溶藻弧菌对凡纳滨对虾血细胞毒性及细胞凋亡和免疫相关基因的影响

Effects of Vibrio alginolyticus on hemocytes toxicity,apoptosis and immune-related genes in Litopenaeus vannamei

【目的】明确溶藻弧菌对凡纳滨对虾的毒性影响及应激情况下对虾的生理响应,为揭示凡纳滨对虾的免疫机理提供参考依据。【方法】采用微量注射器于凡纳滨对虾第二、三步足间注射10.0μL溶藻弧菌悬液(1×108CFU/mL),对照组注射等量灭菌生理盐水,分别于感染后0、1.5、3.0、6.0、12.0、24.0和48.0 h取样,利用流式细胞仪测定对虾血细胞中的活性氧(ROS)和一氧化氮(NO)含量,并以实时荧光定量PCR分析溶藻弧菌感染对血细胞过氧化氢酶基因(CAT)、QM基因、谷氨酰胺转移酶基因(TGase)、细胞色素C基因(CYC)和Caspase-3基因表达的影响。【结果】凡纳滨对虾感染溶藻弧菌后,其血细胞中ROS和NO含量均随感染时间的延长呈持续上升趋势;CAT基因的相对表达量在感染后6.0 h极显著升高至峰值(P<0.01,下同),随后持续下降,至感染后48.0 h极显著低于对照组;QM基因的相对表达量呈先降低后升高再降低的变化趋势,于感染后12.0 h达峰值,而后持续下降,至感染后48.0 h其相对表达量极显著低于对照组;TGase基因的相对表达量分别在感染后1.5和12.0 h出现峰值,从出现第2个峰值后开始快速下降,至感染后48.0 h其相对表达量显著低于对照组(P<0.05,下同);CYC基因相对表达量呈先升高后降低的变化趋势,于感染后1.5 h达峰值,随后持续下降,从感染后12.0 h起低于对照组,但差异不显著(P>0.05);Caspase-3基因相对表达量在感染后1.5 h达峰值,至感染后6.0 h其相对表达量显著低于对照组,随后逐渐恢复,于感染后24.0 h出现第2个峰值,但至感染后48.0 h又降至正常水平以下。【结论】溶藻弧菌感染初期凡纳滨对虾启动免疫机制并产生ROS和NO以对抗病原菌入侵,随感染时间的延长,机体内积累过多ROS和NO,此时抗氧化系统被激活;当过多的ROS和NO无法被抗氧化系统清除时,CYC和Caspase-3等凋亡相关基因大量表达以清除受损细胞。

Abstract:【Objective】The toxic effect of Vibrio alginolyticus on Litopenaeus vannamei and the physiological response of L.vannamei under stress were explored in order to provide referential basis for revealing the immune mechanism of L.vannamei.【Method】10.0μL V.alginolyticus suspension(1×10^8 CFU/mL)were injected in the intermediate section between the second and third feet of L.vannamei with micro injector.Equal volume of sterilized saline was injected in the control group.Hemocytes were sampled at 0,1.5,3.0,6.0,12.0,24.0 and 48.0 h after infection.The contents of reactive oxygen species(ROS)and nitric oxide(NO)in hemocytes of L.vannamei were determined by flow cytometry.Real-time fluorescence quantitative PCR was used to analyze the effect of V.alginolyticus infection on the relative gene expression levels of catalase gene(CAT),QM gene,transglutaminase gene(TGase),cytochrome C gene(CYC)and Caspase-3 gene.【Result】The results revealed that with the extension of infection time,the contents of ROS and NO in hemocytes of L.vannamei after V.alginolyticus infection both kept a rising trend,the relative expression level of CAT gene extremely increased and reached the peak at 6 h after infection(P<0.01,the same below),then decreased continuously and was extremely lower than that in control group at 48.0 h after infection.The relative expression level of QM gene decreased first,and then increased and decreased again,it reached the peak at 12.0 h after infection,then decreased continuously.At 48 h after infection,the relative expression level of QM gene was extremely lower than that in control group.The relative expression level of TGase gene reached the peaks at 1.5 h and 12.0 h,then reduced rapidly after the second peak.At 48.0 h after infection,the relative expression level of TGase gene was significantly lower than that in control group(P<0.05,the same below).The relative expression level of CYC gene increased first and then decreased,and reach the peak at 1.5 h after infection,and then decreased continuously.At 12.0 h after infection,the relative expression level of CYC gene was lower than that in control group,but the difference was not significant(P>0.05).The relative expression level of Caspase-3 gene reached the peak at 1.5 h after infection.At 6.0 h after infection,the relative expression level of Caspase-3 gene was significantly lower than that in control group,then gradually returned to normal level.At 24.0 h after infection,the relative expression level of Caspase-3 gene reached the second peak,and then decreased to below the normal level 48.0 h after infection.【Conclusion】These results indicate that at the initial stage after V.alginolyticus infection,the immune mechanism of L.vannamei is activated and the production of ROS and NO are induced to fight against the invasion of pathogenic bacteria.As the extension of infection time,the excessive ROS and NO are accumulated in organism,and the antioxidant system is activated to eliminate ROS and NO.However,when the production of ROS and NO are too excessive to be removed timely by antioxidant system,then high expressions of apoptosis-related genes such as CYC and Caspase-3 are induced to eliminate damaged cells.