Cloning And Functional Study Of The Pine Elegans Sorbitol Dehydrogenase Gene

Pine wood nematode(PWN), Bursaphelenchus xylophilus, as the causal agent of pine wilt disease( PWD),causes tremendous damage by devastating the resources of pine forest worldwide. In our previous study, one pathogenesis-related gene, sodh-1, of B. xylophilus was identified and its partial sequence was cloned. In this paper, the open reading frame(ORF) encoding sorbitol dehydrogenase was cloned from cDNA library of B. xylophilus based on the partial sequence of sodh-1 through transcriptome analysis of PWN. The ORF was 1059 bp in length and encoded a protein of 352 amino acid residues. The ORF was recovered and ligated into expressing vector pET-15 b to construct recombinant plasmid pET-15b-sodh-1, and this plasmid was transformed into Escherichia coli BL21(DE3) to construct engineering bacteria. Recombinant sorbitol dehydrogenase was overexpressed in engineering bacteria after induced by isopropy-β-D-thiogalactoside(IPTG) as judged by SDS-PAGE, and appeared mainly in the form of inclusion bodies. Recombinant sorbitol dehydrogenase in the inclusion bodies was dissolved in 8mol/L urea, refolded by dilution and purified by Ni2+-charged NTA affinity chromatography. The relative molecular weight of recombinant sorbitol dehydrogenase with a N-terminal His-tag was 41 kDa as verified by SDS-PAGE.Bioassay results revealed that purified recombinant sorbitol dehydrogenase could catalyze the dehydrogen reaction of eight alcohols, but the specific activity of SODH-1 to1-propanol was higher than to the other seven alcohols. The optimal temperature and pH value were 30°C and pH8.0, respectively, when propanol was used as substrate. qRT-PCR was used to investigate the effects of Pseudomonas fluorescens GcM5-1A carried by PWN and ethanol on sodh-1 gene expression level of B. xylophilus. The result confirmed that both P. fluorescens GcM5-1A and ethanol could up-regulate sodh-1 gene expression. The sodh-1expression level of PWN treated with P. fluorescens GcM5-1A was 2.65 times of that of aseptic PWN. The sodh-1 expression level of PWN treated with 4% and 6% of the ethanol was 1.11 times and 1.45 times of that in control. This study analyzed the molecular characterization and functions ofsodh-1gene of B. xylophilus for the firs time and provided fundamental information to illuminate the molecular pathogenic mechanism of B.xylophilus.