天山雪莲RAPD反应条件的优化及再生体系DNA多态性分析

Optimization of RAPD Reaction Conditions and DNA Polymorphic Analysis of the Regeneration System for Saussurea involucrata Kar. et Kir.

目的:用RAPD法研究天山雪莲再生过程中DNA的变异。方法:优化RAPD反应体系中各影响因素,分别以天山雪莲种子苗、愈伤组织、丛生芽及再生植株的总DNA为模板,进行多态性分析。结果:优化反应体系为:Mg2+2.50mmol·L-1、Taq DNA聚合酶1.00U、引物0.30μmol·L-1、模板DNA 20ng、dNTP0.20mmol·L-1,退火温度为37℃。20条随机引物共扩增出174条带,平均每条引物扩增8.7条带,其中多态性条带18条,多态性位点比率为10.3%。天山雪莲再生体系与种子苗之间相似性系数为0.75～0.80。结论:天山雪莲再生过程中在DNA水平上发生了一定程度的变异。

Objective： To study the DNA variation level of tissue cultures of Saussurea involucrata Kar. et Kir. using RAPD. Methods： Some essential factors that affect the result of RAPD were discussed and the optimal RAPD system for Saussurea involucrata Kar. et Kir. was established. The seed plantlet, callus, cluster buds and regenerated plants of Saussurea involucrata Kar. et Kir. were used as the analysis materials of DNA. Results： The optimal system with Mg^2＋ 2. 50mmol.L^-1, Taq DNA polymerase1. 00U, primer 0. 30μmol.L^-1, template DNA 20ng, dNTP 0. 20mmol.L^-1, and annealing temperature 37℃ were suitable for Saussurea involucrata Kar. et Kir. in amplification reaction. 174 bands were detected with 20 primers, with the average of 8.7 bands per primer. 18 polymorphic bands were detected among them, and the percentage of polymorphic loci was 10. 3%. The similarity coefficient between re- generation system and seed plantlet was in the range from 0. 75 to 0. 80. Conclusion ： The results showed that there was genetic variation in Saussurea involucrata Kar. et Kir. regeneration system.