基于RUBY及CaREF1的辣椒高效遗传转化体系构建

doi:10.16420/j.issn.0513-353x.2025-0368

摘要/Abstract

摘要：

以新型可视化报告系统RUBY为标记，筛选获得基因递送高效辣椒受体材料3份，并结合促再生因子小肽CaREF1，构建了辣椒高效遗传转化体系。结果表明，线椒‘L27’、朝天椒‘遵辣1号’及珠子椒‘1-51’的基因瞬时表达效率显著高于其他材料；利用搭载RUBY报告系统的发根农杆菌K599进行遗传转化，于不定芽再生分化培养基（MS + 5 mg · L^-16-BA + 1.2 mg · L^-1 IAA + 10 nmol · L^-1 CaREF1）及壮芽培养基[MS +（5 ~ 10）mg · L^-1 6-BA + 0.5 mg · L^-1 IAA + 3 mg · L^-1 GA₃ + 10 nmol · L^-1 CaREF1]培养60 d后，平均再生效率可达84% ± 7%，平均再生芽数约为5.52个，阳性转化率约为5‰。基于该体系建立了辣椒农杆菌介导的可视化遗传转化体系，且有效在多个辣椒种质中实现了CRISPR/Cas9介导的基因编辑，T₀代植株编辑效率高达100%。

关键词: 辣椒, 遗传转化, RUBY, 再生因子REF1, 基因编辑

Abstract:

Using the novel visual reporter system RUBY as a screening marker，combined with the regeneration-promoting peptide CaREF1，a pepper genetic transformation system were constructed and identified three efficient recipient materials for gene delivery. The transient expression efficiency of RUBY was significantly higher in the pepper genotypes‘L27’（line pepper），‘Zunla-1’（chili pepper），and‘1-51’（bead pepper）compared to other materials. Agrobacterium rhizogenes K599，carrying the pKSE401-RUBY vector，was used for transformation. After 60 days of culturing on the adventitious bud regeneration medium（MS + 5 mg · L^-1 6-BA + 1.2 mg · L^-1 IAA + 10 nmol · L^-1 CaREF1）and shoot proliferation medium[MS + （5-10） mg · L^-1 6-BA + 0.5 mg · L^-1 IAA + 3 mg · L^-1 GA₃ + 10 nmol · L^-1 CaREF1]，the average regeneration efficiency of cotyledon explants was 84% ± 7%，with 5.52 regenerated shoots per explant and a positive transformation rate about 5‰. This system enabled the first Agrobacterium-mediated visual genetic transformation in pepper and facilitated CRISPR/Cas9-mediated gene editing in multiple pepper germplasms，with editing efficiency 100% in T₀ generation plants.

Key words: pepper, genetic transformation, RUBY, regeneration factor REF1, gene editing

王中一, 刘熠, 胡博文, 朱凡, 刘峰, 杨莎, 熊程, 欧立军, 戴雄泽, 邹学校. 基于RUBY及CaREF1的辣椒高效遗传转化体系构建[J]. 园艺学报, 2025, 52(4): 1093-1104.

WANG Zhongyi, LIU Yi, HU Bowen, ZHU Fan, LIU Feng, YANG Sha, XIONG Cheng, OU Lijun, DAI Xiongze, ZOU Xuexiao. Construction of a High-Efficiency Genetic Transformation System in Pepper Leveraging RUBY and CaREF1[J]. Acta Horticulturae Sinica, 2025, 52(4): 1093-1104.

图/表 5

表1 遗传转化各流程的培养基配方

Table 1 Stage-specific culture medium formulations for genetic transformation

培养基 Culture medium	g · L^-1			乙酰丁香酮/（μmol · L^-1） AS	mg · L^-1					小肽/（nmol · L^-1）CaREF1	特美汀/（mg · L^-1） Timentin
培养基 Culture medium	MS	蔗糖 Sucrose	琼脂 Agar	乙酰丁香酮/（μmol · L^-1） AS	细胞分裂素6-BA	生长素 IAA	赤霉素GA₃	卡那霉素Kan	硝酸银 AgNO₃	小肽/（nmol · L^-1）CaREF1	特美汀/（mg · L^-1） Timentin
播种Sowing	2.22	30	7	/	/	/	/	/	/	/	/
预培养Pre-culture	2.22	30	7	/	3	0.5	/	/	/	/	/
侵染液 Inoculation	2.22	30	/	200	2	0.1	/	/	/	/	/
共培养 Co-culture	4.44	30	7	200	2	0.1	/	/	/	/	/
分化Shoot regeneration	4.44	30	7	/	5	1.2	/	50	5	10	300
壮芽Shoot elongation	4.44	30	7	/	5 ~ 10	0.5	3	50	5	10	300
生根Rooting	2.22	30	7	/	/	0.1	/	/	/	/	200

图1 pKSE-RUBY载体示意图及其瞬时表达验证 A：pKSE-RUBY载体示意图；B：烟草瞬时表达48 h的表现；C：辣椒子叶瞬时表达48 h和72 h的表现

Fig 1. Schematic representation of the pKSE-RUBY vector and transient expression validation A：Schematic diagram of the pKSE-RUBY vector；B：Phenotypic observation of agroinfiltration-mediated co-expression in tobacco leaves at 48 hpi （hours post-injection）；C：Transient expression dynamics in pepper cotyledons at 48 hpi and 72 hpi

图2 辣椒高基因递送效率材料的筛选

Fig. 2 Screening of pepper germplasm with high-efficiency gene deliver

图3 辣椒遗传转化体流程及诱导效率分析 A ~ H：辣椒遗传转化体各阶段流程图；I和J：添加10 nmol · L-1 CaREF1小肽后的再生效率和平均再生芽数

Fig. 3 Genetic transformation workflow and transformation efficiency analysis in pepper A-H：Schematic illustration of the pepper genetic transformation workflow；I and J：CaREF1 peptide（10 nmol · L-1） elevates regeneration efficiency and shoot proliferation. t-Test，*** P < 0.001；**** P < 0.0001

图4 辣椒基因编辑植株的鉴定（A ~ D）及植株表现（E ~ F）

Fig. 4 Molecular identification（A-D）and phenotypic characterization（E-F）of gene-edited pepper plants

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