受体细胞胚胎发生体系促进CRISPR/Cas基因编辑
辽宁省农业科学院, 高粱研究所, 辽宁沈阳 110161
| 摘 要： | 体细胞胚胎发生（Somatic Embryogenesis，SE）是植物再生途径之一，可用于构建基因编辑受体系统。构建并优化SE体系，对转化后的体细胞再生成植株尤为重要。本研究以拟南芥野生型Col-0和ABA生物合成双突变体Nced6/9为实验材料，构建并优化了2,4-D诱导的成熟种子体细胞胚胎发生体系。另一方面，用含有载体Pbi121-U6-26-Cas9-sgRNA-ELF6-FastR的农杆菌C58蘸花转化拟南芥。利用FastR荧光标记选出转基因阳性种子，通过体细胞胚胎发生体系或直接播种获得植株，测序比较两者的基因编辑效率。结果显示，始花后25°C条件下生长的Nced6/9采种植株，其种子胚胎发生率显著低于15°C生长的植株。转基因阳性种子直接播种，其植株并未发现目标序列被编辑；而阳性种子通过体细胞胚胎发生体系再生后，其植株PAM附近发生了基因编辑事件。结果表明，低温条件下生长的采种植株，可能通过ABA含量影响种子的体胚诱导率。成熟种子体细胞胚胎发生体系可促进获得基因编辑突变体。 |
| 关 键 词： | 体细胞胚胎发生; CRISPR; 基因编辑; 受体系统 |
| DOI： | 10.57237/j.life.2023.03.001 |
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收稿日期: 2023-03-28
接受日期: 2023-05-23
在线出版日期: 2023-06-15
Recipient Somatic Embryogenesis System Promotes CRISPR/Cas Genome Editing
Sorghum Research Institute, Liaoning Academy of Agricultural Sciences, Shenyang 110161, China
| Abstract: | Somatic embryogenesis (SE) is an important type of plant regeneration, which can be used as a recipient system of genome editing. It is vital to optimize the SE system to improve the mutated somatic cells regenerated into fertile plants after transformation. 2,4-D was used as inducer of somatic embryogenesis from mature seeds. Arabidopsis wild type Col-0 and ABA biosynthesis double mutant nced6 / 9 were grown in 15°C and 25°C respectively, to investigate the effect of sampled plants growth conditions on the SE rate of their seeds and to optimize SE protocol. On the other hand, construct Pbi121-U6-26-Cas9-sgRNA-ELF6-FastR was used for genome editing. The positive seeds after floral dip were separated into two parts: some for sowing and the rest seeds went though SE induction. The efficiency of gene editing was compared by sequencing. Our results show that the SE rate of nced6 / 9 seeds from 25°C was significantly lower than that from 15°C. When transgenic positive seeds were sown directly, the target sequence was not found to be edited; However, when seeds went though somatic embryogenesis protocol, gene editing were found around PAM sequence, suggesting that SE system could improve gene editing after transformation. The sampled plants grown at low temperature (15°C) may affect SE ability of their seeds by ABA level. The mature seed somatic embryogenesis system can improve the efficiency of gene editing. |
| Keywords: | Somatic Embryogenesis; CRISPR; Genome Editing; Recipient System |
基金
| 1. | 中国博士后科学基金面上项目 (2021M693846) |
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