| Pine wilt disease,which is caused by the pine wood nematode Bursaphelenchus xylophilus,is a devastating forest disease that has resulted in enormous economic losses and social impacts in some Asian countries and regions as well as in North American and European countries.Since the beginning of the 1980s in nanjing,this disease has spread quickly in our country,and caused serious damage on our country's forest resources and ecological environment.Currently,chemical controls,including chemical fumigation and chemical trunk implantations,are the primary methods used to manage this disease.However,the high toxicity of the chemical nematicides is problematic,causing increased nematode resistance,ecological damage and human health issues.Therefore,using a high efficiency,environmental protection,no pollution for prevention and control of botanical pesticides become the research hot spot and focus in recent years.In addition,"Return to nature" and pesticides harmless is the inevitable trend of development of social and natural sciences.With scientific and technological advancements,non-pollutant pesticides and molecular biological methods have been widely used to control nematodes in effective and safe ways.The research on mechanism of botanical nematicides may lead to find new molecular targets.The genes in nematodes invovled in the growth,reproduction,stress response,immune defense and so on,are usually the ideal targets of the botanical nematicides.Insulin signaling/IGF-1(IIS)pathway is one of the nematode' innate immune pathways,which not only participate in nematode' dauer formation,but also paly important roles in nematode longevity,fat metabolism,stress responses,innate immunity,etc.It is great significant to study the IIS pathway related genes for finding new targets for the pollution-free control of nematodes.In this study,the B.xylophilus is selected as our research object for the in-depth study of the mechanism of botanical nematicides.Firstly,the transcriptomes were constructed by the high through put sequencing technology,and the B.xylophilus was treated with rotenone medicament.Analysis the differentially expressed genes between the treatment group transcriptome and the control group transcriptome.IIS pathway related genes in B.xylophilus were screened by analyzing the DEGs.The functions of BxDaf-16?BxAge-1?BxAkt?BxSgk-1 genes and the response mechanism of these genes to pesticide stress were specially analyzed.The ain results are as the follows:1.Transcriptome analysis of treatment group and control group based on HiSeq 4000 high-throughput sequencingThe transcriptomes data base of the mixed-stages nematodes treated by rotenone(LC30=1.35 ?g/mL?LC50=2.60 pg/mL)and the control were constructed;69.33 Gb data were obtained,of which Q30 base was over 93.27%.A total of 14115 Unigene were obtained and the average GC content was 48.82%?New gene discovery and analysis showed that 711 new genes were obtained by Mapped Reads splicing.Gene function annotation showed that the number of genes annotated to COG,COG,GO,KEGG,KOG,Pfam,Swiss-Prot,eggNOG and NR library was 25,64,57,114,133,96,169,195,respectively.DEG analysis showed that there were 172 differential genes between the rotenone treatment group(LC30=1.35?g/mL)transcriptome and the control group transcriptome,and there were 614 differential genes between the rotenone treatment group(LC30=2.60 ?g/mL)transcriptome and the control group transcriptome.A large number of detoxification related genes,growth and development related genes,immune related genes,and signal pathway genes were identified from the constructed transcriptomes data base.It laid the foundation for the follow-up study of functional genes and the search for new drug targets.2.Molecular cloning and gene analysis of IIS pathway related genes in B.xylophilusIn this paper,the four IIS pathway related genes BxDaf-16,BxAge-1,BxAkt,BxSgk-1 were obtained by RT-PCR.(1)The complete CDS of BxDaf-16 gene was 1,533 bp,encoding 510 amino acids.The MW was 57.95 kDa and p1 was 6.55.There was no signal peptide and no transmembrane region.Sequence analysis revealed that BxDaf-16 contains six DNA binding sites and the Forkhead conserved domain,implying that this protein is a FoxO transcriptional factor.(2)The complete CDS of BxAge-1 gene was 1539 bp,encoding 512 amino acids.The MW was 60.14 kDa and p1 was 7.22.There was no signal peptide and no transmembrane region.Sequence analysis revealed that BxAge-1 protein contains the TEL1 conserved domain,and belongs to the PKc like superfamily.(3)The complete CDS of BxAkt gene was 1707 bp,encoding 568 amino acids.The MW was 65.54 kDa and p1 was 5.63.There was no signal peptide and no transmembrane region.Sequence analysis revealed that BxAkt protein contains eleven phosphoinositide binding sites,nineteen ATP binding sites.BxAkt protein has the PH and S_TKc conserved domains,and belongs to the PH-like superfamily and the PKc like superfamily.(4)The complete CDS of BxSgk-1 gene was 1281 bp,encoding 426 amino acids.The MW was 49.90 kDa and p1 was 8.79.There was no signal peptide and no transmembrane region in this protein.Sequence analysis revealed that BxAge-1 protein contains nineteen ATP binding sites and the STKc_SGK conserved domain,implying that this protein belongs to the PKc_like superfamily.Multiple sequence alignment analysis showed that the four genes are highly conserved with other nematode homologous proteins.3.Expression analysis of IIS pathway related genes in B.xylophilusISH was used to analysis the location of the four genes expression in B.xylophilus.Microscopic observations were as follows:BxDaf-16 mRNA was constitutively expressed in the circum pharyngeal nerve ring and intestine of males,and the neurons in the head and the ovaries of females;the BxAge-1 mRNA was mainly expressed in the genital area of nematode;the BxAkt mRNA was mainly expressed in the genital area and the nerve of males,and the pharyngeal nerve of females;the BxSgk-1 mRNA was mainly expressed in the dorsal neural notochord of males,and the anterior pharyngeal nerve and the middle esophageal nerve of females.qPCR was used to analysis the expression levels of the four genes under three kinds medicament treatment.The qPCR results showed that the BxDaf-16 gene expression were up-regulated after three pesticides treatment for 12 h and 24 h,suggesting that BxDaf-16 gene may plays important roles in nematode resistance stress;the BxAge-1 gene expression was down-regulated under the levamisole hydrochloride stress for 12 h,and the gene differential expression under rotenone and matrine stress were not obvious;the BxAkt gene expression was down-regulated under the levamisole hydrochloride and rotenone stress for 12 h,while the gene differential expression under other treatments were not obvious;the BxSgk-1 gene expression was down-regulated under the levamisole hydrochloride for 12 h,while the gene expression levels under other treatments were up-regulated.In short,as potential drug target genes,the four IIS pathway genes paly key roles in the insecticidal mechanism mechanism research and new pesticides research.4.RNAi analysis of the four IIS pathway related genesRNAi was used to explore the biological functions of the four IIS pathway related genes in B.xylophilus.Microscopic observation,qPCR experiment and mortality rate assays were used to analysis the gene silencing.Microscopic observations showed that the dsBxDafl6-treated nematode displayed the knotting phenomenon compared with the control nematodes;The dsBxAge1-treated,dsBxAkt-treated and dsBSgk1-treated nematodes displayed no significant difference with the control nematodes.qPCR results showed that the expression levels of all the four genes in dsRNA-treated nematodes were down-regulated compared with the control nematodes,implying that the four genes were successfully inhibited by RNAi.Bioassay experiment showed that the mortality rates of dsBxDaf16-treated nematodes under three drug stress were significantly higher than the control treated nematodes;after the medicament treatment,the mortality rates of dsBAge1-treated,dsBxAkt-treated and dsBxSgk1-treated nematodes were declined in general compared with the no-dsRNA treated nematodes.5.Multi-target gene RNAi analysis of the four genesBased on the research of single-gene RNAi,in vitro dsRNA method was used to study the muti-target gene RNAi.A detailed study of the four genes function in nematode resistant stress and immune defense,and explore the relationship between the four genes in IIS pathway.qPCR results showed that the target genes expression levels in dsDaf-16&Age-1,dsDaf-16&Akt,dsDaf-16&Sgk-1,dsAge-1&Akt,dsAge-1&Sgk-1,dsAkt&Sgk-1 treated nematodes were all down-regulated compared with the control nematodes.Bioassay experiment showed that the mortality rates of dsDaf-16&Age-1,dsDaf-16&Akt,dsDaf-16&Sgk-1 treated nematodes under three drug stress were all decreased than the dsBxDaf-16 treated nematodes;the difference in the mortality rates of dsAge-1&Akt,dsAge-1&Sgk-1,dsAkt&Sgk-1 treated nematodes under drug stress were no regular pattern compared with single gene RNAi. |
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