新疆贝母DNA提取及ISSR-PCR体系的建立与优化

DNA Extraction and Optimization of ISSR-PCRReaction System for Fritillaria walujewii

目的:从新疆贝母干叶片中提取高质量的DNA,建立ISSR-PCR体系并进行优化。方法:通过改进的CTAB法提取基因组DNA;综合利用单因素实验和L9(34)正交实验2种方法,对镁离子、dNTP、模板DNA含量、TaqDNA聚合酶量、引物浓度等因素进行优化,并采用温度梯度筛选引物的退火温度。结果:新疆贝母20μL ISSR-PCR最适反应体系dNTP为0.3 mmol/L,Mg2+为2.0 mmol/L,模板浓度为40～100 ng/(20μL),引物为1.2 mmol/L,Taq DNA聚合酶为1.0 U/20μL,引物UBC 859的最适退火温度为50.7℃。结论:此提取方法得到的DNA纯度高,ISSR-PCR扩增效果显著,为新疆贝母种质资源鉴定、遗传多样性分析奠定研究基础。

Taking dried leaves of Fritillaria walujewii as material, optimizing a DNA extraction method, establishing and optimizing ISSR-PCR Reaction System. Methods DNA was Extracted by optimized DNA extraction method;The ISSR-PCRreaction system for Fritillaria walujewii was optimized in five levels of five factors （DNA template, My^2＋ , primer, dNTPs and DNA Taq polymerase）by single-factor experiment and in three levels of four factors by orthogonal experiment, and the gradient PCR was used to determine the annealing temperature of primer. Result The optimal PCR（20μL） mixture contained 0.2 mmol/L dNTP,2.0 mmol/L Mg^2＋ ,60 -80 ng template DNA, 1.2/xmoLZL primer, 1 U Taq DNA polymerase. The suitable annealing temperature of primer UBC 859 was 50.7℃. Conclusion By the method of DNA Extraction,we get high purity template DNA, which can be used for ISSR-PCR amplification. The establishment of ISSR-PCR system could laid foundation for ISSR analysis on studies of germplasm resources and the genetic diversity of FritiUaria walujewii.