中华补血草ISSR-PCR体系的构建与正交优化  

Establishment and Optimization of the Method of ISSR Fingerprinting Marker on Limonium Sinense

作　　者：丁鸽[1] 张代臻[2] 于延球[2] 赵玲玲[1] 张蓓蓓[1] 

机构地区：[1]盐城工学院化学与生物工程学院,江苏盐城224003 [2]江苏省滩涂生物资源与环境保护重点建设实验室,盐城师范学院,江苏盐城224002

出　　处：《南京师大学报（自然科学版）》2012年第3期93-97,共5页Journal of Nanjing Normal University(Natural Science Edition)

基　　金：国家自然科学基金(31000142);江苏省自然科学基金(BK2011421);盐城工学院科研项目基金;盐城工学院博士启动基金(XKR2010003)

摘　　要：为获得中华补血草ISSR的最佳反应体系,采用正交试验设计,对影响ISSR反应的模板含量、Mg2+浓度、dNTP浓度、Taq酶浓度、扩增程序及退火温度等多种因子进行优化筛选.通过各因子的组合研究,分析非特异性条带的产生原因并进行条件优化,建立了可用于中华补血ISSR-PCR分析的稳定可靠的反应体系:25μLPCR反应体积,10×Buffer 2.5μL,2 mmol/L MgCl2,dNTP 100μmol/L,模板DNA为20 ng,Taq聚合酶1.5 U,随机引物0.4μmol/L,退火温度52℃～58℃.所建立的中华补血草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,可以较好地应用于中华补血草的遗传多样性及物种保护的研究.To obtain the optimal amplification of ISSR conditions for Limonium sinense, the effects of the concentrations of DNA templates, magesium, dNTP, Taq DNA polymerase and annealing tempreture were tested and optimized by orthogonal designed experiment. The reliable ISSR - PCR systems for L. sinense was established by analyzing the reasons for occurrence of differential bands and optimizing reaction conditions. The optimal amplification conditions for L. sinense was 25 μL PCR reaction volume, 10× Buffer was 2. 5μL, the concentration of magnesium was 2 mmol/L, dNTP was 100μmol/L, the concentration of DNA templates was 20 ng, Taq DNA polymerase was 1. 5 U, primer was 0.4 μmol/L. The appropriate annealing temperature was 52℃ -58℃. The ISSR-PCR systems, which were established in this paper for studying L. sinense, could provide clear reliable abundant polymorphisms molecular markers and were proved suitable for studying genetic diversity and conservation of L. sinense.

关 键 词：中华补血草 ISSR 反应体系 正交优化 

分 类 号：Q946[生物学—植物学]