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以小麦品种扬麦158为材料,根据NCBI中小麦TaWRKY46基因序列设计引物,采用RT-PCR技术从小麦总RNA中分离了大小为870 bp的cDNA片段。测序表明,该片段与NCBI中已克隆的小麦TaWRKY46基因高度同源,包含完整的读码框,值得注意的是此蛋白保守氨基酸序列WRKYGQK突变为WRKYGEK,为TaWRKY46的新等位基因,命名为TaWRKY46-1。采用Gateway克隆技术构建了该基因的可诱导型超量表达载体,并转化农杆菌,为接下来转基因验证TaWRKY46-1的功能奠定了基础。
Based on the NCBI wheat TaWRKY46 gene sequence, the wheat variety Yangmai 158 was used as the material, and the 870 bp cDNA fragment was isolated from total RNA of wheat by RT-PCR. Sequencing showed that the fragment was highly homologous to the cloned TaWRKY46 gene in NCBI and contained a complete reading frame. It is noteworthy that the conserved amino acid sequence of WRKYGQK was WRKYGEK, a new allele of TaWRKY46 named TaWRKY46 -1. The inducible overexpression vector of this gene was constructed by Gateway cloning technology and transformed into Agrobacterium tumefaciens, which laid the foundation for the subsequent verification of the function of TaWRKY46-1 by the transgene.