菲律宾蛤仔两种谷氧还蛋白基因对微生物侵染和重金属胁迫的应答

摘要

研究了菲律宾蛤仔(Venerupis philippinarum)两种谷氧还蛋白VpGrx1和VpGrx2 mRNA在鳗弧菌(Vibrio an-guillarum)、藤黄微球菌(Micrococcus luteus)和重金属镉(Cd)作用下的表达调控规律。结果表明,正常菲律宾蛤仔VpGrx1和VpGrx2在所检测组织中均有表达,其中VpGrx1在鳃中的表达量最高,而VpGrx2在蛤仔血淋巴细胞、鳃和肝胰腺中的表达量均较高。鳗弧菌和藤黄微球菌侵染在某些时段可显著诱导VpGrx1和VpGrx2基因的表达水平(P<0.05)。另外,0.8μg/L、8μg/L和80μg/L Cd处理30 d后,菲律宾蛤仔肝胰腺中VpGrx1和VpGrx2基因转录水平显著升高(P<0.05),血淋巴细胞中 VpGrx1基因表达被显著抑制(P<0.05)而 VpGrx2基因表达被显著诱导(P<0.05)。以上结果表明, VpGrx1和VpGrx2在菲律宾蛤仔的固有免疫反应和抗氧化胁迫中发挥着重要作用。%Glutaredoxins (Grxs) are ubiquitous small disulfide oxidoreductases that play a crucial role in response to oxidative stress and regulation of growth and development. However, the structural features and function of Grxs in marine invertebrates are still unknown compared with those of Escherichia coli, yeast, mammals, and plants. In this study, quantitative RT-PCR was performed to assay the temporal expression patterns of VpGrx1 and VpGrx2 post Vibrio anguillarum, Micrococcus luteus and Cd challenge. VpGrx1 and VpGrx2 transcripts could be detected in all the ex-amined tissues, including gills, hepatopancreas, adductor muscles, mantle, and hemocytes. VpGrx1 was found to be most abundantly expressed in gills, while VpGrx2 was highly expressed in haemocytes, hepatopancreas and gills, which is consistent with the viewpoint that the hepatopancreas and gills are the most important organs in the host immune de-fense and detoxification systems. Bacterial challenging significantly up-regulated mRNA expression of VpGrx1 and VpGrx2at different time intervals, with a maximum 2.96 fold rise. The increased VpGrx1 and VpGrx2 transcripts may be associated with increasing reactive oxygen species (ROS) that is generated after apathogen challenge. In addition, the expression levels of VpGrx1 and VpGrx2 in the hepatopancreas of Venerupi sphilippinarum were also significantly up-regulated by 0.8, 8, and 80μg/L Cd with a larger amplitude in VpGrx2, and the results showed an obviously concen-tration-dependent increase. The highest expression level for VpGrx2 (34.87 fold) in hepatopancreas was observed after 80μg/L Cd exposure for 30 days. More or less compatible transcriptional modulation patterns were also experienced in several organisms, such as Pisum sativum, Glomus intraradices, and Homo sapiens. It has been demonstrated that Cd can induce oxidative stress by increasing levels of ROS. The increase in VpGrx1 and VpGrx2 expression in the hepato-pancreas of V. philippinarum could be induced by the production of ROS generated by Cd, suggesting aGrx protective role in the defense against oxidative stress. With regard to hemocytes, the expression of VpGrx1 mRNA decreased sharply (0.03 fold at 0.8 and 80μg/L Cd, 0.16 fold at 8μg/L Cd), while the expression levels of VpGrx2 were increased significantly (4.77 fold at 0.8μg/L Cd, 4.05 fold at 8μg/L Cd, and 4.63 fold at 80μg/L Cd) after 30 d. The selective up-or down-regulation of VpGrxs gene expression highlighted the fact that these isoenzymes probably played divergent physiological roles during the detoxification of Cd in hemocytes of V. philippinarum. Future studies should explore the diverse expression patterns of VpGrx1 and VpGrx2 genes in hemocytes of V. philippinarum after exposure to Cd. In conclusion, all these results suggested that VpGrx1 and VpGrx2 may be important proteins involved in host immune defense and heavy metal detoxification.