Cloning of extracellular chitinase gene AO-379 in Arthrobotrys oligospora and its expression

ObjectiveThe biological function of nematode-trapping fungus chitinase was researched thoroughly to lay a foundation for developing nematodosis biological control agents by Arthrobotrys oligospora recombinant chitinase AO-379.MethodThe full-length cDNA sequence of A.oligospora XJ-A1 chitinase gene AO-379 was cloned,and bioin-formatics analysis was carried out using on-line analysis softwares such as SignalP 4.1 Server,InterPro and ExPASy.The recombinant plasmid vector pET32a-AO-379 was constructed and transformed into Escherichia coli BL21(DE3)compe-tent cells.The recombinant protein was induced by IPTG and the obtained fusion proteins were analyzed by SDS-PAGE and Western blotting.ResultThe full length of A.oligospora chitinase gene AO-379 was 1475 bp,contained a 1203 bp open reading frame(ORF)and encoded 400 amino acids.The homology between chitinase AO-379 gene sequence with that of standard A.oligospora strain(ATCC 24927)was 97.08%,and the deduced amino acid homology was 98.75%.Chi-tinase AO-379 belonged to the family of glycoside hydrolase 18 with two catalytic region conserved sequences: SXGG and VDGFDLDFE.Located in the 127thto 131stposition,SLGGS was substrate binding point.VDGFDLDFE was located in 173rdto 181stposition as hydrolase catalytic active site.The relative molecular mass of fusion protein AO-379 induced by E.coli was 60.0 kD,and the protein could react with polyclonal antibodies against A.oligospora.ConclusionA.oli-gospora chitinase AO-379 can be induced successfully in E.coli.The obtained fusion protein AO-379 can react with poly-clonal antibodies against A.oligospora,and therefore it can be used in developing nematodosis biological control agents.