论文部分内容阅读
目的建立细胞色素P450CYP3A65荧光标记转基因斑马鱼用于快速、直观检测重金属(铜、镉、锌)及类二噁英化合物五氯联苯(PCB126)等环境污染物。方法 PCR方法钓取斑马鱼CYP3A65基因调控序列,构建于p T2AL200R150G转座载体克隆位点,将p Tol2-CYP3A65-EGFP质粒与p CS-TP转座酶m RNA共同注射入斑马鱼胚胎单细胞,通过荧光筛选,遗传选育和建立荧光标记转基因斑马鱼品系Tg(CYP3A65-EGFP);利用该转基因斑马鱼胚胎及幼鱼检测上述3种重金属及PCB126的生物学响应。结果成功建立了CYP3A65荧光标记转基因斑马鱼品系,绿色荧光在肝和消化道表达;该转基因斑马鱼对3种重金属及PCB126产生生物学响应。染毒96 h后,铜0.1和0.2μmol·L-1、镉0.35和0.7μmol·L-1组、锌1.5和3μmol·L-1组、PCB126所有浓度组荧光相对表达量显著增强(P<0.01);铜0.9μmol·L-1组、镉2.7和5.4μmol·L-1组及锌24μmol·L-1组荧光相对表达量显著减弱(P<0.01);染毒168 h后,铜0.1和0.2μmol·L-1组、镉0.35和0.7μmol·L-1组、锌1.5和3μmol·L-1组及PCB126 2~32μmol·L-1浓度组荧光相对表达量显著增强(P<0.01),铜0.9μmol·L-1组、镉2.7和5.4μmol·L-1组、锌12和24μmol·L-1组荧光相对表达量减弱(P<0.05),随着浓度增加荧光减弱,呈现出量效关系。结论 CYP3A65荧光标记转基因斑马鱼模型可以用来检测铜、镉、锌及PCB126等环境污染物。
Objective To establish a cytochrome P450CYP3A65 fluorescent labeled transgenic zebrafish for rapid and visual detection of environmental pollutants such as heavy metal (copper, cadmium, zinc) and dioxin - like compound PCB126. Methods The regulatory sequences of CYP3A65 gene in zebrafish were obtained by PCR. The constructed plasmid pTAL2-CYP3A65-EGFP was co-injected into pZAL200R150G transposon. The fluorescence-labeled transgenic zebrafish strain Tg (CYP3A65-EGFP) was selected by fluorescence screening and the biological response of the three heavy metals and PCB126 was detected by using the transgenic zebrafish embryo and juvenile fish. Results CYP3A65 fluorescently labeled transgenic zebrafish strains were successfully established. The green fluorescence was expressed in liver and gastrointestinal tract. The transgenic zebrafish had biological responses to three heavy metals and PCB126. After exposure to 96 h, the relative fluorescence intensity of all the concentrations of PCB126 in groups of 0.1 and 0.2 μmol·L-1 Cu, 0.35 and 0.7 μmol·L-1 groups, 1.5 and 3 μmol·L-1 groups of zinc increased significantly (P < 0.01). Compared with 0.9 μmol·L-1 copper group, the relative fluorescence intensity of cadmium in 2.7 and 5.4 μmol·L-1 groups and 24 μmol·L-1 groups decreased significantly (P <0.01) And 0.2μmol·L-1 group, the fluorescence relative expression levels of cadmium 0.35 and 0.7μmol·L-1 group, zinc 1.5 and 3μmol·L-1 group and PCB1262 ~ 32μmol·L-1 group were significantly increased (P <0.01 ), 0.9μmol·L-1 copper, 2.7μmol·L-1 and 2.7μmol·L-1 cadmium groups, and the relative fluorescence intensity of 12 and 24μmol·L-1 groups were weakened (P <0.05) The amount of effective relations. Conclusion CYP3A65 fluorescently labeled transgenic zebrafish model can be used to detect copper, cadmium, zinc and PCB126 and other environmental pollutants.