五氯酚钠诱导斑马鱼体细胞p53基因突变的克隆测序研究

A study on the cloning and sequencing of mutation of p53 gene induced by sodium pentachlorophenate in somatic cell of zebrafish

目的研究五氯酚钠(NaPCP)对斑马鱼体细胞p53基因的致突变作用。方法设置5个不同浓度的NaPCP暴露组及空白对照组,采用Tubingen品系斑马鱼进行斑马鱼急性毒性试验计算半数致死浓度(LC50),并依据急性毒性实验所得结果,设置1/10 LC50暴露剂量,暴露组与空白对照组各取10尾斑马鱼,在连续暴露45 d后提取斑马鱼肝脏组织RNA,采用反转录-聚合酶链式反应扩增p53基因c DNA序列,并进行测序分析。结果NaPCP急性毒性实验所得LC50为18.4μg/L,测序分析结果显示斑马鱼p53基因编码区序列长1125 bp,编码374个氨基酸,与Gen Bank已知Tubingen品系斑马鱼p53序列(GI:425876786)同源性为99.2%,与其他生物物种的p53序列存在一定差异。在NaPCP 1.8μg/L浓度下暴露45 d后,与对照组比较,暴露组斑马鱼p53基因序列的8、148和229位密码子发生GAG→AAG、CAT→CAG和CAG→CAA突变,导致所编码的p53蛋白发生Glu→Lys和His→Gln变异。结论 NaPCP是一种致基因突变物,可诱导斑马鱼体细胞p53基因发生突变,对人体可能有潜在的致基因突变风险。

Objective To explore the mutagenic effect of sodium pentachlorophenate（ NaPCP） on zebrafish p53 gene coding sequence（ CDS） in somatic cell. Methods The experiment was carried out using tuebingen strain of zebrafish,according to the results of acute toxicity test to determine the exposure levels in zebrafish. Zebrafish were randomly divided into blank control group and exposed groups,each containing 10 zebrafish. After exposing for 45 d of NaPCP,the RNA was extracted from liver of zebra fish,and the p53 gene including a complete coding sequence of was obtained by RT-PCR.Results LC50 of NaPCP was 18. 4 μg/L. Sequence analysis showed that the p53 gene CDS length of 1125 bp,encoding 374 amino acids. The percent identity between the published zebrafish sequence of p53（ GI： 425876786） and ours was 99. 2%,with the other biological sequence of p53 existing some differences. After 45 d exposure,zebrafish p53 gene of NaPCP exposure group had mutated at the concentration of 1. 8 μg/L. The base substitution of GAG→AAG at codon 8,CAT→CAG at codon 148 and CAG→CAA at codon 229 were detected by PCR-directed sequencing. This may result in the Glu→Lys and His→Gln of expressed p53 protein. Conclusion NaPCP is a kind of gene mutation,which can induce the mutation of p53 gene in zebrafish somatic cells,that has the potential mutagenic risk for humans.