Cloning and expression in E.coli of polygalacturonase-inhibiting protein gene in Malus sieversii f.neidzwetzkyana

【Objective】 PGIPs(polygalacturonase-inhibiting protein) can specially bind Polygalacturonases(PGs) secreted by Plant Pathogenic fungi and inhibit or reduce its hydrolytic activity,and limit the growth of Plant Pathogens.Disease resistance mechanism was studied by cloning and expression of the pgip gene of Malus sieversii f.neidzwetzkyana.【Method】A fragment which was about 1 kb in size was amplified from the total RNA by reverse transcription PCR(RT-PCR)with a pair of specific primers based on the conserved sequences of pgip gene in 'Golden Delicious'apple.The fused expression plasmid was constructed by inserting the cDNA fragment encoding the mature peptide of PGIP into the prokaryotic expression vector pET 30a(+),and then transformed into E.coli BL21(DE3).【Result】Sequence analysis showed that the fragment contains a full coding region of 993 bp encoding 330 amino acid residues with a molecular mass of 36.6 kD.Its GenBank accession number is JQ001783.The deduced protein has a pI of 7.05,six potential N-glycosylation sites,two LRRs,and a hydrophobic region of 24 amino acid residues in the N-terminal which was considered to be a signal peptide.The protein exhibits a homology of 99% with PGIP in 'Granny Smith','Fuji'and 'Golden Delicious'apple.The SDS-PAGE indicated that the size of expressed protein was consistent with that of expected protein.【Conclusion】A pgip gene of Malus sieversii f.neidzwetzkyana was cloned and expressed in E.coli.