摘要
为建立适宜于赤皮青冈ISSR分析的扩增体系,以赤皮青冈叶片基因组DNA为材料,采用单因素和正交设计相结合的方法系统地测试模板DNA、Mg2+、引物、磷酸碱基脱氧核苷(dNTPs)浓度,Taq DNA聚合酶用量和退火温度这6个因素对ISSR-PCR反应结果的影响。综合分析表明:优化后的最佳反应体系为20μL反应总体系中,含20 ng模板DNA,2.5 mmoL/L Mg2+,0.2 mmoL/L dNTPs,1.0 U Taq DNA聚合酶,0.2μmoL/L引物;UBC 808号作引物最佳退火温度为59.3℃。这一优化体系的建立为进一步开展赤皮青冈遗传多样性的ISSR分析奠定了基础。
In order to establish the optimal system of ISSR-PCR for Cyclobalanopsis gilva,with the genomic DNA of C.gilva leaves as material,single factor test and orthogonal experiment design were used to systematically test six parameters of ISSR-PCR which include template DNA,Mg2＋,primers,dNTPs concentration,dose of Taq DNA polymerase and annealing temperature.Integrated analysis implies that the optimal reaction system of ISSR（20 μL） is：20 ng template DNA,2.5 mmoL/L Mg2＋,0.2 mmoL/L dNTPs,0.2 μmoL/L primers and 1.0 U Taq DNA polymerase,if UBC808 as the primers in ISSR-PCR that the optimized annealing temperature is 59.3℃. The establishment of the optimal system lays an important foundation for using ISSR technology in research C.gilva genetic diversity in future.
出处
《中南林业科技大学学报》
CAS
CSCD
北大核心
2014年第6期61-65,80,共6页
Journal of Central South University of Forestry & Technology
基金
"十二五"国家科技支撑项目"楸树和赤皮青冈珍贵用材林定向培育技术研究与示范"(2012BAD21B03)
