Cloning of PGIP gene from Malus domestica M. and its fusion expression in E. coli.

【Objective】In order to lay a foundation for the study of the biological function of polygalacturonase-inhibiting protein(PGIP),PGIP gene was cloned from apple fruit and its prokaryotic expression was induced by IPTG.【Method】A pair of specific primers were designed according to the conserved sequences of apple PGIP genes in GenBank.The cDNA of apple PGIP gene was amplified from fruit by RT-PCR and cloned into plasmid of pMD18-T,then identified by sequencing.The full-length cDNA and the cDNA excluding signal peptide were respectively constructed into expression plasmid pET-32a(+),and the recons were respectively named pET-PGIP and pET-PGIP-X.Then the recombinant plasmid pET-PGIP and pET-PGIP-X were transformed into host bacterium BL21 and their expression were induced by different concentrations of IPTG.Expression products were analyzed by SDS-PAGE.The pET-PGIP-X expression product was detected by solubility analysis.【Result】The results of sequencing and bioinformatical analysis showed that this fragment was 1 091 bp,including a 993 bp complete ORF,encoding 330 amino acid,which was named MdPGIP.The results of SDS-PAGE showed that the recombinant plasmid pET-PGIP and pET-PGIP-X could express interest protein and the molecular weight of which was 49.6 ku and 46.1 ku.Signal peptide excised protein expressed by pET-PGIP-X mainly existed in the form of inclusion bodies.【Conclusion】Apple PGIP gene was successfully cloned and high level expression existed in E.coli.