重金属离子胁迫下花斑裸鲤钙调蛋白磷酸酶(Calcineurin)的应答及其分子调节机理研究

Gymnocypris eckloni, the naked carp species distributed in the Upper Yellow River,is an important wildlife fish germ plasm resource and a significant commercial fish resource of Qinghai province. The result of water quality survey of the Upper Yellow River and monitoring of fish resource in the past 10 years showed that the pollution of heavy metal is worthy of attention. In order to investigate the inner physiological response and molecular regulating mechanism of Gymnocypris eckloni under the stress of heavy metals including copper, zinc, lead, chromium and mercury, the response of Calcineurin (CN) to heavy metal stress will be detected using Real Time PCR, western blotting and enzyme activity determination methods. The responses of Metallothionein(MT) to heavy metal stress will be detected under the condition of CN inhibited or not by CsA, and the correlation between CN and MT responding to heavy metal stress will be established. The responding sensitive tissues of CN and MT to heavy metal stress will be found. The responses of some correlated immunological responsive factors including IL-1,IL-6,TNF-α(the targetd genes of CN)to metal stress will be assayed. The correlation among the heavy metal, CN, MT and immunological responsive factors will be analyzed. The molecular regulating mechanism of CN and its signals transductions pathway responding to heavy metal stress will be primarily elucidated. In this research, the feasibility of making CN, MT and immunological responsive factors as biomarker for predicting the pollution of heavy metal will be probed into, which will play an important role in formulating preventive measures, monitoring of environment pollution, the conservation and restoration of endemic fish species in the Upper Yellow River.

花斑裸鲤(Gymnocypris eckloni)是黄河上游特有鱼种，是重要的水生野生动物种质资源，也是青海省重要的经济鱼类。近十年的黄河上游水域水质和鱼类资源监测结果显示重金属污染的问题不可忽视。为研究重金属离子胁迫条件下花斑裸鲤应答和分子调节机制，将以铜、锌、铅、镉、汞等为金属污染源，以花斑裸鲤钙调蛋白磷酸酶（Calcineurin，CN）在mRNA、蛋白和酶活性水平的变化确立CN与重金属胁迫间的相关性；以CN在被CsA抑制和非抑制条件下金属硫蛋白（Metallothionein，MT）表达量确立重金属胁迫下CN与MT间的相关性；找到CN和MT应答敏感组织；通过检测CN下游靶基因（IL-1、IL-6和TNF-α）表达量，分析CN及其信号通路的调节机制。探讨建立CN、MT及细胞因子等生物标志物作为重金属污染早期预警体系的可行性，这对制定预防性的管理措施、监控环境污染和资源保护恢复意义重大

花斑裸鲤（Gymnocypris eckloni）是黄河上游特有鱼种，是重要的水生野生动物种质资源，也是青海省重要的经济鱼类。本研究分析近十年的黄河上游水域水质和鱼类资源监测结果发现，黄河上游22个监测断面重金属离子水质监测结果基本符合国家渔业水质标准，但是少数采样点的检测值偶尔接近或超过国家标准，这种偶尔出现的瞬时的重金属污染或者长期低浓度的重金属污染对花斑裸鲤繁育、生长乃至种群分布及其水生生态系统的潜在危害不可忽视。通过基因克隆研究技术获得了CNs、MT、IL-1β、TNF-α、Cu/Zn-SOD、Mn-SOD、CAT、Hsp70等基因的全长cDNA序列和Hsp90、NFκB、NFAT和Nrf2等基因的部分cDNA序列，并分析了cDNA序列特征和组织分布表达特异性；通过Cu2+、Pb2+、Cd2+、Hg2+和Zn2+等5种重金属离子胁迫后，分析了花斑裸鲤不同组织中CNs及相关基因的应答模式，获得的5种重金属离子胁迫下花斑裸鲤不同组织的早期上调和明显上调的一些功能基因，可以作为水质监测、评价以及重金属污染早期预警的标记基因的候选生物标记物；运用聚类分析法获得了热休克、金属螯合、抗氧化应激和免疫应答共4类基因的应答层次，初步掌握了花斑裸鲤对重金属胁迫的分层次应答调节机制。通过进一步筛选和验证本研究获得的不同组织的潜在的生物标记物，为土著鱼类保护和水质监测提供理论依据，并为低浓度重金属离子胁迫下鱼类的分子毒理学研究提供基础数据。