龙眼SCoT-PCR反应体系的优化

The Optimization of SCoT-PCR System of Longan (Dimocarpus longan)

目标起始密码子多态（start condon tardeted polymorphism,SCoT）分子标记结合了ISSR标记和RAPD标记的优点,具有操作简单﹑成本低廉﹑多态性丰富﹑重复性好﹑引物设计简单且通用性良好等诸多优点。本实验以石硖龙眼的新鲜叶片为试材,采用单因素试验的方法分别研究模版、引物、rTaqDNA聚合酶、dNTPs、缓冲液（Mg2＋）及退火温度共6因素各8个水平对龙眼SCoT-PCR扩增结果的影响。结果表明：龙眼SCoT标记的20μL优化反应体系为：10×BufferⅡ（含Mg2＋）2.0μL、DNA模板30ng、引物浓度为30μmol/L、rTaqD-NA聚合酶用量为0.45U、dNTP浓度为4.0mmol/L。适宜的扩增程序为：94℃预变性4min;95℃变性50s,49.5℃退火40s,72℃复性2min,36个循环;最后72℃延伸10min。利用24个龙眼品种验证该反应体系,1.5%琼脂糖电泳检测结果显示,扩增产物在400-2200bp之间,不同品种间DNA谱带具有多态性,反应体系具有良好的稳定性和可重复性。

SCoT （start codon target polymorphism） which possesses advantages of both ISSR and RAPD, owns extraordinary characters such as simple operation, low cost, high polymorphism, good reproducibility simple design of primers, and good universality ect. In this paper, we tried to establish the optimal SCOT- PCR amplifying condition in for Longan by using template DNA from the flesh leaves of ＇ Shi Xia＇ longan. Firstly, the suitable concentration of template DNA, primer, rTaqDNA polymerase, dNTPs, buffer and anneal temperature were determined through single factor test, then the amplifying system was optimized through design. According to our experiment, we established a SCoT-PCR system most suitable for longan, which came out total 20 μL reaction system containing 2.0 μL with 10x Buffer, 30 ng template DNA, 30 μmol/L primer, 0.45 U rTaqDNA polymerase and 4.0 mmol/L dNTPs. The most suitable PCR amplification procedure is one cycle of pre-denaturing at 94℃ for 4 min, 36 cycles of denaturing at 95℃ for 50 s, annealing at the primer＇s temperature 49.5℃ for 40 s and extending at 72℃ for 2 min, extending at 72℃ for 10 min finally. Based on the 1.5 percent agarose gelelectrophoresis of 24 longan germplasms, we get that the PCR products obtained are in the range of 400 bp to 2 200 bp, and the DNA among different germplasm are polymorphic, The agarose gelelectrophoresis also illustrates that the SCoT reaction system was quite stable and reproducible. This optimized SCoT-PCR system might facilitate to applying for further research on longan.