CRISPR/Cas9介导的高效四倍体马铃薯试管薯基因编辑体系的建立

doi:10.13560/j.cnki.biotech.bull.1985.2024-0536

摘要/Abstract

摘要：

【目的】基因编辑技术的发展使得马铃薯实现精准分子育种成为了可能，试管薯作为遗传转化的理想材料，但其诱导和转化具有基因型依赖性，建立高效且普遍适用的试管薯基因编辑技术体系可为其精准分子育种提供技术支撑。【方法】以四倍体栽培马铃薯品种青薯9号和并薯6号为材料，对试管薯诱导及其遗传转化体系进行摸索；同时，转化CRISPR/Cas9基因编辑载体进行基因组编辑。另外，利用筛选的条件对其他3个马铃薯品种进行测试。【结果】全黑暗条件下，采用2叶/段的扩繁方式，在含有10%蔗糖和5 mg/L激动素的培养基上，5个马铃薯品种均可成功诱导出试管薯，但诱导效果存在差异。青薯9号的最佳分化激素配方为0.5 mg/L 6-苄氨基腺嘌呤、0.2 mg/L 吲哚-3-乙酸、0.2 mg/L赤霉素、2 mg/L玉米素，再生效率、转化频率和基因编辑效率分别为41.5%、51.9%和82.1%。利用上述筛选的激素配方，在其他4个四倍体马铃薯品种中均可高效地实现试管薯的遗传转化再生，其中并薯6号、Desiree和晋薯16号的编辑效率分别为63.2%、33.3%和10%。【结论】建立了5个不同基因型四倍体马铃薯高效的试管薯遗传转化再生体系，其中的4份材料成功地实现了基因组编辑。

关键词: 马铃薯, 试管薯诱导, 遗传转化再生, GFP, 基因组编辑

Abstract:

【Objective】 The development of gene editing technology has made it possible to achieve precise molecular breeding in potato. Microtubers are ideal materials for genetic transformation, but its induction and genetic transformation are of genotype-dependence. Thus, establishing an efficient and widely used gene editing technology may provide technical support for precise molecular breeding for potatoes in future. 【Method】Cultivated tetraploid potato variety Qingshu No. 9 and Bingshu No. 6 were used to explore microtuber induction and genetic transformation. Concurrently, the CRISPR/Cas9 gene editing vector was transformed for genome editing. In addition, the screening system was tested in another three potato varieties. 【Result】 The efficient tuberizations of five potato varieties were obtained using stem segment with two leaves on solid media including 10% sucrose and 5 mg/L kinetin under dark condition, while induction effect varied. The optimal ratio of hormones for the regeneration of Qingshu No.9 was 0.5 mg/L 6-benzylaminopurine, 0.2 mg/L indole-3-acetic acid, 0.2 mg/L gibberellin, and 2 mg/L zeatin, under which the regeneration rate, the transformation frequency, genome editing rate was 41.5%, 51.9%, and 82.1%, respectively. Using the above screened the formula of hormones, genetic transformation and regeneration was efficiently achieved in another four potato varieties, and the genome editing rate was 63.2%, 33.3%, and 10% for Bingshu No.6, Desiree, and Jingshu No. 16, respectively. 【Conclusion】We established the highly efficient genetic transformation and regeneration system for five potato varieties, and genome editing can be completed for four potato varieties.

Key words: potato, microtubers inducing, genetic transformation and regeneration, GFP, genome editing

宋倩娜, 段永红, 冯瑞云. CRISPR/Cas9介导的高效四倍体马铃薯试管薯基因编辑体系的建立[J]. 生物技术通报, 2024, 40(9): 33-41.

SONG Qian-na, DUAN Yong-hong, FENG Rui-yun. Establishment of CRISPR/Cas9-mediated Highly Efficient Gene Editing System in Microtubers of Potatoes[J]. Biotechnology Bulletin, 2024, 40(9): 33-41.

图/表 9

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蔗糖浓度 Sucrose concentration/%	并薯6号 Bingshu No. 6		青薯9号 Qingshu No.9
蔗糖浓度 Sucrose concentration/%	单瓶平均薯数Number of microtubers per bottle/粒	单瓶平均薯重Weight of microtubers per bottle/g	单瓶平均薯数Number of microtubers per bottle/粒	单瓶平均薯重Weight of microtubers per bottle/g
6	0.17±0.40b	0.02±0.04c	0.20±0.45c	0.02±0.03b
8	2.83±1.17a	0.29±0.19ab	1.80±0.84ab	0.19±0.06a
10	3.50±1.64a	0.48±0.20a	2.60±1.34a	0.28±0.13a
12	0.67±0.82b	0.09±0.10bc	0.80±0.84bc	0.05±0.05b
15	0.67±1.03b	0.06±0.11c	0.40±0.55c	0.02±0.03b

蔗糖浓度 Sucrose concentration/%	并薯6号 Bingshu No. 6		青薯9号 Qingshu No.9
蔗糖浓度 Sucrose concentration/%	单瓶平均薯数Number of microtubers per bottle/粒	单瓶平均薯重Weight of microtubers per bottle/g	单瓶平均薯数Number of microtubers per bottle/粒	单瓶平均薯重Weight of microtubers per bottle/g
6	0.17±0.40b	0.02±0.04c	0.20±0.45c	0.02±0.03b
8	2.83±1.17a	0.29±0.19ab	1.80±0.84ab	0.19±0.06a
10	3.50±1.64a	0.48±0.20a	2.60±1.34a	0.28±0.13a
12	0.67±0.82b	0.09±0.10bc	0.80±0.84bc	0.05±0.05b
15	0.67±1.03b	0.06±0.11c	0.40±0.55c	0.02±0.03b

不同激素配比 Different hormone combination	并薯6号 Bingshu No. 6		青薯9号 Qingshu No.9
不同激素配比 Different hormone combination	单瓶平均薯数/粒Number of microtubers per bottle	单瓶平均薯重Weight of microtubers per bottle/g	单瓶平均薯数Number of microtubers per bottle/粒	单瓶平均薯重Weight of microtubers per bottle/g
0	3.50±1.64a	0.48±0.20a	2.60±1.34a	0.28±0.13a
4 mg/L 6-BA	0.67±0.58b	0.04±0.03b	0.33±0.58b	0.02±0.03b
4 mg/L 6-BA + 3.5 mg/L NAA	0.67±0.58b	0.03±0.03b	0.00±0.00b	0.00±0.00b
5 mg/L KT	4.67±0.58a	0.62±0.07a	3.30±0.82a	0.37±0.15a

不同激素配比 Different hormone combination	并薯6号 Bingshu No. 6		青薯9号 Qingshu No.9
不同激素配比 Different hormone combination	单瓶平均薯数/粒Number of microtubers per bottle	单瓶平均薯重Weight of microtubers per bottle/g	单瓶平均薯数Number of microtubers per bottle/粒	单瓶平均薯重Weight of microtubers per bottle/g
0	3.50±1.64a	0.48±0.20a	2.60±1.34a	0.28±0.13a
4 mg/L 6-BA	0.67±0.58b	0.04±0.03b	0.33±0.58b	0.02±0.03b
4 mg/L 6-BA + 3.5 mg/L NAA	0.67±0.58b	0.03±0.03b	0.00±0.00b	0.00±0.00b
5 mg/L KT	4.67±0.58a	0.62±0.07a	3.30±0.82a	0.37±0.15a

品种 Variety	处理 Treatment	单瓶平均结薯数Number of microtubers per bottle/粒	单瓶平均薯重Weight of microtubers per bottle/g	大薯重Weight of the large tuber/g	大薯直径 Diameter of the large tuber/cm
并薯6号	顶部1个节段	2.00±2.00a	0.13±0.15a	0.08±0.08ab	0.43±0.38a
	底部1个节段	0.67±0.58a	0.04±0.04a	0.04±0.04b	0.38±0.34a
	顶部2个节段	3.33±0.58a	0.23±0.05a	0.09±0.05ab	0.65±0.09a
	底部2个节段	2.67±1.53a	0.45±0.37a	0.22±0.13a	0.80±0.18a
青薯9号	顶部1个节段	1.33±2.16a	0.10±0.20b	0.02±0.04b	0.18±0.29b
	底部1个节段	2.83±1.72a	0.20±0.17ab	0.10±0.07ab	0.44±0.06ab
	顶部2个节段	3.17±1.60a	0.21±0.13ab	0.10±0.04ab	0.55±0.07a
	底部2个节段	3.33±0.82a	0.37±0.15a	0.15±0.05a	0.59±0.06a