过氧化氢酶在豌豆蚜抵御藤黄微球菌和大肠杆菌感染引起的氧化胁迫中的作用

Role of catalase in the antioxidative response induced by Micrococcus luteus and Escherichia coli infection in the pea aphid,Acyrthosiphon pisum(Hemiptera:Aphididae)

【目的】研究细菌-过氧化氢酶-豌豆蚜Acyrthosiphon pisum三者之间的关系,探索过氧化氢酶在豌豆蚜免疫防御反应中的作用。【方法】用体外培养结合菌落计数的方法检测藤黄微球菌Micrococcus luteus和大肠杆菌Escherichia coli感染豌豆蚜后在蚜虫体内的增殖;分别用微孔板荧光检测和实时定量PCR的方法检测藤黄微球菌和大肠杆菌感染豌豆蚜之后,蚜虫体内H_2O_2水平和过氧化氢酶基因的转录水平;通过RNA干扰技术对豌豆蚜过氧化氢酶基因(Cat)进行沉默,并检测沉默后感染藤黄微球菌和大肠杆菌的豌豆蚜体内H_2O_2浓度和细菌数目以及豌豆蚜存活率。【结果】藤黄微球菌感染12 h后,豌豆蚜体内H_2O_2水平以及过氧化氢酶基因表达水平显著升高;大肠杆菌感染12 h后,豌豆蚜过氧化氢酶基因表达水平上升,但H_2O_2水平无明显变化。沉默过氧化氢酶基因后,感染藤黄微球菌导致豌豆蚜体内H_2O_2含量在12 h显著上升,细菌数目在24 h约为对照组的一半,但对豌豆蚜存活率无影响;而过氧化氢酶基因沉默并未引起感染大肠杆菌的豌豆蚜体内H_2O_2水平、细菌数目以及豌豆蚜存活率发生明显变化。【结论】过氧化氢酶参与豌豆蚜对藤黄微球菌的防御过程,但不是该防御反应过程中的关键酶;而针对大肠杆菌感染,豌豆蚜可能通过其他途径来应对。

【Aim】 To investigate the relationship between bacteria, catalase （CAT） and the pea aphid, Acyrthosiphon pisum, and the role of catalase in the immune responses of the pea aphid. 【Methods】 The bacterial load in the aphid was determined by in vitro culturing and colony counting after Micrococcus luteus and Escherichia coli infection. H2O2 concentration and catalase gene transcriptional level of the aphid after bacterial infection were measured by microwell-plate fluorescence assay and real-time quantitative PCR, respectively. The catalase gene （Cat） of the pea aphid was knock-downed by RNA interference, and the H2O2 concentration, bacterial load and survival rate of the aphids were assayed after bacterial infection. 【Results】 The H2O2 level and the expression level of the catalase gene in the pea aphid increased markedly 12 h post infection by M. luteus. After E. coli infection, the expression level of the catalase gene increased 12 h post infection, but the H2O2 level had no obvious change. After the catalase gene was knock-downed, the H2O2 level of the aphid was up-regulated significantly 12 h post M. luteus infection, the bacterial load of dsCat-injected pea aphid was about half that of dsGFP-injected pea aphid 24 h post M. luteus infection, but the survival rate of aphids had no change after M. luteus infection. However, knock-down of the catalase gene had no effects on the H2O2 level of the aphid, proliferation of bacteria inside the aphid, and the survival rate of the aphids infected by E. coli. 【Conclusion】 The catalase is involved in the immune defense of the pea aphid against infection by M. luteus, but is not a key enzyme in this process. To cope with E. coli infection, however, the aphid may have different defense strategies.