甜菜夜蛾半胱天冬酶基因的克隆及对细胞凋亡诱导剂和病原微生物感染的表达响应

Cloning of Spodoptera exigua(Lepidoptera:Noctuidae)caspase genes and their expression in response to apoptosis inducers and pathogenic microorganism infection

【目的】本研究旨在明确甜菜夜蛾Spodoptera exigua半胱天冬酶(caspase)在细胞凋亡诱导剂诱导和病原微生物胁迫下的表达模式,为丰富鳞翅目昆虫细胞凋亡机制研究奠定基础。【方法】利用RT-PCR技术从甜菜夜蛾3龄幼虫体内扩增两个半胱天冬酶基因(SeCasp-3和SeCasp-4)编码区的全长;利用qPCR技术分别检测两个半胱天冬酶基因在细胞凋亡诱导剂过氧化氢(hydrogen peroxide,H2O2)(100μmol/L)、放线菌素D(actinomycin D,ActD)(10μg/mL)和地塞米松(dexamethasone,DEX)(50μg/mL)诱导后甜菜夜蛾脂肪体细胞中及病原菌苏云金芽孢杆菌Bacillus thuringiensis kurstaki(Btk)(108个细菌/mL)、大肠杆菌TG1菌株Escherichia coli TG1(E.coli TG1)(108个细菌/mL)、烟芽夜蛾囊泡病毒3h株(Heliothis virescens ascovirus 3h,HvAV-3h)(1.16×1011个基因组拷贝/mL)和苜蓿银纹夜蛾核型多角体病毒(Autographa californica multiple nucleopolyhedrovirus,AcMNPV)(5000 OBs/μL)感染后甜菜夜蛾3龄幼虫体内的表达模式。【结果】SeCasp-3(GenBank登录号:MW183334)的编码区长942 bp,共编码313个氨基酸;SeCasp-4(GenBank登录号:MW183335)的编码区长843 bp,共编码280个氨基酸。SeCasp-3和SeCasp-4的假定蛋白序列与家蚕Bombyx mori Dronc的氨基酸序列一致性分别为45.54%和58.46%,且SeCasp-3和SeCasp-4之间具有较高的同源性。SeCasp-3和SeCasp-4在不同化学物质诱导下的甜菜夜蛾脂肪体细胞中的表达模式存在明显差异,在100μmol/L H2O2和10μg/mL ActD处理后24和48 h,脂肪体细胞中SeCasp-3和SeCasp-4的相对表达量均显著升高;50μg/mL DEX处理后24和48 h,SeCasp-3的相对表达量未见显著变化,但SeCasp-4的相对表达量升高了数千倍。在不同病原微生物感染后的甜菜夜蛾3龄幼虫体内,SeCasp-3和SeCasp-4的表达模式基本相同。一般线性模型的分析结果表明,Btk和E.coli TG1的感染不造成SeCasp-3和SeCasp-4相对表达量的显著变化,而HvAV-3h和AcMNPV的感染则显著抑制了这两个基因的表达。【结论】本研究鉴定了两个甜菜夜蛾半胱天冬酶基因,并分析了其对细胞凋亡诱导剂和病原微生物感染的表达响应,为进一步探究半胱天冬酶功能和昆虫细胞凋亡过程提供了重要的理论基础。

【Aim】To identify the expression patterns of caspase in Spodoptera exigua induced by apoptosis inducer and under pathogenic microorganism stress,so as to lay a foundation for further study on the mechanism of apoptosis in Lepidoptera insects.【Methods】RT-PCR was used to amplify the full-length coding regions of two caspase genes(SeCasp-3 and SeCasp-4)from the 3rd instar larvae of S.exigua.qPCR was used to detect the expression patterns of the two SeCasp genes in the fat body cells of S.exigua after induction by apoptosis inducers hydrogen peroxide(H2O2)(100μmol/L),actinomycin D(ActD)(10μg/mL)and dexamethasone(DEX)(50μg/mL),and in the 3rd instar larvae of S.exigua infected by pathogens Bacillus thuringiensis kurstaki(108 colonies/mL),Escherichia coli TG1(108 colonies/mL),Heliothis virescens ascovirus 3h(HvAV-3h)(1.16×1011genome copies/mL)and Autographa californica multiple nucleopolyhedrovirus(AcMNPV)(5000 OBs/μL).【Results】The coding regions of SeCasp-3(GenBank accession no.:MW183334)and SeCasp-4(GenBank accession no.:MW183335)are 942 and 843 bp in length,encoding 313 and 280 amino acids,respectively.The putative protein sequences of SeCasp-3 and SeCasp-4 show 45.54%and 58.46%amino acid sequence identities with Dronc of Bombyx mori,respectively,and SeCasp-3 and SeCasp-4 show high homology.SeCasp-3 and SeCasp-4 exhibited different expression patterns in the fat body cells of S.exigua induced by different chemicals.The relative expression levels of both SeCasp-3 and SeCasp-4 in the fat body cells treated by 100μmol/L H2O2 and 10μg/mL ActD for 24 and 48 h were significantly up-regulated.In the fat body cells of S.exigua treated by 50μg/mL DEX for 24 and 48 h,the relative expression level of SeCasp-3 showed no significant change,but that of SeCasp-4 was significantly enhanced by several thousand folds as compared to the control.The expression patterns of SeCasp-3 and SeCasp-4 in the 3rd instar larvae of S.exigua infected by different pathogens were basically similar.The general linear model analysis showed that the infection of B.thuringiensis kurstaki and E.coli TG1 caused no significant change in the expression levels of SeCasp-3 and SeCasp-4 in the 3rd instar larvae of S.exigua,while the infection of HvAV-3h and AcMNPV significantly inhibited the expression of the two genes.【Conclusion】Two S.exigua caspase genes were identified and their expression responses to apoptosis inducers and pathogenic microorganism infection were assayed in this study,laying a foundation for further exploring the function of caspase and process of insect apoptosis.