蚬壳花椒ISSR-PCR反应体系的建立及优化

Establishment and optimization of ISSR-PCR reaction system on Zanthoxylum dissitum

关键词:  蚬壳花椒  正交优化  ISSR-PCR

Key words:  Zanthoxylum dissitum  orthogonal design  ISSR-PCR

摘要:

通过单因素试验及正交设计方法对影响蚬壳花椒ISSR-PCR扩增的主要因素(模板DNA、Mg²⁺的浓度、引物、dNTPs,TaqDNA聚合酶的用量以及退火温度)进行优化,以建立蚬壳花椒ISSR-PCR反应的最佳体系。结果表明:最佳反应体系(20 μL)为模板DNA 60 ng、MgCl₂ 2.5 mmol/L、dNTPs 0.15 mmol/L、引物0.6 μmol/L、Taq DNA聚合酶2.4 U。在此基础上,从100条引物中筛选出18条扩增稳定、多态性好的ISSR引物并经过10份蚬壳花椒种质检验,证明该体系具有扩增条带清晰、稳定、重复性好等优点。该反应体系的建立为蚬壳花椒种质资源分类、遗传多样性分析提供了更客观可靠的方法。

Abstract:

In order to establish the optimal ISSR-PCR reaction system of Zanthoxylum dissitum,several factors that mostly affect the amplification of ISSR-PCR reaction of Z.dissitum,including DNA template,Mg²⁺concentration,primers,dNTPs,TaqDNA polymerase dosage and annealing temperature,were investigated and optimized by single-fact test and orthogonal-design method in the experiment. The results indicated the most stable and suitable reaction system had been established when the amount of DNA template was 60 ng,the dosage of Taq DNA polymerase was 2.4 U and the concentration of MgCl₂,dNTPs and primers was 2.5 mmol/L,0.15 mmol/L,0.6 μmol/L respectively. Further more,18 ISSR primers that possessed the characteristics of stable amplification and rich polymorphism were screened from 100 ISSR primers,and they were examined by the germplasm test of 10 Z.dissitum parts. The results of the examination proved that this ISSR reaction system had the advantages of clearness,stability and good repeatability. In conclusion,this established ISSR-PCR reaction system provided an available and reliable method for genetic diversity analysis and germplasm classification of Z.dissitum.