调控近江牡蛎HSP70基因对污染物响应的转录因子研究

Previous studies have illustrated that heat shock protein 70 (HSP70) gene in marine bivalves is in extensive, sensitive, and stable response to various pollutants and their mixture in sea water, then a molecular candidate of early warning biomarker of pollution. However, temperature change can influence expression level of hsp70, and further interferes reliability of warning pollution based on the expression level of hsp70. In order to resolve the issue, in this research project, on the basis of our previous study results of 5'-flaking regulatory region sequence of HSP70 gene in Jinjiang oyster (Crassostrea hongkongensis), and single nucleotide polymorphisms (SNPs) of the regulatory region for high expression of HSP70 in response to various pollutants, the oyster HSP70 transcription factors in unique response to pollutants (not to temperature) will be isolated, purified, and identified. Moreover, the expression patterns of HSP70 and its transcription factors will be further studied in response to heat stimuli and important various pollutants as well as their mixture of marine environment, and be analyzed to evaluate the suitability of the transcription factors taken as supplementary warning molecules of pollution to wipe off the influence of temperature change on expression level of HSP70. This work will lay a strong foundation of HSP70 used as a reliable warning biomarker of pollution, and explore the function and mechanism of the transcription factors in regulation of HSP70 expression in response to pollutants. So far, the HSP70 transcription factors in unique response to pollutants remain unclear, therefore, it is necessary to study the transcription factors, and the research work is full of innovation.

现有研究显示海洋双壳类的HSP70s分子对污染物具有广泛反应性，反应灵敏度高，响应信号持续时间长，是极具潜力的污染预警分子，但温度变化会影响HSP70s表达水平，干扰污染预警准确性。为了解决这一问题，本项目在已经完成的近江牡蛎HSP70基因 5'调控区序列，以及该基因对污染物高表达响应关联位点（遗传标记）的基础上，分离鉴定确认对污染物特异响应的转录因子，研究该转录因子和HSP70分子对热刺激和多种重要海洋环境污染物及其混合物的响应模式，分析两类分子的表达水平及关联性，阐明该转录因子作为污染辅助指示分子消除温度变化对HSP70分子表达水平影响的可行程度，为提高HSP70分子对污染预警的准确性奠定基础；同时，揭示该转录因子在调控HSP70基因对污染物响应中的作用机制。目前还无HSP70基因对污染物特异反应的转录因子的研究报道，所以本项研究不仅具有理论意义，也有显著创新性。

本项目采用磁性DNA亲和提纯技术以及基质辅助激光解析电离飞行时间质谱技术，在非胁迫（对照）和胁迫（温度胁迫、重金属及有机污染物胁迫）条件下，对近江牡蛎ChHsp70及ChHsc70启动子区域结合的转录调控因子分别进行分离提纯，进一步鉴定各处理组与对照间和各处理组间的差异蛋白身份，共鉴定出ChHsp70潜在转录调控因子14个，ChHsc70潜在转录调控因子11个。本项目从中选取6个蛋白（ChDSP1, ChHP1BP3, ChPur-α, ChYB-1, ChGNAQ，ChGATA-4）进行了深入研究。利用E. coli/pET原核表达体系，成功表达提纯得到可溶性融合蛋白His6-ChDSP1、 His6-ChHP1BP3及His6-ChPur-α，并通过EMSA技术在体外验证了目标融合蛋白His6-ChDSP1、His6-ChHP1BP3与ChHsp70启动子及His6-ChPur-α与ChHsc70启动子的特异性结合。利用荧光素酶报告基因表达体系及RNAi技术鉴定出ChDSP1, ChHP1BP3，ChPur-α及ChGATA-4对相应热休克蛋白转录表达的负调控作用，以及ChYB-1和ChGNAQ对ChHsc70转录表达的正调控作用。采用荧光定量RT-PCR技术从6个转录调控因子与热休克蛋白基因ChHsp70或ChHsc70的转录表达模式证实正、负转录调控因子基因与对应热休克蛋白ChHsp70和ChHsc70的转录表达模式相似，且转录因子基因的转录达峰时间点分别比对应的热休克蛋白基因的mRNA表达峰值出现的时间点提早（正调控）和延后（负调控），说明正、负两类型调控因子在ChHsp70或ChHsc70表达过程中分别发挥正或负调控作用。研究阐明了ChYB-1和ChGnaq对温度变化只有短暂反应，对污染物有偏好性响应，通过比值ChHsc70/ChGnaq (2−∆∆Ct)和ChHsc70 / ChYB-1 (4-∆∆Ct)可以矫正温度变化对污染预警分子ChHsc70指示污染发生的干扰作用。本项目的研究成果不仅在理论层面完善了热休克蛋白Hsp70及Hsc70转录表达的调控机制，在应用基础层面找到了辅助污染预警分子ChYB-1和ChGnaq，建立了一种方法来矫正温度变化干扰ChHsc70对污染发生与否的指示作用，为更准确地对海洋环境污染状况进行预警预报提供了理论和技术支撑。