Bioinformatics Analysis Of UGT And Functional Verification Of PbUGT72AJ2 Involved In Lignin Metabolism Within Fruits Of Pyrus Bretschneideri Cv.dangshan Su

Pyrus bretschneideri cv.Dangshan Su is our domestic characteristic pear species.It is one of the most popular fruits for its special crisp tasty and sweet succulence.Which fruit quality can assess from different aspects,for instance,appearance,sweetness,taste,and so on.It is usually believed that which quality is related to the size,the content and the density of the stone cell in pear fruit.The stone cell is the sclerenchyma cell with a lot of lignin deposition on it,which comes from parenchyma cell.The synthesis of lignin is one of the important factors that affect the formation of stone cells.The lignin monomers are synthesized in the cytoplasm,but its polymerization happens outside of the cytomembrane.In recent years,it has been founded that lignin monomers with galactosylated modification phenomenon during its transport process,so it is very important to study glycosyl transferase that is related to the transportation of the pear fruit lignin.In this study,the glycosyl transferase candidate genes that related to the synthesis of lignin were firstly screened out,then analyzed the bioinformatics characteristics of these genes.And the PbUGT72AJ2 was choosed for the genetic transformation into Arabidopsis thaliana to clarify the relationship between PbUGT72AJ2 and monolignol transport,stone cell synthesis,to lay the relevant theoretical foundation for the regulation of Dangshan su pear lignin and stone cell content.The main results are showed as follows:1.139 Dangshan pear UGT candidate genes were screend out and constructed the composite phylogenetic tree combined with the known 40 UGTs in Arabidopsis,then 8 UGTs may be related to the synthesis and transport of lignin were found from the candidate genes.2.PbUGT72AJ2 was choosed for the genetic transformation in Arabidopsis thaliana,which Genbank’s accession number is KP270486,the length of PbUGT72AJ2 nucleotide sequence is 1440bp,coded amino acid is 479.3.qRT-PCR analysis of PbUGT72AJ2 at different developmental stages of fruit in Dangshan su pear showed the highest relative expression of PbUGT72AJ2 was about at 47d.It is basically in the same period with stone cell content peak at 47d and the lignin content peak at 55d.4.The eukaryotic expression vector pCAMBlA1304-Pb UGT72AJ2 was successfully constructed.The subcellular localization experiment found that PbUGT72AJ2 expressed protein located on the cell membrane and cytoplasm.The glycosyltransferase sites may effect on the cell membrane and cytoplasm,further validation the effect of PbUGT72AJ2 modification in lignin to promotes transmembrane transport.5.The pCAMBIA1304-PbUGT72AJ2 recombinant plasmid was conducted,which was transferred into wild type Arabidopsis thaliana via floral dip mediated by Agrobacterium.The chemical staining of stem cross-sections of Arabidopsis thaliana transformed pCAMBIA1304-PbUGT72AJ2 gene revealed that lignin content in stem xylem cells of transgenic Arabidopsis was higher than that in wild type plants.The lignin content of pC AMBIA1304-Pb UGT72AJ2 transgenic Arabidopsis thaliana was significantly higher than that of wild type Arabidopsis thaliana by UV spectrophotometry,indicated that Pb UGT72AJ2 had an effect on lignin content.6.pET32a-PbUGT72AJ2 recombinant expression vector was constructed using E.coli expression vector pET-32a(+).The His-PbUGT72AJ2 protein was induced by adding the inducer IPTG at 28℃,and the molecular weight of the protein was about 43 kD.