橡胶草及其近缘种ISSR反应体系及扩增程序的优化  

Optimization of ISSR Reaction System and Amplification Procedure for Taraxacum kok-saghyz and Its Related Species

作　　者：徐显异 陆婷[1] 田嘉[1] 陈小和 陈艾 Xu Xianyi;Lu Ting;Tian Jia;Chen Xiaohe;Chen Ai(College of Forestry and Horticulture, Xinjiang Agricultural University, Urumqi, 830052)

机构地区：[1]新疆农业大学林学与园艺学院

出　　处：《分子植物育种》2019年第12期4007-4015,共9页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31460631)资助

摘　　要：本研究旨在建立和优化橡胶草及其近缘种ISSR (Inter-simple sequence repeat)反应体系和扩增程序。以橡胶草及其近缘种叶片为供试材料,采用单因素试验方法,正交试验方法 L16(44)和极差分析方法,对橡胶草ISSR-PCR反应中4个主要影响因素(dNTP浓度, DNA浓度,引物浓度和Taq DNA聚合酶浓度)进行优化,并在最优反应体系的基础上进行引物和退火温度的筛选。结果表明:dNTP和DNA对PCR扩增结果有较为显著的影响,引物和Taq酶的浓度变化对扩增结果无显著影响。最后确定橡胶草及其近缘种ISSR-PCR反应体系(20.0μL)为:双蒸水14.2μL,10×Buffer (含Mg2+) 2.0μL,DNA模板50.0 ng,10 mmol/L引物1.2μL,2.5 mmol/L dNTP 1.6μL,5 U Taq DNA聚合酶0.1μL。PCR扩增程序为:94℃5 min,94℃45 s,退火1 min,72℃70 s,45个循环,72℃10 min,4℃保存。本研究为橡胶草及其近缘种的遗传多样性和交配系统等后续研究提供理论参考。The purpose of this study was to establish and optimize the ISSR(Inter-simple sequence repeat) reaction system and amplification procedure for Taraxacum kok-saghyz and its related species. Taraxacum kok-saghyz and its related species leaves were used as test materials, and the single factor test method, orthogonal experiment method L16(44) and range analysis method were used to optimize the four main influencing factors(dNTP concentration, DNA concentration, primer concentration and Taq DNA polymerase concentration) in the ISSR-PCR reaction of Taraxacum kok-saghyz. Also, primer and annealing temperatures were screened based on the optimal reaction system. The results showed that d NTP and DNA had significant effects on PCR amplification results, while the concentration changes of primers and Taq enzyme had no significant effect on the amplification results. Finally,the ISSR reaction system of Taraxacum kok-saghyz and its related species(20.0 μL) was determined as: double distilled water 14.2 μL, 10×Buffer(containing Mg2+) 2.0 μL, DNA template 50.0 ng, 10 mmol/L primer 1.2 μL, 2.5 mmol/L dNTP 1.6 μL, and 5 U Taq DNA polymerase 0.1 μL. The PCR amplification procedure was: 94℃ for 5 min, 94℃for 45 s, annealing 1 min, 72℃ for 70 s, 45 cycles, 72℃ for 10 min, 4℃ for storage. This study could provide a theoretical reference for the follow-up studies on genetic diversity and mating system of Taraxacum kok-saghyz and its related species.

关 键 词：橡胶草(Taraxacum kok-saghyz) ISSR 单因素试验 正交试验 

分 类 号：S576[农业科学—作物学]