镉胁迫下大弹涂鱼肝脏蛋白质组双向电泳分析

2-DE analysis in liver of Boleophthalmus pectinirostris during acclimation to cadmium exposure

采用双向电泳技术,对重金属镉暴露后海洋鱼类大弹涂鱼肝脏组织的差异蛋白质进行了研究。pH=5～8、7 cm胶条电泳图谱经PDquest软件分析,结果表明,对照组、慢性胁迫组和急性胁迫组平均检测到的蛋白点数分别为512±35、509±29和532±22,匹配率约为90%,急性镉胁迫后差异蛋白点数为14个,其中7个蛋白点在胁迫后表达量显著下调,4个蛋白点显著上调,消失1个蛋白点,并新增1个蛋白点。对其中7个差异蛋白点进行肽指纹图谱分析(MALDI-TOFMS),差异蛋白分别为K18,prohibitin,GTP-binding protein Ypt1,two-componentsystem response regulator,similar to T-complex protein 1 subunit theta(TCP-1-theta)(CCT-theta),transcriptional regulator,CopG family和mitogenactivatedprotein kinase homologue。而慢性镉胁迫后差异表达点数为15个,其中8个蛋白点在胁迫后表达量显著上升,5个蛋白点显著下调,消失1个蛋白点,并新增1个蛋白点。对其中6个差异蛋白点进行肽指纹图谱分析(MALDI-TOF-MS),差异蛋白分别为GTP-binding protein Ypt1,two-component system response regulator,hypothetical protein BT-1634,keratin9,anscriptional regulator,CopG family和mitogen-activated protein kinase homologue。通过分析各蛋白的变化,探讨了慢性(0.05 mg/L,30 d)和急性(5mg/L,3 d)重金属镉胁迫的致毒机制。在急慢性镉胁迫胁迫下,不仅大弹涂鱼体内的蛋白表达发生相应的变化,鱼体内环境的动态平衡也被打破。这种变化超过大弹涂鱼的自我解毒和保护能力,就会使得肝脏细胞的正常代谢受到破坏,引起细胞凋亡。

The aim of the present study is to characterize differental protein exposure profiles in the liver of Boleophthalmus pectinirostris after cadmium （Cd） exposure through the proteomic approaches. For this purpose, we have developed a two-dimensional polyacrylamide gel electrophorcsis （2D-PAGE） technique for examining the response of the proteome from the liver tissue of Boleophthalmus pectinirostris to test its extremely severe cadmium toxicity. Actually, about 530 protein spots have been detected from the liver samples to a 2D-PAGE gel in the pH range 5 - 8. Fish of this sort exposed were supposed to be separated into three groups： an acute exposure group （5 mg/L for 3 days）, a chronical exposure （0.05 mg/L for 30 days）, and a control group （no Cd）. The results of our examination show that the total amount of proteins found in the control group, the acute exposure group and chronical exposure group were 512 ~ 35, 509 ＋ 29 and 532 -＋ 22, i＇espectively. The matching rate in average was 90%. For more specific purposes, 14 protein spots were found differentially ex- posed after the acute Cd exposure by two-dimensional gel elec- trophoresis （2-DE）. The exposure of the 8 proteins among them were significantly down-regulated, with four of them being up-regulated, one merely seen in the control, and the one added. Seven of them were thern analyzed by using MALDI-TOF-MS. The results of our ex- amination demonstrates that the proteins were K18, prohibitin, GTP- binding protein Yptl, two-componcntial system response regulator, similar to T-complex protein 1 subunit theta （TCP-l-theta） （CCT- theta）, transcriptional regulator, CopG family and mitogen^activated protein kinase homologue, respectively. The fifteen protein spots were found differentially exposed after the chronic Cd exposure, whereas eight spots among them were found significantly up-regulated,and five down-regulated, with one only noticed in control, and one added. In addition, 6 of them were analyzed by using MALDI-TOF-MS. The re- suits of our examination suggest that the 6 proteins were GTP-binding protein Yptl, the two-componential system response regulator, hypo- thetical protein BT-1634, keratin 9, and inscriptional regulator CopG family and mitogen-activated protein kinase homologue, respectively. Based on their variable levels and trends on the 2D-PAGE gel, this protein exposure examination suggestes that the finding can be utilized as biomarkers to investigate cadmium pollution degrees in the halobios survival, and evaluate the degree of risk of the human fatalities. In addition, the result of this research reveal that the element cadmium can not only result in differences in protein exposure profiles of Boleophthalmus pectinirostris, but also could induce the homeostasis, even further leading to the apoptosis.