油棕SCoT-PCR反应体系的建立与优化

Establishment and Optimization of SCoT-PCR Reaction System for Oil Palm(Elaesis guineensis)

为获得油棕SCoT-PCR反应的最佳反应体系以及筛选适用于该体系的SCoT引物,本研究首先通过利用L16(45)正交设计试验和单因素实验2种方法,对反应体系中的各因子进行优化,并在此基础上确定最优退火温度和循环数。结果表明,各因子对SCoT-PCR扩增影响大小依次为:dNTPs浓度>Mg^(2+)浓度>DNA模板量>Taq DNA聚合酶量>引物浓度;最佳反应体系(25μL)为:DNA模板量为120 ng,dNTPs浓度为0.2 mmol/L,Mg^(2+)浓度为1.5 mmol/L,引物浓度为0.6μmol/L,Taq DNA聚合酶量为2.0 U,其余用灭菌ddH_(2)O补足至25μL。最优扩增程序为:95℃预变性3 min;95℃变性30 s,55℃退火复性30 s,72℃延伸1 min,30个循环;最终72℃延伸5 min,保存于4℃恒温冰箱中。此外,利用优化体系从36条SCoT引物中筛选出30条可从油棕中扩增条带清晰的引物。使用SCoT-2等3条引物对30份油棕材料进行扩增,扩增产物均具有良好的多态性、可靠性和稳定性。本研究结果为油棕种质资源鉴定、遗传多样性分析以及品种遗传图谱构建等提供理论依据。

In order to obtain the best SCoT-PCR reaction system of oil palm(Elaesis guineensis),and select suitable SCoT primers,this study used the L16(45)orthogonal design and single fantor experiment to optimize the influencing factors of the SCoT-PCR reaction system,and based on this,determine the optimal annealing temperature and cycle number.The results showed that the factors affecting SCoT-PCR amplification,in decreasing order of impact,were dNTPs concentration>Mg^(2+)concentration>DNA template amount>Taq DNA polymerase amount>primer concentration;the optimized SCoT-PCR reaction system(25μL)was composed of 120 ng DNA template,0.2 mmol/L dNTPs concentration,1.5 mmol/L Mg^(2+) concentration,0.6μmol/L primer concentration,2.0 U Taq DNA polymerase amount,and sterilized ddH_(2)O supplemented to 25μL.The optimized PCR amplification profile was pre-denaturation at 95℃ for 3 min,denaturing at 95℃ for 30 s,annealing at 55℃ for 30 s,and extension at 72℃ for 1 min.After 30 cycles,extengding at 72℃ for 5 min and stored at 4℃ finally.In addition,30 primers were selected which could amplify clear bands from oil palms based on the optimized reaction system.Using three primers which including SCoT-2 to amplify 30 oil palms materials,the PCR products exhibited good polymorphism,reliability and stability.These results provide a theoretical basis for germplasm resources identification,genetic diversity and variety fingerprint construction of oil palms.