The CDNA Cloning And Functional Study Of Sft-4 Gene From PWN

Pine wilt disease(PWD),also known as pine wood nematode disease,is a destructive disease caused by the pine wilt nematode(PWN)[Bursaphelenchusxylophilus(Steiner&Buhrer)Nickel].It is one of the most serious tree diseases affecting coniferous forest in the world,and has caused huge economic losses and serious ecological environment damage worldwide.Therefore,strengthening the prevention and treatment of this disease is an important task for forestry protection.However,due to the lack of in-depth research on the pathogenic mechanism of PWD,effective methods and means for preventing and controlling this disease have not yet been developed.As a result,the prevention and control of this disease is still in a very passive situation both domestically and internationally.The death of pine trees caused by PWNs is a complex process that involves multiple factors,among which PWNs are the main pathogenic factor.Studying the characteristics of PWNs can provide a certain theoretical basis for the prevention and control of this disease.In this study,by analyzing the transcriptome data of PWN(experimental group)treated with fomepizole for 24 h and PWN(control group)treated with sterilized water,we screened a down-regulated differentially expressed gene sft-4.The sft-4 gene of PWN has a total length of 1,290 bp and consists of 4 exons and 3 introns,the full length of the gene’s open reading frame(ORF)is 852 bp in length,encoding a protein with 283 amino acid residues.Bioinformatics analysis shows that the amino acid sequence of the protein encoded by PWN sft-4 shares 46% and 49% homology with that of Apelenchus avenae(Gen Bank accession number: KAH7713954.1)and that of Pristionchus pacificus(Gen Bank accession number: KAF8382798.1),respectively.The SFT-4 protein of PWN also has 46% homology with that of Bursaphelenchus okinawaensis(Gen Bank login number: CAD5229971.1).The theoretical p I of SFT-4 isolated from PWNs is 7.00.The results of Signal P5.0 and TMHMM analysis showed that the amino acid sequence encoded by sft-4 did not contain the signal peptide sequence,but contained five transmembrane helical structures.After advanced structural prediction,it was found that in the secondary structure of SFT-4,α-helix proportion reached 72% and β-pleated sheet was only 1%,indicating that the structure of this protein was very stable.And three-level structural prediction showed that α-helix was significantly enhanced,while β-pleated sheet and partially random coil had also been significantly improved in SFT-4.The ORF of sft-4 was cloned into the expression vector p ET-15 b to construct the recombinant expression vector p ET-15b-sft-4,and the plasmid was transformed into E.coli BL21(DE3)strain to construct the engineered bacteria.IPTG(Isopropyl-β-d-Thiogalactopyranoside)was used to induce the expression of recombinant protein in engineered bacteria,soluble recombinant SFT-4 was obtained through purification by nickel ion affinity chromatography,and the protein purity was significantly improved.Mass spectrometry and SDS-PAGE analysis were performed and results showed the recombinant SFT-4 had a molecular weight of 33 k Da.RNA interference(RNAi)technology was used to reduce the expression level of sft-4 in order to further study the roles of sft-4 in PWN.The results of RNAi study showed that the mobility,fecundity and feeding ability of PWN to Botrytis cinerea were significantly reduced after the sft-4 gene of PWN was interfered(down regulated).The infection assays showed that the pathogenicity of PWNs decreased after RNAi.PWNs was soaked in purified recombinant SFT-4 protein solution to increase the concentration of protein in the nematode body to mimic overexpression of sft-4,and the experimental results showed that after overexpression of STF-4,the reproduction ability,feeding ability,and oviposition ability of PWNs were significantly improved.Furthermore,in situ hybridization was used to detect the expression patterns of sft-4 in male and female individuals of PWNs,as well as in different developmental stages of PWNs.Results showed that sft-4 could express in various stages from eggs to adult nematode.However,the expression level was very low at eggs and J1 stage,relatively high at head of J2 stage,and sft-4 was mainly expressed in intestine and tail at J3 and J4 stages.The expression of sft-4 was observed in whole bodies both in male and female adult nematodes,with the highest expression in the spicule of adult male nematodes.In order to understand the roles of sft-4 gene in PWN,the transcriptome of PWN of which sft-4 gene wasdown-regulated by RNAi and that of PWN in the control group were analyzed.The functional annotation of differentially expressed genes in NCBI NR,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)databases showed that 122 down-regulated genes and 69 up-regulated genes were found in RNAied PWN through the annotation of differentially expressed genes.There were a total of 100 differentially expressed genes with multiple differences of over 2,including 51 upregulated genes and 49 downregulated genes(FDR<0.001).Some genes were associated with pathogenicity(cytochrome P450(log2Fold Change:-10.67),elongation of very long chain fatty acids protein 6(log2Fold Change:-5.66),cytochrome C oxidase(log2Fold Change:-10.67),-7.00))and other genes were associated with growth and development,such as tyrosinase(log2Fold Change:-2.26),acetyl-Co A synthetase(log2Fold Change:-6.37),and acyl-coenzyme A thioesterase 9(log2Fold Change:-7.62).KEGG analysis showed that there were more differentially expressed genes in the energy metabolism pathway of sft-4 ds RNA treated PWN.In summary,the sft-4 gene of PWNs might mainly affect the pathogenicity of PWNs by regulating the motility and reproductive ability of PWNs.