摘要
mRNA差异显示技术(DDRT-PCR)是分离差异表达基因的有效方法。以陆地棉组织培养过程中不同时期愈伤组织为材料,对RNA提取、PCR反应体系、银染条件等影响mRNA差异显示的关键因素进行优化试验,建立了一套适合棉花组织培养的mRNA差异显示体系。最终确定了优化体系(50μL)为:随机引物0.4μmol/L,锚定引物0.4μmol/L,dNTP 250μmol/L,Mg2+1.5 mmol/L,Taq酶1.5 U,退火温度为39.3℃。利用优化后的体系对材料进行扩增,扩增产物特异性好、条带清晰、背景干净。该体系简便、快捷、灵敏、高效,可用于分离差异表达基因。
The method of mRNA differential display PCR （DDRT- PCR） has been widely used for identifying differentially expressed genes. Taking different periods callus during somatic embryogenesis in cotton （Gossypium hirsutum L. ）, we optimized critical parameters of DDRT - PCR, RNA isolation, PCR reaction system, silver straining. After optimized the reaction, we established the method of mRNA differential display PCR suitable to tissue culture of cotton. The improved reaction system contains： arbitrary primer 0.4 μmol/L, anchor primer 0.4 μmol/L, dNTP 250 μmol/L,MgCl2 1.5 mmol/L, Taq polymerase 1.5U, annealing temperature 39.3℃. The production of amplification shows the characteristics of well specificity, distinct DNA fragments, clearly background under the improved system. This system is very convenient, sensitive, efficient, and can be used to identify differentially expressed genes in cotton.
出处
《河南农业科学》
CSCD
北大核心
2008年第12期31-35,共5页
Journal of Henan Agricultural Sciences
基金
国家科技支撑计划项目(2006BAD01A05-27)
河南省自然科学基金项目(0411034100)
