食蚊鱼(Gambusia afinis)cat、gapdh和gst基因的克隆及其在生态毒理学中的应用

Cloning of cat,gapdh and gst Genes of Gambusia affinis and Its Application in Ecotoxicology

根据Ensembl、Genbank登录的鱼类cat、gapdh和gst基因的CDS序列设计普通PCR扩增引物,寻找食蚊鱼的cat、gapdh和gst基因的c DNA片段,并根据定量引物设计要求设计出相应的SYBR Green I荧光定量RT-q PCR引物,建立了食蚊鱼cat、gapdh和gst基因的SYBR Green I荧光定量RT-q PCR方法。该方法在104~108数量级范围内有良好线性关系(R=0.999~1.000);熔解曲线显示扩增产物特异性良好,均为单一峰值;质粒标准品最高浓度与最低浓度的批内试验变异系数与批间试验变异系数均低于2%。利用该方法监测和评价环境污染物对水生生物的影响,选择了水体中常见典型药物污染物——双氯芬酸,研究其对食蚊鱼抗氧化基因表达的影响。结果表明,雌性食蚊鱼暴露在不同浓度双氯芬酸钠(0.005、0.05、0.5和5 mg·L-1)24 h后,其肝脏cat、gapdh和gst的mRNA呈现显著变化,相对于对照组,在低浓度0.005 mg·L-1时,cat与gst mRNA的表达量均有极显著上升(p<0.01),而其它浓度均极显著下降(p<0.01)。试验表明该方法具有快速、精确、灵敏度高的优点,可为利用该类小型鱼类的原位污染物的生物监测和生态毒理评价提供有力的技术支持。

According to the coding sequences of cat gapdh and gst genes in fish species published in Ensembl and GenBank, three pairs of primers were designed to find the eDNA sequences of cat, gapdh and gst in mosquitofish （Gambusia afffnis）.The primers for SYBR Green I real-time quantitative PCR was employed based upon the design requirements of quantitative primer. A new method of SYBR Green I real-time quantitative PCR for determination of cat, gapdh and gst genes in G. affinis was successfully established. The results showed that a good linear rela- tionship existed in the range from 104 to 108copies：μLI（R= 0.999~ 1.000） and the melt curve exhibited good speci- ficity of amplification products. The co-efficiencies of variation for both intra- and inter-experimental reproducibili-ty were less than 2%. Furthermore, in order to validate and assess the utilization and feasibility of the new method in ecotoxicology, diclofenac, a typical pharmaceutical detected frequently in water, was employed to investigate its effects on the expression of antioxidant genes of G. aft^nis. Results showed that the cat and gst mRNA expressions were very significantly induced （P〈0.01） in 0,005 mg·L1 when female individuals were exposed to diclofenac so- dium salt （0.005~ 5 mg·L-1） for 24 h, but decreased very significantly （P〈0.01） with the increasing concentrations. It indicates that this new method is feasible, accurate and highly sensitive, and may provide a methodological basis for the utilization of mosquitofish as a sentinel in molecular eco-toxicological assessment of environmental pollution.