| Pyrus bretschneideri cv. Dangshan Su, which originated in Dangshan in Anhui Province, China, has a long cultivation history and remains the main export product of this region. However, the local economy associated with this fruit is under threat. It has been confirmed recently that the content of stone cells is increasing in this fruit due to natural environment changes, extensive field management, and other issues. Stone cell is a type of sclerenchyma cells formed by the secondary depositionof lignin on the primary walls of parenchama cells. The development of stone cell is closely related to the synthesis, transfer and deposition of lignin. Caffeic acid o-methyltransferase (COMT) is a key enzyme in the lignin biosynthesis pathway. It mainly participates in the synthesise of S-lignin. It has not been elucidated that how COMT gene affects the polymerization of lignin and the formation of stone cell so far. So the study of COMT in Pyrus bretschneideri cv. Dangshan Su is of particular importance. It could facilitate further studies and be applied to improve the quality of Pyrus bretschneideri cv. Dangshan Su fruit in genetic engineering technology cultivation and production practice breeding in future.In this study, we Successfully cloned the COMT gene from Pyrus bretschneideri cv. Dangshan Su fruit, and do some bioinformatics anaysis. We also studied the prokaryotic expression and real-time fluorescence quantitative expression of this gene. In addition, we constructed eukaryotic expression vector, and this laid the foundation for the research of protein Subcellular localization and genetic transformation. The main research reSults were as follows:1. Using RT-PCR and RACE method, we got COMT gene cDNA from Pyrus bretschneideri cv. Dangshan Su, named PbCOMT(GenBank. accession number KC905086). The full-length PbCOMT cDNA comprised 1371 bp, with an ORF of 1098 bp which encoded a putative protein containning 365 amino acid residues.2. The structure and function of PbCOMT protein were analyzed and predicted by bioinformatic methods. The oretical molecular mass of PbCOMT was 40 KD and the oretical isoelectric point was 5.61. The phylogenetic tree was constructed to further identify the relationship between the amino acid sequence of PbCOMT and that of other COMTs. The amino acid sequence of Pyrus bretschneideri cv. Dangshan Su had higher homology with the COMT of Rosaxeae plant, and had 94% similarity in amino acid compared with Malus domestica. By the secondary structure prediction of PbCOMT protien, we knew that it had 34.25% a helix,16.44% extended strand and 49.32% random coil. Morever, PbCOMT was hydrophobic protein, and it had no signal peptide at its N terminal. PbCOMT, which belonged to non-secreted protein, might be located in cytoplasmic matrix. PbCOMT protein had dimerisation function domain and methyltransferase domain at its N terminal.3. The PbCOMT gene was cloned into the prokaryotic expression vector pET-32a(+). With IPTG induction and then SDS-PAGE detection, PbCOMT gene was constructed and transformed into E.coli BL21(DE3). Then a fusion protein which molecular mass was 60 KD was produced. Its molecular mass matched that of prediction protein.4. Using real-time fluorescence quantitative expression analysis, we got the reSult that PbCOMT gene expression increased first and then decreased during the fruit development(23,39,47,63 and 125 day). And the peak of PbCOMT gene expression level occurred on day 63. Moreover, on day 47, the expression level of PbCOMT gene in the intermidiate pulp was clearly higher than that the pulp near the core and then the pulp near the peel. However, on day 63, the expression level of PbCOMT gene in the pulp near the core was higher than that the pulp near the peel and then the pulp near the core.5. With eukaryotic expression vector plasmid pCAMBIA1301-EGFP, we Successfully constructed fusion expression vector 1301-PbCOMT-EGFP and did some research of the protein subcellular localization. |
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