不同截短U3启动子在棉花中的功能分析.pdf

棉 花 学 报 Cotton Science 20 16, 28(3): 307―3 14 同截短U3 启动子在棉花中的功能分析 1 2 1 1 1* 1* 建峰 徐新霞 代培红 李继洋 张巨松 刘晓东 1. 疆农业大学农学院/ 新疆农业大学农业生物技术重点实验室袁 疆乌鲁木齐830052 曰2. 新疆巴州农业技术推广中心袁 疆库尔勒84 1000冤 摘要院从海岛棉中克隆了2 种GbU3-2P 尧GbU3-3P 启动子袁对它们进行2 种长度截短袁长度分别为436尧245 bp 和384尧233 bp 袁同时构建了4 个相应启动子驱动 的融合植物表达载体遥 利用农杆菌真空渗透转化法将4 个GbU3Ps GUS-sgRNA-P 1300 植物表达载体分别转染棉花胚性愈伤组织遥 经GUS 组织化学染色显示院克隆 得到的2 种截短大小的GbU3-2P 和GbU3-3P 启动子均能驱动 基因在棉花愈伤组织中表达袁棉花愈伤组 织被染成蓝色袁但颜色深浅没有显著差异遥 本研究表明袁同一GbU3 启动子截短后袁较短的启动子和较长的启 动子具有相同的转录活性遥 这为构建棉花CRISPR/Cas9 基因组编辑载体系统提供了更多理想的启动子遥 关键词院棉花曰GbU3 启动子曰截短克隆曰真空渗透曰愈伤组织 中图分类号院S562.03 文献标志码院A 文章编号院1002-7807 20 16冤03-0307-08 10.11963/issn.1002-7807.20 16030 15 1 2 1 1* 1* Lei Jianfeng , Xu Xinxia , Dai Peihong , Li Jiyang1, Zhang Jusong , Liu Xiaodong (1. 830052, 2. 84 1000, The U3 promoter is an important element for the transcription of sgRNA in the CRISPR/Cas9 genome editing system. Two rounds of PCR were performed to clone different truncated GbU3-2P and GbU3-3P promoters from L.; lengths were 436 and 245 bp, and 384 and 233 bp, respectively. GUS fusion expression vectors were constructed using the four GbU3 promoters. Next, four GbU3Ps GUS-sgRNA-P1300 plant expression vectors were transformed into cotton embryo- genic callus by vacuum infiltration transformation. GUS histochemical staining results revealed that both truncated GbU3-2P and GbU3-3P promoters could drive gene expression in cotto