玉米鞘腐病菌ISSR-PCR反应体系优化及引物筛选
为建立玉米鞘腐病菌ISSR-PCR最佳反应体系,本研究以玉米鞘腐病菌基因组DNA为模板,筛选出各影响因子比较适宜的浓度范围,再利用正交试验,对玉米鞘腐病菌ISSR-PCR反应有影响的模板DNA、dNTPs、Mg2+、Taq DNA聚合酶和引物浓度等5种因素4个水平进行了优化试验。玉米鞘腐病菌的ISSR-PCR最佳反应体系为：在25 μL的反应体系中,DNA模板是80 ng,Mg2+浓度为2.5 mmol/L,引物浓度为0.4 μmol/L,Taq DNA聚合酶用量为2.5 U,dNTPs浓度为0.2 mmol/L。利用该体系从21条ISSR引物中筛选出了扩增条带清晰、多态性好的2条引物。这一体系的建立及引物的筛选为利用ISSR分子标记技术进行玉米鞘腐病菌的有关研究提供了科学依据。
英文摘要:
In order to establish an optimal reaction system of ISSR-PCR in corn sheath rot. the suitable concentration ranges of the different influential factors were firstly screened with genome DNA as the templates.And then the 5 factors(DNA Template, Mg2+, dNTPs. The polymerase and primer) that affected the ISSR-PCR amplification system were optimized through the orthogonal experiment design(L16(45)). The results showed that a suitable ISSR-PCR reaction system established: 80 ng DNA template, 2.5 mmol/L Mg2+, 0.4 μmol/L primer, 2.5 U Taq polymerase, 0.2 mmol/L dNTPs were contained in 25 μL reaction solution. Using this system 2 primers with resolution and multiple polymerase bands were selected from 21 primers. The optimal ISSR-PCR system and polymorphism primers could be provide the scientific basis for related research using ISSR molecular marker technology in corn sheath rot.
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