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选择标记基因的剔除是转基因植物商品化种植的重要基础。为建立小麦(Triticum aestivum)低温诱导的位点特异性标记基因删除体系,本研究以小麦西农928基因组DNA为模板,通过PCR扩增获得小麦低温诱导表达wcs120启动子,序列分析表明,该序列与Gen Bank中序列AF031235相比有一段21 bp插入,与AY493570.1序列完全一致。以含有Cin H/RS2单向位点特异性重组系统的植物表达载体p XL5513为框架,成功构建了包含有抗旱相关性基因PYL5(pyrabactin-resistance like gene 5)表达框的低温诱导的位点特异性重组植物表达载体p XL5513-fwcs-RDA;并对小麦绵阳19(M19)幼胚愈伤组织进行基因枪转化,1 745块愈伤组织经筛选分化共获得9株转基因植株,经PYL5基因特异引物PCR检测,6株为阳性植株,低温春化处理后经删除引物PCR检测,6株阳性植株初步证实成功删除了选择标记基因。本研究为利用低温诱导的位点特异性重组系统进行小麦无标记基因转化奠定了基础。
Elimination of selectable marker genes is an important basis for the commercialization of transgenic plants. In order to establish a low-temperature-induced site-specific marker gene deletion system for wheat (Triticum aestivum), we used the wheat XN928 genomic DNA as a template to obtain the wheat wc120 promoter induced by low temperature induction. The sequence analysis showed that this sequence A 21 bp insert compared to sequence AF031235 in Gen Bank, identical to the AY493570.1 sequence. The site-directed mutagenesis of the expression cassette containing a drought-related gene PYL5 (pyrabactin-resistance like gene 5) was successfully constructed using the plant expression vector p XL5513 containing the Cin H / RS2 unidirectional site-specific recombination system as a framework The recombinant plant expression vector p XL5513-fwcs-RDA was constructed. The immature embryos callus of Mianyang 19 (M19) was transformed by gene gun. Seven 745 callus were screened and differentiated to obtain 9 transgenic plants. The PYL5 gene-specific Primer PCR detection, 6 strains were positive plants, after low temperature vernalization PCR primers detected by deletion, 6 positive plants initially confirmed the successful deletion of the selectable marker gene. This study laid the foundation for the use of site-specific recombination system induced by low temperature for wheat marker-free gene transformation.