不同截短U3启动子在棉花中的功能分析

Functional Analysis of Different Truncated U3 Promoters in Cotton

从海岛棉中克隆了2种GbU3-2P、GbU3-3P启动子,对它们进行2种长度截短,长度分别为436、245 bp和384、233 bp,同时构建了4个相应启动子驱动GUS的融合植物表达载体。利用农杆菌真空渗透转化法将4个GbU3Ps∷GUS-sg RNA-P1300植物表达载体分别转染棉花胚性愈伤组织。经GUS组织化学染色显示:克隆得到的2种截短大小的GbU3-2P和GbU3-3P启动子均能驱动GUS基因在棉花愈伤组织中表达,棉花愈伤组织被染成蓝色,但颜色深浅没有显著差异。本研究表明,同一GbU3启动子截短后,较短的启动子和较长的启动子具有相同的转录活性。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。

The U3 promoter is an important element for the transcription of sg RNA in the CRISPR/Cas9 genome editing system.Two rounds of PCR were performed to clone different truncated GbU3-2P and GbU3-3P promoters from Gossypium barbadense 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-sg RNA-P1300 plant expression vectors were transformed into cotton embryogenic callus by vacuum infiltration transformation. GUS histochemical staining results revealed that both truncated GbU3-2P and GbU3-3P promoters could drive GUS gene expression in cotton callus, and all corresponding cotton callus could be stained blue without significant differences in color depth. These results demonstrated that the two truncated GbU3 promoters had the same transcriptional activity regardless of the promoter being long or short. This work provides ideal promoters for the construction of a CRISPR/Cas9 genome editing system for use in cotton functional genomics.