柞蚕微粒子病潜在生物标志物的挖掘

Mining of potential biomarkers of pebrine disease of the Chinese oak silkworm,Antheraea pernyi(Lepidoptera:Saturniidae)

【目的】挖掘柞蚕Antheraea pernyi微粒子病的潜在生物标志物,为开发该病害检测方法及研究柞蚕被微孢子虫Nosema pernyi侵染后体内代谢产物的差异及功能奠定基础。【方法】利用高效液相色谱和高分辨率质谱进行非靶向代谢组学分析,调查健康和患微粒子病柞蚕雌成虫血淋巴中代谢物差异。【结果】正离子模式下从健康和患微粒子病柞蚕雌成虫血淋巴中共获得8870个代谢物,注释代谢物5390个,筛选到差异表达代谢物472个(上调260个,下调212个),其中二级鉴定差异表达代谢物12个(上调8个,下调4个);负离子模式下获得6716个代谢物,注释代谢物3848个,筛选到差异表达代谢物301个(上调207个,下调94个),其中二级鉴定差异表达代谢物9个(上调8个,下调1个)。正离子模式下二级鉴定的差异表达代谢物包括缬氨酸(valine)、苯并噻唑(benzothiazole)、3-脱羟基肉碱(3-dehydroxycarnitine)、1-甲基鸟嘌呤(1-methylguanine)、2-乙氧基萘(2-ethoxynaphthalene)、N6-乙酰基-L-赖氨酸(N6-acetyl-L-lysine)、生物素(biotin)、桑色素(morin)、噻吗洛尔(timolol)、酰基肉碱15∶0(acylcarnitine 15∶0)、酰基肉碱18∶4(acylcarnitine 18∶4)和异槲皮苷(isoquercitrin);负离子模式下二级鉴定的差异表达代谢物包括二甲基丙二酸(dimethylmalonic acid)、戊二酸(glutaric acid)、2,5-二羟基苯甲酸(2,5-dihydroxybenzoic acid)、1,3-二乙酰基丙烷(1,3-diacetylpropane)、3-(4-羟基苯基)乳酸(DL-p-hydroxyphenyllactic acid)、泛酸(pantothenate)、荧光素(fluorescein)、飞燕草素-3-O-beta-吡喃葡萄糖苷(delphinidin-3-O-beta-glucopyranoside)和溶血磷酯酰肌醇16∶1(lysoPI 16∶1)。【结论】健康与患柞蚕微粒子病的柞蚕雌成虫血淋巴内代谢物具有显著差异,通过代谢组学挖掘出21个二级鉴定的差异表达代谢物,这些代谢物可作为潜在生物标志物用于开发柞蚕微粒子病的检测方法。

【Aim】The potential biomarkers of pebrine disease of the Chinese oak silkworm,Antheraea pernyi,are to be identified in this study,so as to lay a foundation for the development of detection methods of the disease and the study of the differences and functions of metabolites of A.pernyi infected by Nosema pernyi.【Methods】The differences of metabolites in the haemolymph between healthy and pebrine-infected female adults of A.pernyi were assayed via non-targeted metabolomics analysis by means of high performance liquid chromatography(HPLC)and high resolution mass spectrometer(MS).【Results】Tested in the positive ion model,8870 metabolites were identified from the haemolymph of healthy and pebrine-infected female adults of A.pernyi and 5390 metabolites were annotated.In response to the infection of N.pernyi,472 differentially expressed metabolites were screened,including 260 up-regulated and 212 down-regulated.Furthermore,12 differentially expressed metabolites were secondarily identified,including 8 up-regulated and 4 down-regulated.Tested in the negative ion model,6716 metabolites were identified,and 3848 metabolites were then annotated.In total,301 differentially expressed metabolites were identified including 207 up-regulated and 94 down-regulated.Furthermore,9 differentially expressed metabolites were secondarily identified including 8 up-regulated and 1 down-regulated.Moreover,differentially expressed metabolites secondarily identified in the positive ion model included valine,benzothiazole,3-dehydroxycarnitine,1-methylguanine,2-ethoxynaphthalene,N6-acetyl-L-lysine,biotin,morin,timolol,acylcarnitine 15∶0,acylcarnitine 18∶4 and isoquercitrin,and those in the negative ion model included dimethylmalonic acid,glutaric acid,2,5-dihydroxybenzoic acid,1,3-diacetylpropane,DL-p-hydroxyphenyllactic acid,pantothenate,fluorescein,delphinidin-3-O-beta-glucopyranoside and lysoPI 16∶1.【Conclusion】There are significant differences in the metabolites of haemolymph collected from healthy and pebrine-infected female adults of A.pernyi.Twenty-one differentially expressed metabolites secondarily identified have been mined by metabolomics,and these metabolites can be used as potential biomarkers for developing the detection methods for pebrine disease in A.pernyi.