摘要： 以扁蓿豆(Medicago ruthenica)为试材,采用改良的CTAB法提取DNA,利用正交设计L16(45)探讨10×PCRBuffer(含Mg2+)、dNTP_s、引物、TaqDNA聚合酶及模板DNA用量对扁蓿豆ISSR-PCR反应的影响,正交试验的结果采用直观分析和方差分析相结合。建立了扁蓿豆的ISSR-PCR优化反应体系,在25μL反应体系中,TaqDNA聚合酶1.5U,10×PCR Buffer(含Mg2+)2.0 mmol/L,模板DNA0.5 ng/μL,dNTP_s 0.6 mmol/L,引物0.9μmol/L。同时探讨引物HZD09211的最适退火温度为52.3℃。2个不同引物对20份扁蓿豆材料DNA进行ISSR-PCR扩增,结果显示该体系具有较高的稳定性。

关键词: 扁蓿豆, ISSR, 正交设计, 反应体系, 优化

Abstract: Medicago ruthenica genomic DNA,extracted by modified CTAB method Orthogonal design was applied to optimize ISSR-PCR amplification system of Medicago ruthenica in five factors such as Mg2+,dNTPs,primer,TaqDNA polymerase and template DNA at four levels.The result of PCR was analyzed by tuitive analysis and variance analysis.An optimal reaction system was established,in a total volume of 25 μL ISSR-PCR system,it contained TaqDNA polymerase 1.5 U,10×Buffer(Mg2+)2.0 mmol/L,template DNA 0.5 ns/μL,dNTP 0.6 mmol/L and primer 0.9 μmol/L.The annealing temperature was also investigated.52.3℃ was the most suitable annealing temperature of HZD09211 primer.20 Medicago ruthenica germplasm materials were used to test the stabilization of the optimized reaction system.The results indicated that the optimized reaction system was very stable.

Key words: Medicago ruthenica, ISSR, Orthogonal design, Reaction system, Optimization

中图分类号: 

• S541.9