茄子ISSR-PCR反应体系优化及验证

以茄子为试验材料,采用改良CTAB法提取茄子嫩叶总DNA,结合单因素设计和正交设计L9(34)的优点,探讨Mg2+、dNTP、引物及TaqDNA聚合酶等因素对茄子ISSR-PCR反应体系的影响,利用16份广西茄子主要栽培品种对最优处理组合进行验证。结果表明,在25μL反应体系中,含2.5μL10×buffer、2.0～3.0mmol·L-1Mg2+、0.32～0.36mmol·L-1dNTP、0.4μmol·L-1引物、50ng模板DNA、0.6UTaqDNA聚合酶等成分,可以获得比较稳定、清晰和丰富的条带。

Optimization and Verification of ISSR Reaction System of Eggplant

In this study, eggplant（Solanum melongena L.）was the test material. The total DNA was extracted from its fresh young leaves by modified CTAB method. The advantage of orthogonal design and single factor design were applied to optimize ISSR-PCR amplification system of eggplant in 4 factors, such as Mg2+, dNTP, primer and Taq DNA polymerase at 3 levels, and the optimal treatment was verified by 16 cultivate eggplants from Guangxi. The results indicated that an optimal reaction system（25 μL）was established. It was consisted of 2.5 μL 10×buffer, 2.0-3.0 mmol&;#8226;L-1 Mg2+, 0.32-0.36 mmol&;#8226;L-1 dNTPs, 0.4 μmol&;#8226;L-1 primer, 50 ng template DNA, 0.6 U Taq DNA polymerase. The sore band of the optimized treatment showed stabilization, distinct and abundant.