Study On CDNA Cloning And Enzymatic Properties Of An Aldehyde Dehydrogenase From Bursaphelenchus Xylophilus

Pinewood nematode（Bursaphelenchus xylophilus）is a dangerous invasive species which is highly pathogenic to several species of pine trees.Pine wilt disease（PWD）has caused severe damage to pine forests and huge economic losses all over the world.The pathogenic mechanism of the disease is complex,symptoms occur rapidly,and prevention and treatment are difficult.At present,the pathogenic mechanism of PWD is still unclear,and no effective measure has been explored in prevention and treatment of PWD.The traditionally methods include erasing and burning the diseased trees,fumigating pine wood with organic solvents,killing dissemination medium-Monochamus alternatus with pesticides,and injecting drugs such as aloperine and avermectin into pine trunks.In recent,more and more researches focus on exploring of specific drugs based on target enzymes,which can affect the physiological process of PWN and kill nematodes in the end.In previous experiments in our laboratory,transcriptome analysis was performed for PWN treated with alcohol dehydrogenase inhibitor-fomepizole,and Bx-aldh,encoding an aldehyde dehydrogenase enzyme with unknown functions,was found to be obviously up-regulated.In this study,total RNAs were extracted from PWN and reverse-transcripted into c DNAs.A pair of primers were designed according to the data obtained from transcriptome analysis.Bx-aldh from a pinewood nematode was amplified using c DNAs as template and two primers designed.Sequence analysis revealed that it was 1353 bp and encoded 451 amino acid residues.Bx-aldh was cloned into p ET-15b to construct the expression vector p ET-15b-aldh.The expressing plasmid was transformed into Escherichia coli BL21（DE3）to construct engineered bacteria.The recombinant aldehyde dehydrogenase was overexpressed in engineered bacteria induced by isopro-β-D-thiogalactoside（IPTG）.The recombinant aldehyde dehydrogenase was purified by Ni-NTA affinity chromatography to yield an approximately 51 k Da protein in size estimated by SDS-PAGE,which was consistent with the predicted size.The biochemical properties of the recombinant aldehyde dehydrogenase,which include Km,optimum p H,optimum temperature,effects of metal ions and substrate specificity on catalytic activity,were studied.Results showed that the km of aldehyde dehydrogenase was27.87 mmol/L determined by Lineweaver-Burk plot using formaldehyde as substrate and NAD as coenzyme.The optimal temperature for the aldehyde dehydrogenase was 25℃,and the optimal p H value was 7.5.When different metal ions were present in the reaction system,the activity of the aldehyde dehydrogenase was clearly affected.The presence of Fe³⁺and Ni²⁺obviously increased the enzyme activity compared with control,while Ca²⁺,Mn²⁺,Na⁺and K⁺inhibited the activity to different extents.The catalytic ability of the recombinant aldehyde dehydrogenase towards different substrates was examined using formaldehyde,acetaldehyde,glutaraldehyde,vanillin,and 4-（dimethylamino）-benzaldehyde as substrates,respectively,and it was found that the catalytic activity of this aldehyde dehydrogenase towards vanillin was highest,followed by formaldehyde,glutaraldehyde and acetaldehyde.No catalytic ability was detected when 4-（dimethylamino）-benzaldehyde was used as substrate.This study offered a reference for studying on pathogenesis mechanism of PWN and exploring of nematicides based on this enzyme.