马氏珠母贝4种壳色选育系RAPD反应体系优化

旨在筛选出马氏珠母贝RAPD反应的最佳体系，为不同壳色马氏珠母贝种质资源遗传多样性和亲缘关系的研究奠定基础。试验以4种壳色马氏珠母贝为材料，采用SDS法进行闭壳肌的总DNA提取并对影响RAPD扩增效果的因素进行优化。通过单因素优化筛选出最佳的25μL反应体系：10×Taqbuffer2.5μL，DNA2.0ng/μL，Mg^2＋3.0mmol/L，引物0.25μmol/L，0.2mmol/LdNTP，Taq酶1.0U；反应程序：94℃预变性5min，然后进行40个循环：94℃变性45s，39℃退火60s，72℃延伸90s，最后72℃延伸10min，4℃终止反应。结果发现，采用SDS法所提取马氏珠母贝的DNA达到RAPD反应的要求，筛选体系扩增出清晰稳定的多态性条带，可用于对马氏珠母贝遗传育种方面的研究。

Pinctada Martensii Four Shell Color Breeding Department of RAPD Reaction System Optimization

In this paper the optimum RAPD reaction system of Pinctada martensii was selected by experiment. It is the basis for preparation of genetic diversity and relatives of different shell color pearl oyster germplasm resource study. SDS method was used to extract adductor muscle of four shell color Species of Pinctada martensii DNA and factors which affect the RAPD amplification were optimized and selected. A single-factor optimization method used showed that a total volume of 25 μ L RAPD system of Pinctada martensii consisted of 2.5 μ L 10 × Taq buffer, 2.0 ng/μ L DNA, 3.0 mmol/L Mg^2＋, 0.25 μ mol/L primer, 0.2 mmol/L dNTP and 1.0U Taq DNA polymerase. The suitable PCR procedure is one eycle denaturing at 94℃ for 5rain, 40 cycles which involves denaturing at 94℃ for 45 s, annealing at 39℃ for 60 s and extending at 72℃for 90 s, one cycle extending at 72℃ for 10 min, and then kept at 4℃. The experiment shows that SDS method could extract adductor muscle of Pinctada martensii DNA which could meet the requirement of RAPD reaction. The selected RAPD system amplified the polymorphism and its bands are clear.