运用RAPD技术检测除草剂对草鱼的致突变作用

Detection on Mutagenicity of Herbicides to Grass Carp by RAPD Technique

用使它隆和嗪草酮注射草鱼,抽取注射前后的草鱼血液提取基因组DNA,选用20个随机引物对草鱼基因组DNA进行RAPD扩增。结果表明,11个引物能产生2～9条扩增带,扩增产物分子大小在200～1900bp之间。其中引物S10能检出用使它隆染毒前后草鱼基因组DNA的差异,引物S17能检出用嗪草酮染毒前后草鱼基因组DNA的差异。RAPD技术可运用于环境水质监测。

Two kinds of herbicides, Starane and metribuzin, were intraperitone ally injected into grass carps at the dosages of 24 mg·kg-1 and 170 mg·kg-1, respectively. Blood samples were drawn from caudal vein before and after inj ection with heparin sodium anti-coagulated syringes. Then 30 ìL of blood wer e added to 470 ìL of saline EDTA-Tris buffer. After adding final concentrati ons of 0.5%SDS and 200 ìg穖L-1 proteinase K, the mixture was incubated overnight at 55 ℃. The DNA was extracted and purified by successive extraction s with phenol, phenol∶chloroform∶isoamyl alcohol (25∶24∶1) and chlorof orm: isoamyl alcohol (24∶1). Then the DNA was precipitated with ice-cold abs olute ethanol and washed with 70%ethanol. The DNA pellet was dried and re-s uspended in Tris-EDTA buffer (pH 8.0). The DNAs in every experimental groups w ere mixed and diluted 80～100 times for PCR template. 20 arbitrary primers were used to amplify genomic DNA of grass carp. The amplification reaction was perfo rmed in 10 mmol·L-1 Tris-HCl (pH 9.0), 50 mmol·L-1 KCl, 0.1%Triton X-100, 2.5 mmol·L-1 MgCl2, 0.2 mmol·L-1 dNTPs, 15 ng primer, 1.2 units Taq DNA polymerase and 20 ng template DNA in a final volume of 25 ìL. The rea ction parameters were: predenaturing for 3 min at 95 ℃, then denaturing for 1 min at 94 ℃, annealing for 1 min at 36 ℃, prolonging for 2 min at 72 ℃, re action recycling 45 times, and finally prolonging for 10 min at 72 ℃. The ampl ified products were separated on 1.4%agarose gel containing ethidium bromide, and were observed and photographed under UV light. The result showed that 2～9 amplification fragments were generated by using 11 primers. The molecular size of amplified products was between 200～1 900 bp. The primer S10 produced differ ent amplified fragments of grass carp genomic DNA between before and after injec tion of Starane and S17 produced different amplified fragments of grass carp gen omic DNA between before and after injection of metribuzin. The result indicated that the mutagenicity of herbicides could be detected by using RAPD technique. I t was inferred that RAPD technique could be potentially used in monitoring of wa ter quality.