大豆花芽mRNA差异显示技术体系反应条件的优化

摘要

为建立适用于大豆花芽的mRNA差异显示体系,以“吉农18”花芽突变体为材料,采用RNAiso Plus试剂盒提取了大豆花芽的总RNA,并对DDRT-PCR体系中的Mg2+、dNTP及Taq酶的用量进行了优化.试验结果表明:适合大豆花芽mRNA差异显示的PCR体系(25 μL)为反转录产物用量2.0μL,锚定引物50 pmol/L,随机引物50 pmol/L,Mg2+ 1.5 mmol/L,dNTP 0.2 mmol/L,Taq酶1.5U.%In this study,we chose the JN18 of flower buds,and established a high efficient mRNA differential display system.Using RNAiso Plus kit extraction soybean of flower buds total RNA,reverse transcriptase cDNA synthesis,chain DDRT-PCR amplification reaction system optimization,we established a system suitable for reverse transcription of mRNA differential analysis of soybean.Conditions appropriate to analyze the soybean PCR reactions (25 μL) were 2.0 μL RT reaction first strand,50 pmol/L anchor primer,50 pmol/L random primer,1.5 mmol/L Mg2+,0.2 mmol/L dNTP and 1.5 U Taq.