【摘要】 为探讨咪唑类物质KK-42提高感染嗜水气单孢菌的日本沼虾幼虾成活率的机制,首次克隆了日本沼虾几丁质酶基因-3部分序列,研究了其时空表达及酶活力的变化。将体长3.5~5.0cm的日本沼虾幼虾随机分为两组,分别用1.95×10-4 mol/L的咪唑类物质KK-42溶液(试验组1)或不含咪唑类物质KK-42的溶液(对照组1)浸泡处理1min,12h后,分别注射嗜水气单胞菌菌悬液(菌攻毒试验组和菌攻毒对照组)或生理盐溶液(试验组2和对照组2),不同时间测定幼虾的成活率,分析几丁质酶基因-3mRNA水平及其酶活力的变化。结果显示,12~48h时,菌攻毒试验组的成活率比菌攻毒对照组增加133%以上。克隆的日本沼虾几丁质酶基因-3部分mRNA序列与其他甲壳类具有90%以上同源性。几丁质酶基因-3表达水平在肝胰腺中最高,以蜕皮前期最为显著。与对照组1相比,在12h,试验组1几丁质酶基因-3mRNA水平和酶活力分别增高了150%和75%;菌攻毒试验组mRNA水平在12~48h明显高于菌攻毒对照组,尤其在24h,增幅高达304%,酶活力在24h比菌攻毒对照组增高48%。咪唑类物质KK-42预处理能显著上调感染嗜水气单胞菌的日本沼虾幼虾肝胰腺几丁质酶基因-3的转录、诱导酶活力增强,这可能是咪唑类物质KK-42提高幼虾成活率的机制之一。更多还原

【Abstract】 This study was designed to determine the possible molecular mechanism of imidazole derivative KK-42 acting for increasing survival rate of oriental river prawn Macrobrachium nipponense infected with Aeromonas hydrophila.The part sequence of oriental river prawn chitinase-3was firstly cloned and the spatio-temporal expression of the gene as well as its activity were measured.The prawn with body length3.5—5.0cm was soaked for 1min in KK-42 solution at a concentration of 1.95×10-4 mol/L(KK-42 treatment group 1)or 0(control group 1),separatively.The prawn in KK-42 treatment group 1was injected individually with A.hydrophila suspension(KK-42 treatment group-A.hydrophila)or saline(KK-42 treatment group 2)into the ventral sinus at 12 hafter KK-42 treatment and the prawn in control group 1was divided into control group-A.hydrophilaand control group 2,according to the same way.Then the survival rate was surveyed at different time,and the prawn chitinase-3 mRNA level and activity derived from different tissues were measured.Results showed that the survival rate was dramatically increased by133%in KK-42 treatment group-A.hydrophilathan that in the control group-A.hydrophila.Sequence comparison indicated that the prawn chitinase-3deduced amino acid sequence of M.nipponense had an overall similarity of 90%to that of other crustaceans.Real-time PCR analysis revealed that the prawn chitinase-3was mainly expressed in hepatopancreas,and higher at premolt.Compared with the control group1,the changes in the prawm chitinase-3mRNA level and activity from KK-42 treatment group was found to be an up-regulation about 150%and 75%,respectively.In KK-42treatment-A.hydrophilagroup,the prawn chitinase-3mRNA transcripts were significantly higher at 12—48h,the maximum at 24 hwith an increase by 304%,than that in control group-A.hydrophilaand its activity increased by 48%at 24 h.In conclusion,imidazole derivative KK-42 pretreatment can significantly up-regulate the prawn chitinase-3expression as well as its activity in hepatopancreas of prawn infected with A.hydrophila,which is likely one of the molecular mechanisms of imidazole derivative KK-42 acting for increasing survival rate of the prawn infected with A.hydrophila.更多还原