郭静远 赵辉 屈静 张丽丽 郭安平

摘  要：狗尾草是重要的模式植物，是研究C4光合作用的优秀模型，具有热带植物的典型特征。U6启动子主要用于构建RNAi和CRISPR表达载体，有启动干扰发夹结构的表达和基因编辑引导序列复合结构的表达的作用，该启动子具有特殊的启动位点G，能够保证转录后RNA结构的完整性。随着CRISPR 技术的发展，利用狗尾草进行基因编辑的研究日益增多和深入，目前还没有针对狗尾草U6启动子的克隆及功能鉴定。U6启动子具有种属特异性，在转基因技术中，使用转化物种本身或亲缘物种的U6启动子能取得更高的的启动效率，本研究克隆了狗尾草2个U6启动子基因，并构建不同序列长度的U6启动子序列驱动GUS基因的表达，GUS融合表达载体转化狗尾草胚性愈伤，瞬时表达验证表明，克隆出的2个狗尾草 U6启动子在狗尾草上具有很强的启动效率;并且将该载体转化狗尾草后，获得了能够稳定表达GUS基因的转基因植株;将该U6启动子用于构建狗尾草PDS基因CRISPR编辑载体，获得能够稳定表达的白化苗，说明克隆的狗尾草U6启动子能够很好的启动基因编辑载体的转录。

关键词：狗尾草;U6启动子;CRISPR系统;RNA干扰

中图分类号：Q949.748.5      文献标识码：A

Abstract： Setaria viridis is an important model plant and an excellent model for C4 photosynthesis， which has typical characteristics of tropical plants. U6 promoter is mainly used to construct RNAi and CRISPR expression vectors. It has the function of initiating the expression of the interference hairpin structure and the expression of the compound struc-ture of the gene editing guide sequence. The promoter has a special start site G， which can ensure the integrity of the RNA structure after transcription. With the development of CRISPR technology， there are more and more researches on gene editing by Setaria viridis. At present， there is no cloning and functional identification of U6 promoter of S. viridis. The U6 promoter is species-specific. In transgenic technology， using the U6 promoter of the transformed species itself or relative species can achieve higher activation efficiency. In this study， two U6 promoter genes of S. viridis were cloned， and U6 promoter sequences with different length were constructed to drive GUS gene expression. GUS fusion expression vector was transformed into embryogenic callus of S. viridis， and the transient expression verification showed that the two cloned U6 promoters of S. viridis had strong activation efficiency on S. viridis. After the vector was transformed into S. viridis， the transgenic plants were obtained which could stably expressing GUS gene. The U6 promoter was used to construct CRISPR editing vector of PDS gene of S. viridis， and the albino seedlings with stably expressing GUS gene were obtained， which indicated that the cloned U6 promoter of S. viridis could well start the tran-scription of gene editing vector.

Keywords： Setaria viridis; U6 promoter; CRISPR system; RNA interference

DOI： 10.3969/j.issn.1000-2561.2021.11.0014

由于許多粮食作物为禾本科、单子叶植物，而传统的模式植物拟南芥则为双子叶植物，与单子叶植物亲缘关系较远，在双子叶模式植物中研究单子叶植物的基因功能时有时会出现同家族基因功能的严重分化，甚至基因功能完全相反。……