坛紫菜ISSR反应体系的建立与优化  

Establishment and optimization of ISSR reaction system for Porphyra haitanensis

作　　者：纪德华[1] 谢潮添[1] 陈昌生[1] 徐燕[1] 张元[1] 刘冰[1] 

机构地区：[1]集美大学水产学院,福建厦门361021

出　　处：《海洋科学》2008年第6期20-24,29,共6页Marine Sciences

基　　金：福建省科技重大专项资助项目(2004NZ03);厦门市科技局资助项目(3502Z20041051);厦门市海洋与渔业局资助项目

摘　　要：为了利用ISSR分子标记技术对坛紫菜(Porphyra haitanensis)种质资源进行遗传分析及种质鉴定,获得清晰稳定、重复性好、多态性高的扩增结果,对影响ISSR-PCR扩增的多个因素,包括DNA模板含量、Mg2+浓度、TaqDNA聚合酶含量、引物浓度、dNTP浓度以及复性温度进行了全面比较和优化,建立了坛紫菜种质资源ISSR-PCR扩增的最佳反应体系:25μL的反应体系中含2.5μL 10×PCR缓冲液,5 ng模板DNA,2.5 mmol/L Mg2+,1.5 UTaqDNA聚合酶,200 nmol/L引物,200μmol/L dNTP。最佳PCR扩增程序:94℃预变性7 min;每个循环94℃变性1 min,48℃复性45 s,72℃延伸2 min,共进行35个循环;循环结束后72℃再延伸7 min。Porphyra haitanensis is the most economically important seaweeds in south China. The factors which affect the ISSR analysis in the study of the genetic diversity and variety identification of P. haitanensis, such as the amount of template DNA, Taq DNA polymerase, primers concentration, Mg^2＋ concentration, dNTP concentration and annealing temperature, were studied for optimizing conditions of the ISSR- PCR. The results showed that the optimal conditions being suitable for ISSR- PCR of P. haitanensis were as follows. 25 μL reaction system containing 2. 5 μL 10×PCR Buffer, 5 ng template DNA, 2.5 mmol/L Mg^2＋ ,1.5 U Taq DNA polymerase （produced by Takara company）, 200 nmol/L primers and 200 μmol/L dNTP. With an MJ thermal cycle optimal amplification program was 1 cycle of 7 min at 94℃ ; 35 cycles of 1 minat 94℃, 45 s at 48℃, and 2 minat 72℃; 1 cycle7 minat 72℃, using block control style. All these results provided a standardizing ISSR-PCR program for the analysis of genetic diversity and variety identification of P. haitanensis.

关 键 词：坛紫菜(Porphyra haitanensis) ISSR 实验条件 优化 

分 类 号：S917.3[农业科学—水产科学]