TAIL-PCR的改良及其在分离小麦基因启动子中的应用

A Modified TAIL-PCR and Its Application in Isolating Gene Promoter of Wheat

在经典TAIL-PCR(Thermal asymmetric interlaced PCR)的基础上,对其进行了如下四处改进:用10个碱基的RAPD引物代替16个碱基的随机兼并引物作为PCR中的随机引物;将较低特异性循环的复性温度由44℃降至29℃;增加5个高特异性反应循环,减少5个较低特异性反应循环;用单引物对第三轮PCR产物进行初步鉴定。利用改进的TAIL-PCR方法分离了小麦X基因的5′未知的侧翼序列,与GUS基因融合后转入拟南芥,通过组织化学检测分析表明分离到的5′侧翼序列具有启动子功能,同时说明改进的TAIL-PCR能更好地应用到较复杂基因启动子的分离。

Using a modified TAIL-PCR technique, the 5′-flanking region of the X gene in wheat was successfully isolated. Two novel modifications of the TAIL-PCR were introduced here： using a battery of random 10-mers as the short arbitrary primers instead of three degenerate 16-mers; using 29℃ instead of 44℃ as the annealing temperature for the low-stringency cycle; increasing five high-stringency cycles and reducing five low-stringency cycles; and using single primers for the third round of product identification. Isolated 5 ′ -flanking region was fused to the GUS gene, and tested for expression in Arabidopsis plants. Histochemicai analysis of the transgenic plants showed the report gene was driven by isolated 5′-flanking region. Modified TAIL-PCR technique could isolate rapidly the promoter of any gene from organisms with large genomes.