双酚A诱导斑马鱼P53基因CDS突变的RT-PCR及序列分析

Bisphenol A induced zebrafish P53 gene CDS mutation of RT-PCR and sequence analysis

目的:研究双酚A(bisphenol A,BPA)对斑马鱼肝脏p53基因编码序列(CDS)的致突变作用。方法:试验设空白对照组与BPA暴露组,连续染毒30 d后,从斑马鱼肝脏组织中提取RNA,通过RT-PCR方法扩增得到有完整编码区序列的p53基因,并进行测序分析。结果:序列分析表明,斑马鱼P53基因编码区序列长1122 bp,编码373个氨基酸,与网上已经登陆的斑马鱼p53序列有99.9%的相似系数,而与其他生物的P53序列存在一定的差异。在0.6 mg/L浓度下染毒30 d后,BPA暴露组斑马鱼p53基因序列发生了突变。直接测序证明,它们分别在135位、311位密码子发生GAG→GGG、AGG→CGG转变,使其编码的p53蛋白发生Glu→Gly变异。结论:试验结果表明BPA对斑马鱼p53基因CDS有致突变作用,采用RT-PCR技术检测BPA诱导p53基因突变是可行的。

Objective：To study the mutagenic effect of bisphenol A（BPA） on zebrafish p53 gene coding sequence（CDS） in liver cells.Methods： 20 zebra fish were randomly divided into blank control group and exposed groups,each containing 10 fish.After exposing for 30 days of BPA,the RNA was extracted from Zebrafish liver tissue,the p53 gene including a complete coding sequence of was obtained by RT-PCR.Results： Sequence analysis showed that the P53 gene CDS length of 1122bp,encoding 373 amino acids,the percent identity between the published zebrafish sequence of p53 and ours′ was 99.9%,with the other biological sequence of p53 existing some differences.After 30 days exposure,BPA exposure group zebrafish p53 gene had mutated at the concentration of 0.6 mg/L.The base substitution of GAG→GGG at codon 135,AGG→CGG at codon 311 were detected by PCR-directed sequencing.This may result in the Glu→Gly of expressed p53 protein.Conclusion： The results showed that BPA on zebrafish p53 gene CDS is mutagenicity and using RT-PCR detection of p53 mutations induced by BPA is feasible.