铁皮石斛居群差异的研究ⅡISSR指纹标记方法的建立与优化  

Studies on population difference of Dendrobium officinale Ⅱestablishment and optimization of the method of ISSR fingerprinting marker

作　　者：沈洁[1] 丁小余[1] 丁鸽[1] 刘冬扬[1] 唐凤[1] 贺佳[1] 

机构地区：[1]南京师范大学生命科学学院江苏省生物多样性与生物技术重点实验室,江苏南京210097

出　　处：《中国中药杂志》2006年第4期291-294,共4页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金资助项目(30370144)

摘　　要：目的：针对铁皮石斛ISSR的反应特点，建立稳定可靠的ISSR分子指纹标记反应体系，为进一步研究铁皮石斛的居群差异奠定基础。方法：通过筛选引物并设定影响铁皮石斛ISSR反应的诸因子的不同浓度，检测ISSR不同反应体系的扩增效果；通过分析非特异性条带的产生原因并进行条件优化，建立铁皮石斛ISSR稳定可靠的反应体系。结果与结论：首次建立了可用于铁皮石斛ISSR-PCR分析的最适宜的反应体系：25μL．PCR反应体系中，10×Taq酶配套缓冲液，1．5UTaq DNA聚合酶，1．2—1．8mmol·L^-1 MgCl2，80μmol·L^-1 dNTP，0．2μmol·L^-1引物，DNA模板约20ng，退火温度52-60℃；实验表明：Taq酶质量、DNA模板品质、退火温度等对ISSR反应结果具有较大影响。所建立的铁皮石斛ISSR反应体系具有标记位点清晰、反应系统稳定、检测多态性能力较强、重复性好等特点，可以较好地应用于铁皮石斛的居群鉴别及居群分子生态的研究。Objective： To establish and optimize ISSR-PCR system of Dendrobium officinale according to the ISSR-PCR characters of D. officinale. Methyl： The effects of ISSR-PCRs was examined by selecting primers and designing different concentrations of the factors in the ISSR-PCRs, the reliable ISSR-PCR systems for D. officinale populations re,arching was established by analyzing the reasons for occurrence of differential bands and optimizing reaction conditions. Result and Condusion： The optimal ISSR-PCR system in D. officinale was reported for the first time, and 25μL ISSR-PCR system contained 10× Taq buffer, 1.5 U Taq DNA polymerase, 1.2-1.8 mmol·L^-1 MgCl2, 80μmol·L^-1 dNTP, 0.2μ mol·L^-1 primer and 20 ng template DNA. The appropriate annealing temperature was among 52-60 ℃. ISSR PCRs were significantly influenced by Taq DNA polymerase, template DNA quantity and annealing temperature, etc. The ISSR-PCR systems, which were established in this paper for studying D. offtcinale, could provide clear reliable abundant polymorphisms molecular markers and were proved suitable for studying population authentication and population molecular ecology of D. offtcinale.

关 键 词：铁皮石斛居群 ISSR反应体系 建立与优化 

分 类 号：S567.239[农业科学—中草药栽培]