全缘叶绿绒蒿ISSR体系的建立与优化

Establishment and Optimization of ISSR-PCR Reaction System for Meconopsis integrifolia

以全缘叶绿绒蒿的DNA为模板,利用正交实验设计,研究了Mg^(2+)、dNTPs、引物、Taq DNA聚合酶、DNA这5种PCR反应成分对反应体系的影响,以期建立并优化一个适合全缘叶绿绒蒿的ISSR反应体系,并用优化的体系对UBC公布的100条ISSR引物进行筛选。结果表明:在50μL优化后的反应体系中,含3 mmol·L^(-1) Mg^(2+)、2mmol·L^(-1)dNTPs、0.4mmol·L^(-1)引物、1.25UTaq DNA聚合酶、60ng DNA。通过梯度PCR仪检验,确定了适宜的退火温度、延伸时间和循环次数。利用该体系共筛选出16条ISSR引物,并对全缘叶绿绒蒿共10株个体材料进行扩增检验,获得了较好的扩增效果。利用该体系,可为今后ISSR标记在绿绒蒿属植物的种质鉴定和遗传多样性分析等方面的应用提供一定的试验基础。

The M.integrifolia was used as template in this study.And the effects of five PCR reaction components including Mg^2＋,dNTPs,primers,Taq DNA polymerase,and DNA on the reaction system were studied by using the orthogonal design.The purpose was establishment and optimization of ISSRPCR reaction system for M.integrifolia,and screened 100 ISSR primers published by UBC with this system.The results showed that the 50 μL reaction system,including 3 mmol· L^-1 Mg^2＋,2 mmol·L^-1 dNTPs,0.4 mmol·L^-1 primers,1.25 U Taq DNA polymerase,60 ng DNA.And the suitable annealing temperature,extension time and cycle times were determined by gradient PCR.A total of 16 ISSR primers were screened by the system,and 10 individual materials were tested for amplification,which obtained good amplification results.This system could provide some experimental basis for the future study on germplasm identification and genetic diversity analysis of M.Vig.by using ISSR marker.