作物学报 ›› 2021, Vol. 47 ›› Issue (3): 427-437.doi: 10.3724/SP.J.1006.2021.04178
周冠彤(), 雷建峰, 代培红, 刘超, 李月, 刘晓东*()
ZHOU Guan-Tong(), LEI Jian-Feng, DAI Pei-Hong, LIU Chao, LI Yue, LIU Xiao-Dong*()
摘要:
单向导RNA (sgRNA)是CRISPR/Cas9基因组编辑技术体系的重要元件之一。然而研究显示, 很多sgRNA不能有效工作, 因此需要对多个设计的候选sgRNA进行筛选, 以验证它们的有效性。早期对sgRNA有效性的验证采用的是完整编辑载体瞬时转化原生质体或者叶片的方法。这些方法费时费力, 成功率不高, 尤其是对于原生质体制备效率比较低的棉花。本研究针对GhMAPKKK2和GhAE基因分别设计靶序列, 构建了只转录sgRNA的载体: GhU6-5P::MAPKKK2-sgRNA-1300和GhU6-5P::AE-sgRNA-1300, 并通过农杆菌注射YZ-1 Cas9转基因棉花植株叶片; 与此同时, 构建了对应完整的CRISPR/Cas9 基因组编辑载体: GhU6-5P::MAPKKK2-sgRNA-Cas9和GhU6-5P::AE-sgRNA-Cas9, 并通过农杆菌注射YZ-1野生型棉花植株的叶片。另外, 针对GhPDS、GhCLA1、GhMAPKKK2和GhAE基因分别设计靶序列并构建了GhU6-5P-2::PDS-sgRNA-CLCrVA、GhU6-5P-2::CLA1- sgRNA-CLCrVA、GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA和GhU6-5P-2::AE-sgRNA-CLCrVA病毒投送载体, 通过农杆菌注射YZ-1 Cas9转基因棉花植株叶片。以上试验均以转化对应空载体的植株为对照。对转化后的棉花叶片基因组DNA进行PCR扩增后酶切, 并对未完全消化的PCR产物进行克隆测序, 结果显示, 转化GhU6-5P::AE-sgRNA- 1300、GhU6-5P::MAPKKK2-sgRNA-Cas9、GhU6-5P::AE-sgRNA-Cas9载体的棉花植株均未检测到靶基因突变, 而转化GhU6-5P::MAPKKK2-sgRNA-1300、GhU6-5P-2::PDS-sgRNA-CLCrVA、GhU6-5P-2::CLA1-sgRNA-CLCrVA、GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA和GhU6-5P-2::AE-sgRNA-CLCrVA载体的Cas9转基因阳性植株基因序列发生了改变, 突变类型包括碱基替换、碱基缺失和碱基插入。表明以Cas9转基因阳性植株为转化受体的策略可以高效真实地验证sgRNA的有效性, 排除了因转化效率低而带来的假阴性的结果, 且病毒载体投送sgRNA的策略更高效、更准确。该sgRNA高效验证体系的建立, 为棉花功能基因组学研究提供了重要的技术基础。
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