,and their structural variation,evolutionary history and expansion patterns in Rosaceae genome were analyzed.The RNA-seq data indicated that PbBZR1,PbBZR2 and PBBZR3 expressed higher levels during the P.bretschneideri fruits,suggesting that regulation may be involved in the fruit development of P.bretschneideri.6.The promoters of PbBZR1,PbBZR2 and PbBZR3,and the lignin metabolic pathways PbHCT6,PbCES9,PbCCOAMT1,PbCOMT3 and PbCCR20 were cloned from P.bretschneideri fruit.Silencing of PbBZR1,PbBZR2 and PbBZR3 was found to result in a significant increase in lignin content.Transcriptional activation and subcellular localization indicated that these three genes localize to the nucleus and act as transcriptional inhibitors.The dual luciferase assay confirmed that PbBZR1 can bind to the promoters of PbHCT6,PbCES9,PbCCOAMT1,PbCOMT3 and PbCCR20 to regulate the biosynthesis of lignin in P.bretschneideri fruit.