作物学报2016，Vol.42Issue(5)：675-683,9.DOI:10.3724/SP.J.1006.2016.00675
棉花不同GbU6启动子截短克隆及功能鉴定
Cloning and Functional Analysis of Different Truncated GbU6 Promoters in Cotton
摘要
Abstract
U6 promoter is an important element for the transcription of sgRNA in the CRISPR/Cas9 genome editing system. Using a short promoter is also one of the basic requirements for the construction of CRISPR/Cas9 genome editing vector. According to the sequence of GbU6-5P promoter cloned, six different truncated U6 promoters were successfully cloned using Transfer PCR method and their length were 672, 468, 358, 280, 202, and 105 bp, respectively. Together with GbU6-5P::GUS-pCAMBIA1300, GUS fusion expression vectors driven by corresponding truncated promoter were constructed and transformed into cotton pollen by the vacuum in filtration transformation method. Results of GUS histochemical staining showed that the cloned seven truncated GbU6-5P promoters could drive GUS expression in cotton pollen and all corresponding cotton pollen could be stained blue, but there exist different shades among them. Results showed that the shorter promoter, the stronger tran-scription activity, and so did the two other GbU6 promoter. In this study three short GbU6 promoters with transcription activity in cotton pollen were cloned. GUS staining results showed that the shorter U6 promoter had higher transcriptional activity, which was the common characteristics in different U6 promoter. The above results indicated that using shorter U6 promoter not only conform to the requirement of constructing CRISPR/Cas9 genome editing vector, but also improve the transcription of sgRNA, which may enhance the efficiency of genome editing finally.关键词
棉花/GbU6启动子/截短克隆/真空渗透/棉花花粉Key words
Cotton/GbU6 promoter/Truncated cloning/Vacuum infiltration/Cotton pollen引用本文复制引用
雷建峰,李月,徐新霞,阿尔祖古丽·塔什,蒲艳,张巨松,刘晓东..棉花不同GbU6启动子截短克隆及功能鉴定[J].作物学报,2016,42(5):675-683,9.基金项目
本研究由国家自然科学基金项目(31560534)和新疆维吾尔自治区研究生科研创新项目(XJGRI2015084)资助. This study was supported by the National Natural Science Foundation of China (31560534) and the Science and Technology Innovation Pro-jects for Graduate Students in Xinjiang Uygur Autonomous Region (XJGRI2015084). （31560534）
