以玉米自交系QCL1094为材料，用RNAiso Reagent、柱式试剂盒法、异硫氰酸胍法等3种方法提取苗期叶片总RNA，用PrimeScriptTM 1st Strand cDNA Synthesis Kit反转录获得第一链cDNA，用RNase H、E.coli DNA聚合酶I和E.coli DNA连接酶合成第二链cDNA，用T4 DNA聚合酶进行末端平滑得到双链cDNA，最后，用双低频酶EcoR I和Pst I酶切双链cDNA，用T4 DNA连接酶连接接头，用引物EA00和P00进行预扩增，用引物EA01和P01进行选择性扩增。经1%琼脂糖凝胶和6%变性聚丙烯酰胺凝胶电泳检测，结果表明，RNA提取及cDNA-AFLP预扩增和选择性扩增的效果良好。

The RNAiso Reagent, GenerayGK3092 kit, Guanidine Thiocyanate were used to extract total RNA from seedling leaves of QCL1094, a maize inbred line. With the PrimeScript^TM 1st Strand cDNA Synthesis Kit, the first single strand cDNA was gained through the reverse transcription from the total RNA. The second strand cDNA was synthesized by using RNase H, E.coil DNA polymerase I and E.coil DNA ligase, and filled in the ends by T4 DNA polymerase. The double-strand cDNA was cut with two low-frequency cut enzyme, EcoR I and Pst I, and ligated to two adapters by T4 DNA ligase. The cDNA fragments were pre-amplified with a pair of primers EA00/P00 and detected by 1% agarose gel electrophoresis. Then the pre-amplified fragments were select-amplified with another pair of primers EA01/P01 and detected by 6% denaturing PAGE. The results indicated that the RNA extraction, cDNA synthesis, cDNA-AFLP pre-amplification and select- amplification by above procedures were effective. The technical system established is economical and effective, and a good reference to the application of cDNA-AFLP technology.

国家重点基础研究发展计划项目(2006CB708206)