摘要
对草鱼腹腔注射了CuSO4,抽取注射前后其血液以提取基因组DNA。选用10个随机引物对草鱼基因组DNA进行了RAPD扩增,结果表明:5个引物能产生1～5条扩增带,扩增产物分子大小在200～3500bp之间,其中引物S15和S16能检出用CuSO4染毒前后草鱼基因组DNA的差异。CuSO4可使草鱼基因组DNA发生改变,应对其使用加以限制。
CuSO4 were intraperitoneally injected into grass carps with the dosage of 0.02 mg/kg. The DNA was extracted and purified by successive extractions with phenol, phenohchloroform：isoamyl alcohol （25：24：1） and chloroform. Then the DNA was precipitated with ice-cold absolute ethanol and washed with 70% ethanol. The DNA pellet was dried and resuspended in Tris-EDTA buffer （pH 8.0）. The DNAs extracted from the blood which was drawn before injection of CuSO4 were mixed and diluted 80-100 times for the PCR template before injection, and that extracted after injection of CuSO4 were mixed and diluted 80-100 times for the PCR template after injection . 10 arbitrary primers （purchased from Shanghai Sangon Biotechnology Corporation, product code： S11-S20） were used to amplify the genomic DNA of grass carp. The amplification reaction was performed in 2.5 μL 10×buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 15 ng primer, 1.2 units Taq DNA polymerase and 20 ng template DNA in a final volume of 25 μL. The reaction parameters were： predenaturing for 5 min at 94℃, then denaturing for 1 min at 94℃, annealing for 1 min at 36℃, prolonging for 2 min at 72℃, reaction recycling 45 times, and finally prolonging for 10 min at 72℃. The amplified products were separated on 1.4% agarose gel containing ethidium bromide, and were observed and photographed under UV light. The result showed that 1-5 amplification fragments were generated by using 5 primers. The molecular size of amplified products was between 200-3 500 bp. The primer S15 and S16 produced different amplified fragments of grass carp genomie DNA between before and after injection of CuSO4. So CuSO4 can make changes with genome of grass carp,it should be limited to a certain extent.
出处
《浙江海洋学院学报（自然科学版）》
CAS
2007年第1期41-43,47,共4页
Journal of Zhejiang Ocean University(Natural Science Edition)
