摘要
以红千层(Callistemon rigidus R．Br．)的幼叶为材料,利用正交试验设计,对ISSR反应体系中的主要影响因子进行优化筛选,研究了Mg2+、TaqDNA聚合酶、dNTPs、引物、模板DNA浓度5种因素对扩增结果的影响,同时研究了退火温度和循环次数对PCR扩增结果的影响。实验结果表明,在25μL ISSR反应体系中各组分的最适浓度分别为:1×PCR buffer,2．5 mmol／L Mg2+,0．2 mmol／L dNTPs,0．4μmol／L引物,1．2ng／μL模板DNA,0．5UTaqDNA聚合酶。通过梯度PCR测验,适宜的退火温度为50℃,循环次数45次。并且在红千层ISSR分析中,带纹清晰,稳定性好。
The main effectors such as the concentration of Mg^2＋,Taq DNA polymerase, dNTPs, primer and genome DNA were screened to optimize ISSR amplification system in orthogonal design experiments by using young leaves of Callistemon rigidus. The annealing temperature and the number of cycles were also investigated. The results showed that the optimum concentrations of components such as Buffer, Mg^2＋, dNTPs, primer, template DNA , Taq DNA polymerase in 25 ml reaction system were 1 × Buffer （ without Mg^2＋）, 2.5 mmol/L, 0.2 mmol/L, 0.4 mmol/L, 1.2 ng/ml, 0.5 U respectively. Annealing at 50℃, clear and stable bands were obtained after 45 cycles in gradient PCR test.
出处
《西南园艺》
2006年第3期1-4,共4页
Southwest Horticulture
