摘要
目的建立适于柴胡ISSR分析的PCR反应体系；筛选适宜引物，并确定最佳退火温度。方法CTAB法提取叶片DNA，通过单因子实验分别找出合适的ISSR-PCR反应条件，利用梯度PCR确定引物最佳退火温度。结果适于柴胡ISSR分析的PCR反应体系为25μl总体积中含有1×Taq酶缓冲液，2．0mmol／L MgCl2，各0．15mmol／L的dNTPs，0．3μmol／L引物，1．5U Taq酶（上海生工），20ng模板DNA，2％去离子甲酰胺。在100条引物中筛选出30条扩增条带清晰且条带数目较多的引物，最佳退火温度为49．9～63．6℃（因引物不同而异）。结论建立了柴胡ISSR—PCR反应体系，筛选出30条引物并确定了最佳退火温度，为应用ISSR技术鉴定柴胡种质资源、分子标记辅助选择育种及其遗传多样性研究奠定了基础。
Objective To establish and optimize the ISSR - PCR reaction system for Bupleurum chinense DC. ; to screen the suitable primers and determine their optimal annealing temperatures. Methods Genomic DNA was extracted by CTAB method from Bupleurum chinense DC. plant leaves. The main influencing elements in different levels were tested by single factor experiment. The gradient PCR was used to determine the optimal annealing temperatures of each selected primer. Results The optimal reaction system for ISSR analysis contains 1 × Taq DNA polymerase reaction buffer, 20 ng template DNA, 1.5 U Taq DNA polymerase, 2.0 mmol/L MgCl2, 0.15 mmol/L each of dNTPs, 0.3 μmol/L primer, 2% formamide deionized in 25 μl total reaction volume. 30 primers which showed clearer and more bands from 100 primers were selected and the optimal annealing temperatures were 49. 9- 63.6℃ （changed among different primers）. Conclusion The stable and reproducible optimal ISSR -PCR reaction system and suitable annealing temperatures of 30 selected primers from 100 are established for Bupleurum chinense DC. which had laid the good foundation for ISSR analysis on studies of germplasm resources identification, molecular marker assisted breeding and genetic diversity.
出处
《时珍国医国药》
CAS
CSCD
北大核心
2008年第8期1837-1839,共3页
Lishizhen Medicine and Materia Medica Research
基金
国家科技支撑"十一五"计划重点项目(No.2006BAI09B01)
国家中医药管理局科技专项(No.2004ZX06-1)
北京市科技新星计划(No.2004A60)
