Function Analysis Of Adh-1 Of B.xylophilus

Pine wilt disease?PWD?,which is caused by the pine wood nematode?PWN?,Bursaphelenchus xylophilus,has caused huge damage to pine forests around the world.The PWD caused by B.xylophilus is a complex process which involved in B.xylophilus,host pines and bacteria carried by B.xylophilus.More and more studies indicated that B.xylophilus isolated from different resources had different pathogenicity.Some bacterial species such as Pseudomonas fluorescens had a positive effect on B.xylophilus development,however,the underlying molecular mechanism of B.xylophilus pathogenicity is still unclear.In our previous study,we analysed the transcriptome date of aseptic B.xylophilus and B.xylophilus carrying P.fluorescens GcM5-1A and found that the adh-1 gene encoding alcohol dehydrogenase?ADH?was upregulated.The open reading frame?ORF?of adh-1,which encoded a protein of 352 amino acid residues,was cloned from B.xylophilus.Recombinant ADH was overexpressed in Escherichia coli BL21?DE3?.The biochemical assay revealed that recombinant ADH could catalyse the dehydrogen reaction of eight tested alcohols including ethanol in the presence of NAD⁺.Quantitative real-time PCR?qRT-PCR?analysis indicated that ethanol could upregulate adh-1 expression in B.xylophilus.The above analyses indicated that ADH encoded by adh-1 in B.xylophilus could catalyse the oxidation reaction of ethanol,and adh-1 expression level has a close relationship to ethanol existed in environment.In this study,we further analyzed the gene structures and bioinformatic prediction of the adh-1 in B.xylophilus.Sequence analysis showed that the full-length adh-1 gene encoding ADH was 1458 bp,including a 1059 bp ORF which encoded a protein of 352amino acids with a relative molecular mass of 39 kDa.Alignment of the ORF with the genomic sequences of B.xylophilus revealed that B.xylophilus adh-1 contained three exons of 291,189 and 579 bp,two introns of 92 and 206 bp,a 52 bp 5'untranslated region?UTR?and a 48 bp 3'UTR.Sequences of introns corresponded to the“GT-AG rule”for cis-splicing.Protein BLAST showed that the deduced amino acid sequence of adh-1exhibited a relatively high level of identity with the ADH proteins of Caenorhabditis.elegans and Caenorhabditis.briggsae,with identities of 55%and 54%,respectively.The prediction of SignalP 4.0 and TMHMM server 2.0 software indicated that the ADH in B.xylophilus does not contain a signal peptide sequence or a transmembrane domain,suggesting that the ADH is an intracellular enzyme rather than a transmembrane protein.In order to further understand the biological functions of the adh-1,RNA interference?RNAi?and ADH inhibitor fomepizole were used in this study.Results of RNAi and inhibition of ADH treatment indicated that downregulating expression of adh-1 or inhibition of ADH could reduce the ADH activity,ethanol tolerance,vitality,reproduction,and feeding ability of B.xylophilus,suggesting that the adh-1 gene participated in modulating PWN vitality and reproduction.Furthermore,to investigate the effect of ethanol on adh-1 expression level,qRT-PCR was performed in this study,and the result revealed that ethanol could induce adh-1 gene expression level.Compared with B.xylophilus in sterilized water,the adh-1 expression level was upregulated 1.35-,1.76-,and2.07-fold when B.xylophilus were treated with 0.1%,0.5%and 1.0%ethanol,respectively.This result suggested that the adh-1 gene had a close relationship with ethanol metabolism in B.xylophilus.To further investigate the nematicidal activity of fomepizole,the LC₅₀0 and LC₉₀0 values of fomepizole to nematode at 24 h,48 h were calculated.The results indicated that fomepizole showed effective nematicidal activity in vitro,of which the LC₅₀0 values to nematode at 24 h,48 h were 0.026 mM,0.025 mM,respectively.There was no significant difference of fomepizole at LC₅₀0 values at 24 h,48 h,suggesting that fomepizole has significantly rapid nematicidal activity.The LC₉₀0 values of fomepizole to nematode at 24 h,48 h were 0.040 mM,0.038 mM,respectively.In addition,fomepizole showed approximately 100%nematicidal activity at a concentration of 0.055 mM.The above analyses may be useful for providing a novel insight and approach to control the PWN and PWDIn this study,to further understand the underlying molecular mechanism of negative effects of fomepizole on B.xylophilus vitality,reproduction,and development,we performed a transcriptome analysis to investigate differentially expressed genes?DEGs?and metabolic pathways associated with vitality and reproduction to B.xylophilus.Functional annotation of the DEGs was performed by NCBI NR,Gene Ontology?GO?,and Kyoto Encyclopedia of Genes and Genomes?KEGG?database.By comparing the expression profiles of B.xylophilus soaked in fomepizole solution and sterile water,3114DEGs in fomepizole-treated B.xylophilus were obtained,which included 2124up-regulated genes and 490 down-regulated genes.A total of 367 DEGs,which included214 up-regulated genes and 153 down-regulated genes with at least a two-fold change at the expression level and false discovery rate?FDR?<0.001 were obtained.Some key DEGs and metabolic pathways with potential regulatory roles in B.xylophilus vitality and reproductive capacity were evaluated in detail,such as the MAPK7 gene,HSP70 gene and RSK gene in MAPK signaling pathway,40S ribosomal protein S8-1 gene,40S ribosomal protein S12 gene and 40S ribosomal protein S18 gene in ribosome,and AchE gene.Based on the transcriptome data analysis and annotation results of the DEGs obtained,a total of 7genes included 3 up-regulated genes and 4 down-regulated genes were selected at random from the DEGs for verifying expression patterns with qRT-PCR.The results of qRT-PCR revealed that the expression levels of 7 DEGs from the B.xylophilus soaked in 0.020 mM fomepizole solutions were similar to those detected by the transcriptome analysis compared with the control nematodes.These results indicated that the transcriptome analysis data in this study were reliability.Furthermore,based on the transcriptome data analysis and annotation results,a number of DEGs related to development of larvae nematodes,lifespan,germ cell proliferation was obtained,indicating that fomepizole might also has negative influences on these physiological functions.The results revealed that continuous fomepizole exposure could reduce the development of larvae nematodes,lifespan,spawning rate,and hatchability of eggs.The above results indicated that fomepizole had significantly negative influences on biological functions of B.xylophilus.In summary,we firstly analysed the transcriptome from B.xylophilus associated bacterial species Pseudomonas fluorescens and screened out the up-regulated adh-1 gene encoding alcohol dehydrogenase?ADH?which had a close relationship with ethanol metabolism,and then further analyzed gene structures and bioinformatics information of the adh-1 in B.xylophilus.Later,RNAi and ADH inhibitor fomepizole were used to investigate the biological functions of the adh-1,and the results indicated that adh-1 gene could participate in regulating the vitality and reproduction ability of B.xylophilus.We also verified the positive correlation between adh-1 expression level of B.xylophilus and ethanol existed in environment.Finally,we analysed the nematicidal activity of fomepizole and performed a transcriptome analysis of B.xylophilus soaked in fomepizole solution and sterile water.Some key DEGs and metabolic pathways with potential regulatory roles in B.xylophilus vitality and reproductive capacity were evaluated in detail.qRT-PCR was used to validate the results of transcriptome analysis data in this study.Simultaneously,we verified that continuous fomepizole exposure had significantly negative influences on development of larvae nematodes,lifespan,spawning rate,and hatchability of eggs.This study preliminarily confirmed that the adh-1 gene,which participated in regulating B.xylophilus vitality and reproduction,had a great influence on proliferation of B.xylophilus,which could become an effective target for controlling the B.xylophilus and PWD.As an ADH inhibitor,low concentration of fomepizole solution??M order?could significantly affect key genes expression of B.xylophilus and cause metabolic disorders of B.xylophilus,and ultimately result in B.xylophilus death.The fomepizole and its derivatives could be used as a novel alternative agent against nematodes.This study not only helps us understand the underlying molecular mechanism for development and pathogenicity of B.xylophilus,but also contributes to provide a novel choice to control B.xylophilus and PWD.