伊氏线虫真菌碳氮条件下的表型和毒力基因表达差异

Phenotypic and Virulence Gene Expression Difference of Esteya vermicola,A Biocontrol Fungus for Pine Wood Nematode under Carbon and Nitrogen Conditions

【目的】研究松材线虫生防真菌伊氏线虫菌(EV)碳氮条件下表型和毒力基因表达差异,为培养高毒力菌株提供依据。【方法】分别选取碳、氮培养基培养EV,测定生长速度及产孢量。收获菌丝体并提取RNA进行建库和差异分析。结合经FDR法校正后的P值及以2为底的倍数变化的对数的绝对值(|log_(2)FoldChange|)判断差异表达的显著性。【结果】EV在氮培养条件下生长速度更快,且基因表达的总量和特异表达的数量均高于碳培养条件下表达的数量。氮组相对碳组显著差异表达的基因共有7138个,其中3571个显著上调,3567个显著下调;474个基因在氮组中特异表达,295个基因在碳组中特异表达,且氮组特异表达的基因涉及杀线虫基因的数量高于碳组。经过聚类分析,差异基因表达水平变化趋势分为4个类群,其中,在氮组表达远高于碳组表达的基因数量为165个;在碳组表达远高于氮组表达的基因数量仅有65个。枯草杆菌蛋白酶及毒素合成相关基因在氮组中显著上调表达,其中枯草杆菌蛋白酶在氮组中表达量为相对于碳组表达的2.29~363.52倍。【结论】碳、氮培养基对EV生防真菌的转录具有显著影响,氮培养条件下表达的总基因数量和特异表达的基因数量高于碳培养条件下的基因表达。具有杀灭和侵染线虫作用的枯草杆菌蛋白酶、毒素合成相关基因在氮培养下显著上调。可为高毒力EV菌株的培养、基因功能研究和菌株改良提供重要依据。

【Objective】Pine wilt disease has been intensifying in China in recent years,and its pathogenic factor is pine wood nematode(Bursaphelenchus xylophilus),and it is a globally recognized quarantine pest.Esteya vermicola(EV)is an important biocontrol fungus infesting the pine wood nematode.The lunate spores produced by EV can infect and kill the pine wood nematode,thus EV has a good application prospect in the biological control of the pine wood nematodes.How to increase the growth rate,sporulation ability,and the virulence of EV to the pine wood nematode through cultivation,and then apply it to the field biological control is an important problem that needs to be solved urgently.【Method】In this study,EV was cultured in carbon and nitrogen medium respectively,and the growth rate and spore production were determined.The mycelia were harvested and RNA was extracted for library construction and differential analysis.The corrected P-value(Padj)and the absolute value of the logarithm of the base 2 fold change(|log_(2)FoldChange|)were used to judge the significance of the differential expression.【Result】EV fungus grew fast under nitrogen culture conditions,and the total number of expressed genes and specifically expressed genes of EV fungi under nitrogen culture conditions were higher than those under carbon culture conditions.A total of 7138 genes were significantly differently expressed in the nitrogen group compared to the carbon group,of which 3571 genes were significantly up-regulated,and 3567 genes were significantly down-regulated.There were 474 genes specifically expressed in the nitrogen group and 295 genes specifically expressed in the carbon group,and the number of genes specifically expressed in the nitrogen group involved nematicidal genes was greater than that in the carbon group.After cluster analysis,the variation trend of differential gene expression levels was divided into 4 clusters,among which there were 165 genes expressed much higher in the nitrogen group than in the carbon group;and 65 genes expressed much higher in the carbon group than in the nitrogen group.The expression of subtilisin and toxin synthesis-related genes involved in the activity of lethal nematodes and in the process of infecting nematodes were significantly up-regulated in the nitrogen group.The expression of subtilisin in the nitrogen group was 2.29-363.52 times higher relative to that in the carbon group.【Conclusion】Carbon and nitrogen media have significant effects on the transcription of EV biocontrol fungi.The total number of genes and specifically expressed genes of EV fungi under nitrogen culture conditions are higher than those under carbon culture conditions.Subtilisin and toxin synthesis-related genes with nematicidal effects are significantly up-regulated under nitrogen culture.The research results will provide an important theoretical basis for the cultivation,gene function research and strain improvement of highly virulent EV strains,and then lay a foundation for improving the control efficacy of EV biocontrol fungi.