温郁金ISSR-PCR反应体系的建立及条件优化

Establishment of ISSR-PCR Reaction System for Curcuma wenyujin and Its Conditions Optimization

[目的]寻找一个可用于温郁金ISSR-PCR的最适宜反应体系。[方法]利用CTAB法提取基因组DNA,同时利用PCR扩增技术和方法,对引物、模板DNA、Mg2+d、NTP、Taq聚合酶等反应条件进行优化。[结果]反应体系的最佳条件是总体积为25μl,其中Mg2+浓度(25mmol/L)2.2μl,Taq聚合酶(5 U/μl)0.4μl,引物浓度(20μmol/L)1.5μl,模板DNA(5 ng/μl)1.5μl,dNTP(2.5 mmol/L)2.2μl,10×PCRbuffer2.5μl;PCR扩增程序为:1个循环的94℃预变性5 min;94℃变性35 s,相对应的引物退火温度退火1 min,72℃复性1.5 min,共36个循环;最后72℃延伸10 min。[结论]该体系是适合温郁金ISSR-PCR反应的最适宜体系,具有省时、经济、简便以及扩增条带清晰而稳定等特点,为今后温郁金遗传多样性的研究奠定了基础。

[ Objective ] The research aimed to seek the optimum ISSR-PCR amplification reaction system for Curcuma wertyujirt. [ Method]The genomic DNA was extracted by CATB method. And the reaction conditions such as primers, template DNA,Mg^2＋ ,dNTP, Taq polymerase were optimized by using amplification technology and method. [ Result] The optimum conditions of the reaction system were as follows： the total volume of 25μl, 2.2μl 25 mol/L Mg^2＋ , 0.4 μl 5 U/μl Taq polymerase, 1.5 μl 20μmol/L primer, 1.5 μl 5 ng/μl template DNA and 2.2 μl 2.5 mol/L dNTP, 2.5 μl 10 × PCR buffer. PCR amplification procedure was as follows：one cycle of pre-denaturing at 94 ℃ for 5 rain; 36 cycles of denaturing at 94 ℃ for 35 s, annealing at the primer＇s annealing temperature for 1 min and renaturing at 72 ℃ for 1.5 min; Extending at 72 ℃ for 10 min finally, [ Conclusion] This system is the optimum system for ISSR-PCR reaction of C. wenyujin. It has the characteristics such as being fast, economic and simple and having clear and stable bands, This research laid the foundation for studying the genetic diversity of C. wenyujin in future.