黑老虎ISSR-PCR反应体系的建立与优化

Establishment and Optimization of ISSR PCR Reaction System for Kadsura coccinea

为建立和优化特色药食两用植物黑老虎(Kadsura coccinea)的ISSR-PCR反应体系和扩增程序,通过正交和单因素试验研究Mg2+、dNTPs、引物、Taq DNA聚合酶、模板DNA、退火温度及循环次数等因素对黑老虎ISSR-PCR反应体系扩增效果的影响。结果表明,在20μL的反应体系中,最佳扩增条件为Mg2+浓度2.5 mmol/L、dNTPs浓度0.1 mmol/L、引物浓度0.6μmol/L、Taq DNA聚合酶用量1.50 U、模板DNA用量45 ng,退火温度50.4℃,循环40次。这一优化体系的建立可为深入开展黑老虎种质资源遗传多样性研究奠定基础。

In order to establish and optimize ISSR-PCR reaction system and amplification procedure of the characteristic medicinal and edible Kadsura coccinea,the effects of Mg2+,dNTPs,primers,Taq DNA polymerase,template DNA,annealing temperature and number of cycles on the amplification of ISSR-PCR reaction system of K.coccinea were studied by orthogonal and single-factor experimental design methods.The results showed that in a 20μL reaction system,the optimal reaction system was the system with Mg2+of 2.5 mmol/L,dNTPs of 0.1 mmol/L,primer of 0.6μmol/L,Taq DNA polymerase dosage of 1.50 U and template DNA dosage of 45 ng.The annealing temperature was 50.4℃,and the cycle was 40 times.The establishment of this optimization system can lay a foundation for further research on genetic diversity of K.coccinea germplasm resources.