注射磷脂酰丝氨酸对凡纳滨对虾血蓝蛋白合成、酚氧化酶活性的影响

Hemocyanin synthesis and hemocyanin-derived phenol oxidase in white shrimp(Litopenaeus vannamei)injected with phosphatidyl serine

  • 摘要: 为研究磷脂酰丝氨酸(PS)对凡纳滨对虾血蓝蛋白合成、酚氧化酶活性的影响,并探讨PS激活凡纳滨对虾血蓝蛋白发挥酚氧化酶活性的机制,实验采用5、10和20 μg/mL 3个PS浓度对凡纳滨对虾尾节肌肉进行注射,注射量为50 μL,对照组注射生理盐水,各处理组设3个平行组,取样时间为0、6、12、24、36、48和60 h,分别测定了血浆血蓝蛋白含量,肝胰脏血蓝蛋白p75、p77亚基和mRNA的表达以及血浆血蓝蛋白酚氧化酶活性。结果显示:与对照组相比,血蓝蛋白含量在6 h内略有下降,6~36 h内呈峰值变化,24 h时达到最大值(P<0.05);血蓝蛋白亚基p75、p77和mRNA表达在0~36 h内呈峰值变化,12 h时达到最大值(P<0.05);血蓝蛋白酚氧化酶活性在36 h内呈峰值变化,12 h时达到最大值(P<0.05)。研究表明,PS能够激活凡纳滨对虾血蓝蛋白的酚氧化酶活性,表现出明显的时间剂量效应,同时在短时间内引起血蓝蛋白含量下降,随后机体启动血蓝蛋白的合成机制,证实PS是凡纳滨对虾血蓝蛋白发挥酚氧化酶活性的激活因子,为对虾血蓝蛋白免疫活性的研究奠定了理论基础。

     

    Abstract: In order to investigate the effects of injecting phosphatidyl serine(PS)on hemocyanin synthesis and hemocyanin-derived phenol oxidase(Hd-PO)and probe into the pathway and activating mechanism of PS as a possible native elicitor for Hd-PO in white shrimp(Litopenaeus vannamei),we injected adult white shrimps different PS with 5,10,20 μg/mL,and assayed hemocyanin concentration in plasma,expression of hemocyanin subunits(p75 and p77)and mRNA in hepatopancreas,along with Hd-PO activity in plasma from white shrimp.They were investigated by spectrophotometry,native-PAGE,SDS-PAGE and RT-PCR in vivo with different sampling time at 0,6,12,24,36,48 and 60 h respectively,and we set the control group and parallel group.The results showed that hemocyanin concentration declined slightly during 6 h after injecting PS and afterwards changed as a peak by 36th h after injection with a maximum at 24th h(P<0.05);expression of hemocyanin subunits and mRNA all had peak changes during 36 h after injecting PS,and likewise,all reaching their ceiling values at 12th h(P<0.05);Hd-PO activity had a peak change within 36 h after injecting PS,with its climax at 12th h(P<0.05).The research revealed that PS could activate hemocyanin to show phenol oxidase activity with significant time-dose effects,and meanwhile brought down hemocyanin concentration in a short time at first and subsequently started the hemocyanin synthesis,indicating that PS is the elicitor of Hd-PO in white shrimp,which lays the theoretical foundation for the study on immunological function of hemocyanin in shrimp.

     

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