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^-1 6-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

中图分类号:

S 641.3

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.

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