| The low temperature stress resistance of pine wood nematode(Bursaphelenchus xylophilus)is closely related to the long-term survival of its third-stage dispersal juvenile(LⅢ).LⅢ can survive unfavorable conditions such as low temperature,and plays an important role in the wintering of pine wood nematode and its spread to low temperature areas such as Northeast China.Trehalose is closely related to the resistance of organisms to environmental stress.In order to investigate the molecular mechanism of LⅢ formation and low temperature stress resistance and the function of trehalose in this process,21 RNA-Seq were completed,and 9 trehalose metabolism genes of pine wood nematodes were identified.The functions of these 9 genes related to LⅢ formation and low temperature stress resistance were studied.The main research content and results were as follows:1.Identification of LⅢ high-associated gene group by weighted gene co-expression network analysis(WGCNA):WGCNA was used to comprehensively analyze the expression patterns of 15,889 genes from 21 RNA-Seq results of pine wood nematodes at 7 different developmental stages,and a total of 12 co-expression modules were obtained.Among them,magenta module has the highest correlation with LⅢ,which included a total of 652 genes.GO enrichment analysis showed that most of the genes in the magenta module existed in cell or cell part,had catalytic activity or binding function,and participated in biological process,metabolic process or cellular process.KEGG enrichment analysis showed that most of the genes in the magenta module were involved in metabolic processes,which were related to autophagy and longevity regulation.More genes were enriched to carbohydrate metabolism and lipid metabolism in metabolism.The sugar content measurement found that the trehalose content in LⅢ was higher than in the various propagative stages.In order to explore the molecular mechanism of trehalose on pine wood nematode in adverse environmental protection,especially the specific function of trehalose in the formation of LⅢ and the resistance to low temperature,trehalose metabolism genes were selected as the further research object.2.Cloning of 9 trehalose metabolism genes in pine wood nematode and analysis of expression patterns at various developmental stages:In order to explore the function of trehalose in the formation of LⅢ and survival at low temperature,a trehalose-6-phosphate synthase(TPS)-encoding gene(Bx-tps),a trehalose-6-phosphate phosphatase(TPP)-encoding gene(Bx-tpp)and 7 trehalase(TRE)-encoding genes(Bx-tre)were identified in pine wood nematode.The expression patterns of these 9 trehalose metabolism genes were investigated.RT-PCR results showed that the expression levels of Bx-tps and Bx-tpp in LⅢ were significantly higher than in other developmental stages,and the results showed the same trend as the DGE sequencing results.Therefore,TPS and TPP might play a more important role in LⅢ.3.The function of 9 trehalose metabolism genes in pine wood nematode in the formation of LⅢ:After RNAi decreased the expression level of Bx-tps or Bx-tpp,TPS activity decreased,and both the trehalose content and the LⅢ formation rate decreased.After reducing the expression level of Bx-tres by RNAi,TRE activity decreased,trehalose content increased,and LⅢ formation rate decreased.The above results indicated that trehalose metabolism genes expressions were related to the formation of LⅢ.After Bx-tps silencing treatment,the formation rate of LⅢ was the lowest,indicating that the expression of Bx-tps was more closely related to the formation of LⅢ.4.The function of 9 trehalose metabolism genes of pine wood nematode in LⅢresistance to low temperature:After low temperature treatment at-20℃,the expression of Bx-tps and Bx-tpp increased significantly,Bx-trel,Bx-tre2,Bx-tre3,Bx-tre4,Bx-tre5,Bx-tre6 and Bx-tre7 decreased significantly,trehalose content increased,TPS activity increased,but TRE activity decreased,indicating that low temperature stress induced LⅢ to accumulate trehalose.After the trehalose metabolism genes were silenced,compared with the control group,the survival rate of LⅢ in each RNAi-treated group did not change significantly at 250C,but the survival rate at-20℃ decreased significantly,indicating that the expression of trehalose metabolism genes was related to LⅢ resistance to low temperature.After Bx-tps or Bx-tpp silencing treatment,the survival rate of LⅢ decreased more at low temperature,indicating that the expression of Bx-tps or Bx-tpp was more closely related to the survival of LⅢ at low temperature.5.Analysis of trehalose metabolism gene integration pathway during LⅢformation and low-temperature stress resistance:WGCNA results showed that Bx-tps and Bx-tpp were strongly correlated with LⅢ.Therefore,the synthesis of trehalose might play a more important role in the formation and maintenance of LⅢ than the hydrolysis of trehalose.Further analysis found that most genes highly related to Bx-tps,Bx-tpp and LⅢ in the magenta module were involved in metabolic processes,including sugar metabolism,carbon metabolism and fatty acid metabolism.Among them,18 genes were enriched into 15 pathways related to energy conversion and autophagy.These pathways were related to a variety of longevity-related signaling pathways,including AMPK signaling pathway,insulin signaling pathway,glucagon signaling pathway,lysosome and peroxisome.The expression levels of these genes in LⅢ were significantly higher than in the various propagative stages.The above results indicated that trehalose metabolism promoted LⅢ formation and helped LⅢ survive at low temperature.Although trehalose accumulation was favorable for LⅢ to cope with low temperature stress,multiple trehalose metabolism genes needed to work together to complete a dynamic process of trehalose synthesis and degradation to help LⅢ survive at low temperature.The above results suggested that there was a multi-path regulated physiological process involving Bx-tps and Bx-tpp under low-temperature stress resistance.This physiological process might regulate the formation and maintenance of LⅢ through autophagy and energy conversion. |
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