杨树U6启动子转录活性分析及其对基因编辑效率的影响

Transcriptional activity analysis of U6 promoters and their effect on gene editing efficiency in poplar

摘要

【目的】植物CRISPR/Cas基因编辑的效率受到sgRNA转录水平的影响，内源启动子可以提高sgRNA的转录水平，从而提高CRISPR/Cas基因编辑效率。U6启动子是双子叶植物CRISPR/Cas基因编辑系统中驱动sgRNA表达的常用元件，但目前尚缺乏关于杨树U6启动子的研究。本研究旨在挖掘杨树高效内源U6启动子，以优化杨树CRISPR/Cas基因编辑工具。【方法】从‘84K’杨中鉴定并克隆7条内源性U6启动子，构建 proPagU6 ::GUS融合表达载体并遗传转化烟草；通过组织化学染色与qRT-PCR分析不同 PagU6启动子对 GUS转录活性的影响；选取转录活性最高的U6启动子驱动sgRNA表达，构建CRISPR/Cas基因编辑载体并遗传转化杨树。【结果】7条杨树 PagU6启动子均可驱动 GUS基因表达，表现出转录活性，其中 PagU6 -1启动子的活性最高；由 PagU6 -1启动子驱动sgRNA表达的基因编辑载体成功编辑了杨树目标基因，编辑效率达16.22%。【结论】本研究筛选鉴定出具有较高转录活性的 PagU6-1启动子，可用于优化杨树CRISPR/Cas基因编辑工具，为提升杨树基因编辑效率提供了技术支撑。

Abstract

[Objective] The efficiency of CRISPR/Cas-mediated gene editing in plants is influenced by the transcriptional level of sgRNA. Endogenous promoters can enhance sgRNA transcription and improve gene editing efficiency. The U6 promoter is commonly used to drive sgRNA expression in CRISPR/Cas gene editing in dicottyledonous plants, however, studies on the U6 promoter in poplar remain scarce. This study aims to identify potent endogenous U6 promoters in poplar and optimize the CRISPR/Cas toolbox for this species. [Method] Seven endogenous U6 promoters were identified and cloned from the poplar ‘84K’ (Populus alba × P. tremula var. glandulosa). Seven proPagU6 ::GUS fusion expression vectors were constructed and transformed into tobacco. The transcriptional activities of different PagU6 promoters were analyzed using histochemical staining and qRT-PCR. A CRISPR/Cas9 vector utilizing PagU6 to drive sgRNA was constructed and transformed into poplar. [Result] Histochemical staining and qRT-PCR analyses revealed that all seven PagU6 promoters exhibited transcriptional activity, with PagU6-1 showing the highest activity. The gene editing vector, driven by the PagU6-1 promoter for sgRNA expression, successfully edited target genes in poplar, achieving a gene editing efficiency of 16.22%. [Conclusion] This study identified PagU6-1 as an ideal endogenous promoter with high transcriptional activity for optimizing CRISPR/Cas gene editing tools, providing a critical technical foundation for enhancing gene editing efficiency in poplar.

关键词

杨树 / U6启动子 / CRISPR/Cas系统 / 转录活性 / 基因编辑

Key words

Populus spp. / U6 promoter / CRISPR/Cas / transcriptional activity / gene editing

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基金资助

国家重点研发计划(2021YFD2200101)