六溴环十二烷胁迫下红树蚬鳃及肝胰腺线粒体基因表达定量研究
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摘要
红树蚬(Polymesoda erosa)作为一种主要栖息于潮间带的沼泽地或红树林的双壳贝类,近年来很多研究都证明其具有做为海洋污染监测指示物种的潜力。红树蚬受六溴环十二烷(HBCD)不同浓度(0、0. 086、0. 860、8. 600μg/dm~3)及不同天数(1、3、11、15、22 d)胁迫后,从转录组上调文库中挑取6个线粒体编码基因:细胞色素c氧化酶亚基Ⅰ、Ⅲ(COXⅠ、COX Ⅲ),NADH脱氢酶亚基Ⅰ、Ⅲ(ND Ⅰ、ND Ⅲ),细胞色素b(Cyt b),无机焦磷酸酶(PPase),并用实时荧光定量PCR研究各基因在红树蚬各组织内的表达情况,因为基因不稳定性,利用β-actin作为内参对各目的基因所得数据进行均一化处理。结果发现:6个基因在HBCD胁迫后都有转录且差异表达。经生物统计学分析,胁迫后鳃及肝胰腺组织中各基因的表达量较对照组中有显著变化,总体上随着胁迫天数及浓度的增加呈增加趋势,但当胁迫浓度过高时,因线粒体产生的应激能力有限,表达量反而降低。最后从酶复合物在呼吸链、ROS生成、ATP合成中的作用等各方面分析了HBCD胁迫导致线粒体基因表达变化的原因,为进一步开展分子毒理研究和开发分子生物标志物在海洋监测中的应用奠定了基础。
Mangrove mud clam( Polymesoda erosa) has been proved to be an indicator specie in monitoring marine pollution by many studies in recent years. After post-exposure treatment to Hexabromocyclododecane( HBCD) at 1,3,11,15 and 22 days and three concentrations( 0. 086,0. 860 and 8. 600 μg/dm~3),several genes encoded by mitochondria were selected from the upregulated cDNA library of the species,including cytochrome c oxidase subunit Ⅰ,Ⅲ( COX Ⅰ,COX Ⅲ),NADH dehydrogenase subunit Ⅰ,Ⅲ( ND Ⅰ,ND Ⅲ),cytochrome b( Cyt b) and inorganic pyrophosphatase( PPase). Realtime fluorescent quantitative PCR( qRT-PCR) is often used to study the tissue distribution and expression level of mRNA in such studies,however the technique is subject to considerable experimental error and variation. A stably expressed housekeeping gene( β-actin in this study) is needed as a reference gene to normalize the variation of target genes before performing qPCR analysis and comparison with control group. The result showed that expression of 6 genes could be detected and transcribed differently in all tissues after hexabromocyclododecane exposure. After statistic analysis,we found that compared with the control the expression of 6 genes in gill and hepatopancreas changed significantly,increased on the whole with the increase of exposure time and concentrations,but decreased when the exposure concentration was too high for the reason of limited stress ability of mitochondrion probably. Finally from the function of multienzyme complex in respiratory chain,reactive oxygen generation,ATP synthesis,and etc. we analyzed the reason for mitochondrial gene expression changes caused by HBCD exposure in order to lay a foundation for further study of molecular toxicology and development of molecular biomarkers in monitoring marine pollution in the near future.
Mangrove mud clam( Polymesoda erosa) has been proved to be an indicator specie in monitoring marine pollution by many studies in recent years. After post-exposure treatment to Hexabromocyclododecane( HBCD) at 1,3,11,15 and 22 days and three concentrations( 0. 086,0. 860 and 8. 600 μg/dm~3),several genes encoded by mitochondria were selected from the upregulated cDNA library of the species,including cytochrome c oxidase subunit Ⅰ,Ⅲ( COX Ⅰ,COX Ⅲ),NADH dehydrogenase subunit Ⅰ,Ⅲ( ND Ⅰ,ND Ⅲ),cytochrome b( Cyt b) and inorganic pyrophosphatase( PPase). Realtime fluorescent quantitative PCR( qRT-PCR) is often used to study the tissue distribution and expression level of mRNA in such studies,however the technique is subject to considerable experimental error and variation. A stably expressed housekeeping gene( β-actin in this study) is needed as a reference gene to normalize the variation of target genes before performing qPCR analysis and comparison with control group. The result showed that expression of 6 genes could be detected and transcribed differently in all tissues after hexabromocyclododecane exposure. After statistic analysis,we found that compared with the control the expression of 6 genes in gill and hepatopancreas changed significantly,increased on the whole with the increase of exposure time and concentrations,but decreased when the exposure concentration was too high for the reason of limited stress ability of mitochondrion probably. Finally from the function of multienzyme complex in respiratory chain,reactive oxygen generation,ATP synthesis,and etc. we analyzed the reason for mitochondrial gene expression changes caused by HBCD exposure in order to lay a foundation for further study of molecular toxicology and development of molecular biomarkers in monitoring marine pollution in the near future.
引文
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