马铃薯CRISPR/Cas9介导的毛状根高效基因编辑体系的建立

doi:10.16420/j.issn.0513-353x.2024-0690

园艺学报 ›› 2025, Vol. 52 ›› Issue (7): 1758-1768.doi: 10.16420/j.issn.0513-353x.2024-0690

• 遗传育种·种质资源·分子生物学 • 上一篇下一篇

马铃薯CRISPR/Cas9介导的毛状根高效基因编辑体系的建立

宋倩娜¹^,², 宋陆帅¹^,³, 段永红¹, 白小东³^,^*(), 冯瑞云¹^,²^,^*()

¹ 山西农业大学农学院，山西太谷 030801
² 山西省农业科学院作物科学研究所，太原 030031
³ 山西农业大学高寒区作物研究所，马铃薯遗传改良与种质创新山西省重点实验室，山西大同 037008

收稿日期:2024-10-14 修回日期:2025-04-16 出版日期:2025-07-23 发布日期:2025-07-23
通讯作者:
*E-mail：fengruiyun1970@163.com
bxd5561@126.com
基金资助:
山西省农业科学院作物科学研究所博士基金项目(ZB1102); 山西农业大学博士科研启动项目(2021BQ44); 山西省基础研究计划项目(202303021212112); 山西省基础研究计划项目(202203021211272); 山西农业大学农学院育种工程专项(YZ2021-04); 山西省重点研发计划项目(202102140601004); 山西省农业关键核心技术攻关项目(NYGG30-02)

Establishment a System to Obtain CRISPR/Cas9 Genome Editing Plants Based on Hairy Root Transformation in Potatoes

SONG Qianna¹^,², SONG Lushuai¹^,³, DUAN Yonghong¹, BAI Xiaodong³^,^*(), and FENG Ruiyun¹^,²^,^*()

¹ College of Agriculture，Shanxi Agricultural University，Taigu，Shanxi 030801，China
² Institute of Crop Sciences，Shanxi Academy of Agricultural Sciences，Taiyuan 030031，China
³ Shanxi Key Laboratory of Potato Genetic Improvement and Germplasm Innovation，Institute of High Altitude Crop of Shanxi Agricultural University，Datong，Shanxi 037008，China

Received:2024-10-14 Revised:2025-04-16 Published:2025-07-23 Online:2025-07-23

摘要/Abstract

摘要：

以四倍体栽培马铃薯品种‘Desiree’‘青薯9号’和‘晋薯16号’为研究材料，StFBH3、StbHLH52和StPDS为靶基因，构建5个GFP荧光筛选CRISPR/Cas9基因编辑载体。通过发根农杆菌介导的遗传转化法侵染茎段诱导产生毛状根，评估转化频率及编辑效率。进一步探索毛状根再生体系，并对再生马铃薯完整植株进行突变鉴定。测序检测表明5个靶位点在3个品种中均有编辑活性，StPDS单靶点在Desiree和晋薯16号毛状根中的敲除效率分别为87.8%和80.0%，StFBH3单靶点在青薯9号毛状根中的敲除效率为72.7%。StbHLH52的双靶点在Desiree毛状根中的片段删除效率可达19.4%。基因编辑的毛状根经组织培养再生形成植株，Desiree、青薯9号和晋薯16号的再生效率分别为60%、75%和50%，再生植株的编辑效率为100%，且可获得StPDS基因敲除的白化植株。该技术体系的建立可快速验证基因编辑靶位点活性，高效获得基因编辑马铃薯植株，为马铃薯基因功能研究及精准遗传育种提供技术支撑。

关键词: 马铃薯, GFP, 基因编辑, 毛状根, 遗传转化, 植株再生

Abstract:

The StFBH3，StbHLH52 and StPDS genes were targeted by five CRISPR/Cas9 vectors for genome editing with GFP fluorescence screening marker in tetraploid potato cultivar Desiree，Qingshu 9 and Jinshu 16. The stem segments were infected by Agrobacterium rhizogenes-mediated genetic transformation to induce hairy roots，then the transformation rate and gene editing efficiency were evaluated. Further，whole potato plants were regenerated from hairy roots based on the optimized regeneration system and the mutations were identified. The sequencing showed that five editing vectors were all activity in the three potato varieties. Among them，the knockout efficiency for single target of StPDS were 87.8% and 80.0% in hairy roots of Desiree and Jinshu 16，respectively，while that of StFBH3 was 72.7% in Qingshu 9 hairy roots. The fragment deletion efficiency for dual-target of StbHLH52 was 19.4% in Desiree hairy roots. Moreover，the efficiency of plants regenerated from hairy roots in Desiree，Qingshu 9 and Jinshu 16 were 60%，75% and 50%，respectively. The editing efficiency of regenerated plants was 100%，and the StPDS-knockout line displayed the albino phenotype. The establishment of technology system in this study can quickly verify the target site activity and efficiently obtained edited potato plants，which provides a technical support for gene function research and precise genetic breeding in potatoes.

Key words: potato, GFP, gene editing, hairy root, transformation, plant regeneration

宋倩娜, 宋陆帅, 段永红, 白小东, 冯瑞云. 马铃薯CRISPR/Cas9介导的毛状根高效基因编辑体系的建立[J]. 园艺学报, 2025, 52(7): 1758-1768.

SONG Qianna, SONG Lushuai, DUAN Yonghong, BAI Xiaodong, and FENG Ruiyun. Establishment a System to Obtain CRISPR/Cas9 Genome Editing Plants Based on Hairy Root Transformation in Potatoes[J]. Acta Horticulturae Sinica, 2025, 52(7): 1758-1768.

图/表 7

表1 本研究中的引物序列

Table 1 Prime sequences in this study

引物名称 Prime name	引物序列（5′-3′） Sequences of primes
StFBH3-sgRNA-F1	ATTGAGTTGAGAGAATAAACCAGC
StFBH3-sgRNA-R1	AAACGCTGGTTTATTCTCTCAACT
StFBH3-sgRNA-F2	ATTGTCCATAGCTGCTATATCAGC
StFBH3-sgRNA-R2	AAACGCTGATATAGCAGCTATGGA
StPDS-sgRNA-F	ATTGAAGGAGCGGGTAAAGCTTCG
StPDS-sgRNA-R	AAACCGAAGCTTTACCCGCTCCTT
StbHLH52-DT1-BsF	ATATATGGTCTCGATTGAATTGTCAATAGTTTCAACGTT
StbHLH52-DT1-F0	TGAATTGTCAATAGTTTCAACGTTTTAGAGCTAGAAATAGC
StbHLH52-DT2-R0	AACGAGTACTCTGTTTATGAGACAATCTCTTAGTCGACTCTAC
StbHLH52-DT2-BsR	ATTATTGGTCTCGAAACGAGTACTCTGTTTATGAGACAA
StbHLH52-DT3-BsF	ATATATGGTCTCGATTGTCTCAAAAGCAGAGTACTCGTT
StbHLH52-DT3-F0	TGTCTCAAAAGCAGAGTACTCGTTTTAGAGCTAGAAATAGC
StbHLH52-DT4-R0	AACGCCACTCACAATTCGTCATCAATCTCTTAGTCGACTCTAC
StbHLH52-DT4-BsR	TTATTGGTCTCGAAACGCCACTCACAATTCGTCATCAA
StFBH3-F1	CACAGTTGCCACCTCAATATCCAAGG
StFBH3-R1	ATAGTCCCTCAGGCTCAGCTCA
StFBH3-F2	CAGCAGCACTCACAGGCCAC
StFBH3-R2	GCTCTTGTAGCTTTCTCATTCGTTCAC
StPDS-F	GCCTTGCTTTTCCTTTCTCATCTTG
StPDS-R	GGCAGGTAGACAATCTAACACCTC
StbHLH52-F	GGAACCAAATGTTGAAGAATTCAAC
StbHLH52-R	CTCCAGCATTATTGCTGCTAAAA
Cas9-F	CCCTCAAAGAAGTTCAAGGTCCT
Cas9-R	AGATCCCCCTCGATCAGGAAATG

图1 不同共培养时间处理下马铃薯毛状根数（A）和荧光毛状根的诱导效率（B）采用单因素方差分析，不同小写字母表示在0.05水平差异显著。下同

Fig. 1 Numbers of hairy roots（A）and induction efficiency of fluorescent hairy roots（B）under different co-culture time treatments in potato One-way ANOVA Duncan’s test is used for statistical analysis. Different lowercase letters indicate a signification difference at the 0.05 level. The same below

图2 两种AS浓度处理下马铃薯毛状根数（A）和荧光毛状根诱导效率（B）

Fig. 2 Numbers of hairy roots（A）and induction efficiency of fluorescent hairy roots（B）under two kinds of AS concentration treatments in potato

图3 不同卡那霉素浓度处理下的毛状根数量（A）和荧光毛状根诱导效率（B）

Fig. 3 Numbers of hairy roots（A）and induction efficiency of fluorescent hairy roots（B）under different Kan concentration treatments in potato

图4 StFBH3、StbHLH52和StPDS基因编辑载体转化发根农杆菌诱导马铃薯毛状根的鉴定 A：外植体的侵染；B：毛状根的诱导；C：毛状根的扩繁培养；D：荧光毛状根；E：荧光毛状根转化效率；F：CRISPR/Cas9载体示意图；G：StFBH3、StbHLH52和StPDS基因结构示意图和靶点序列；H：毛状根的PCR检测

Fig. 4 Identification of hairy roots induced by Agrobacterium rhizogenes strain with StFBH3，StbHLH52 and StPDS gene editing vectors A：The infected explants；B：Hairy root appear from explants；C：Hairy root further growth；D：Fluorescent hairy roots；E：The Percentage of fluorescent roots；F：Schematic illustration of the CRISPR/Cas9 construct；G：Schematic of the StFBH3，StbHLH52，StPDS gene and the selected target sequences；H：PCR screening of hairy roots

表2 荧光毛状根基因编辑事件的PCR鉴定

Table 2 Identification of gene editing events in fluorescent hairy roots based on PCR

基因 Gene	载体 Vectors	材料 Materials	总毛状根数 No. of total hairy roots	荧光毛状根数 No. of fluorescent hairy roots	突变毛状根数 No. of mutant hairy roots	编辑效率/% Editing efficiency
StPDS		青薯9号 Qingshu 9	62	36	30	83.3
	StPDS-sgRNA	晋薯16号 Jinshu 6	42	15	12	80.0
		Desiree	60	33	29	87.8
StFBH3	StFBH3-sgRNA1	青薯9号 Qingshu 9	68	17	12	70.6
StFBH3	StFBH3-sgRNA2	青薯9号 Qingshu 9	30	11	8	72.7
StbHLH52	StbHLH52-sgRNA14	Desiree	173	42	7	16.7
StbHLH52	StbHLH52-sgRNA23	Desiree	130	31	6	19.4

图5 毛状根再生马铃薯植株系的突变检测 A：突变的毛状根；B：愈伤组织的诱导；C：苗的分化；D ~ E：再生苗的GFP表达；F：获取基因编辑马铃薯植株过程及每一步骤需要的时间；G：PCR/RE和一代测序分析突变类型；H：光漂白马铃薯植株表型

Fig. 5 Mutation detection of regeneration plants from mutated hairy root lines A：Mutated hairy roots；B：Callus induction；C：Seedings differentiation；D - E：GFP expression of regenerated plants；F：Schematic of the process for obtaining mutated potato plants；G：PCR/RE and sanger sequencing to analysis mutant types；H：Photobleaching phenotype of potato plants

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马铃薯CRISPR/Cas9介导的毛状根高效基因编辑体系的建立

Establishment a System to Obtain CRISPR/Cas9 Genome Editing Plants Based on Hairy Root Transformation in Potatoes

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马铃薯CRISPR/Cas9介导的毛状根高效基因编辑体系的建立

Establishment a System to Obtain CRISPR/Cas9 Genome Editing Plants Based on Hairy Root Transformation in Potatoes

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