忍冬U6启动子的克隆及功能验证

Cloning and functional verification of a U6 promoter with high transcriptional activity in Lonicera japonica

以忍冬基因组DNA为模板,克隆并筛选出具有较高转录活性的忍冬U6启动子。采用PCR方法,从忍冬基因组中克隆到4个LjU6启动子,长度分别为336、708、359、602 bp, PlantCARE分析发现4个启动子中均含有TATA框以及CAAT框等典型的启动子顺式元件,且包含与光响应、胁迫响应等相关的调控元件;克隆产物经测序正确后,将LjU6启动子连接至携带β-葡萄糖苷酸酶(GUS)基因的pBI121载体,成功构建4个LjU6-pBI121融合表达载体,通过农杆菌瞬时转化法转化烟草叶片,并对叶片进行GUS组织化学染色,染色结果显示LjU61-F1转录活性最高,本研究初步筛选出转录活性较高的忍冬U6启动子,为忍冬CRISPR/Cas9基因组编辑技术的建立奠定了基础。

Using Lonicera japonica genomic DNA as a template, we cloned Lonicera japonica U6 promoters.Four LjU6 promoters, 336, 708, 359 and 602 bp in length, were cloned by PCR from Lonicera japonica genomic DNA. PlantCARE analysis found that the four promoters contained typical promoter cis-elements, such as a TATA box and CAAT box, and contained regulatory elements related to light response and stress response. After the cloning products were sequenced, the LjU6 promoter was ligated to the pBI121 vector carrying the β-glucuronidase(GUS) gene to construct four LjU6-pBI121 fusion expression vectors. Nicotiana tabacum leaves were transformed by the Agrobacterium transient transformation method and GUS histochemical staining was performed on the leaves. The staining results showed that LjU61-F1 had the highest transcriptional activity. This study thus identified a U6 promoter with high transcriptional activity, providing a basis for the establishment of CRISPR/Cas9 genome editing technology in Lonicera japonica.