花生RGA-SSCP实验条件的优化

Optimization of RGA-SSCP System for Peanut

单链构象多态性(SSCP)技术是一种简便、灵敏的多态性检测方法,近年在植物研究中得到了较多应用。为了检测花生品种间抗病基因同源序列(RGA)的单核苷酸差异,对影响花生RGA-SSCP的电泳温度、电压、胶浓度、交联度、变性剂等因素进行了优化。结果表明,碱变性后的PCR产物(PCR产物与碱变性剂比例1∶2)在不加甘油的8%非变性聚丙烯酰胺凝胶(交联度为29∶1)、1×TBE电泳缓冲液、恒温4℃和600V条件下电泳5 h为最佳优化组合。用上述优化方法分析了抗、感北方根结线虫病的亲本及F2群体部分单株,获得了电泳条带细而清晰的SSCP图。该体系的建立对RGA-SSCP标记用于花生基因定位、遗传图谱构建、标记辅助选择等具有重要意义。

Single strand conformation polymorphism （SSCP） is a quite simple and sensitive method for polymorphism detection, which has been widely used in plant research recently. In order to develop an SSCP analysis method for detecting the single nueleotide changes of resistant gene analogs （RGA） in peanut, the optimization trials were performed in temperature, voltage, gum concentration, crosslinking degree, denaturant and so on. The best separation of SSCP bands of RGA products was obtained with 8% native polyacrylamide gels （29：1 ） not containing glycerin in 1 x TBE buffer under constant 4℃ and 600 V for 5 hours. The opti mized method was used to analyze two parents resistant/susceptible to northern root - knot nematode and the individuals of F2 population, and a SSCP spectrum was obtained with slender and clear brands. The development of RGA - SSCP analysis system would be used for gene mapping, construction of genetic map and marker -assisted selection in peanut.