Molecular Cloning, Prokaryotic Expression and Antibacterial Activity of β-1,3-Glucanase

LI Zhen-gang,ZHAO Ai-chun,WANG Xi-ling,JIN Xiao-yun,LI Jun,YU Mao-de*(State Key Laboratory of Silkworm Genome Biology,College of Biotechnology,Southwest University,Chongqing 400715,China)

glucanase gene;complementary DNA;prokaryotic expression;plant fungal disease;gene cloning

In this study,two β-1,3-glucanase(Glu) genes were amplified from the cDNAs of Triticum aestivum and Oryza sativa by RT-PCR and designated as TaGlu9507 and OsGlu30,respectively.Sequence analysis showed that both TaGlu9507 and OsGlu30 contained a 1002 bp-length ORF encoding two putative peptides of 334 amino acids with 20 and 29 residues signal peptides in length,respectively.DNA fragments from the coding sequences(CDS) of TaGlu9507 and OsGlu30 genes without their signal peptide sequences were cloned into the E.coli expression vector pET-28a(+).The recombinant vectors were transformed into E.coli BL21(DE3) strain,respectively.The obtained transformants were able to express β-1,3-glucanase in a large scale when they were induced with IPTG at a final concentration of 0.5 mmol/L for 3 hours.SDS-PAGE analysis showed that the contents of the expressed β-1,3-glucanase in total soluble protein of E.coli were 49.7% and 26.7%,respectively.The expressed β-1,3-glucanase products had an obvious inhibitory effect on fungi such as Aspergillus niger and Pichia pastoris.These results further revealed that β-1,3-glucanase gene was a potential target gene for the prevention and cure of fungal diseases of plants.