CRISPR/Cas9 介导的转录抑制子SlMYB32突变诱导番茄果实黄酮醇和黄烷酮积累

doi:10.1016/j.jia.2025.11.011

Journal of Integrative Agriculture ›› 2026, Vol. 25 ›› Issue (4): 1463-1474.DOI: 10.1016/j.jia.2025.11.011

CRISPR/Cas9 介导的转录抑制子SlMYB32突变诱导番茄果实黄酮醇和黄烷酮积累

收稿日期:2025-01-31 修回日期:2025-11-13 接受日期:2025-09-25 出版日期:2026-04-20 发布日期:2026-03-10

CRISPR/Cas9-mediated mutagenesis of transcriptional repressor SlMYB32 improves flavonols and flavanones accumulation in tomato fruit

Ruining Zhang^{1, 2}, Yunlin Cao^{1, 2}, Tong Zhang^{1, 2}, Yingyue Ma^{1, 2}, Jiajia Li^{1, 2}, Kunsong Chen^{1, 2}, Xian Li^{1, 2#}

¹ College of Agriculture & Biotechnology, Zhejiang University, Hangzhou 310058, China
² Zhejiang Key Laboratory of Horticultural Crop Quality Improvement, Zhejiang University, Hangzhou 310058, China

Received:2025-01-31 Revised:2025-11-13 Accepted:2025-09-25 Online:2026-04-20 Published:2026-03-10
About author:Ruining Zhang, E-mail: rnzh@zju.edu.cn; #Correspondence Xian Li, E-mail: xianli@zju.edu.cn
Supported by:
The research was supported by the National Natural Science Foundation of China (32372667).

摘要/Abstract

摘要： 黄酮醇和黄烷酮是重要的生物活性物质，具有多样的药理学活性和保健功能。黄酮醇和黄烷酮生物合成的转录激活研究较为广泛，但相关负调节因子信息很少。CRISPR/Cas9 基因编辑技术可以对基因进行精确修饰，是培育生物强化材料和探索潜在分子机制的重要方法。本研究鉴定了番茄果实中的转录抑制子SlMYB32，基因编辑敲除SlMYB32显著提高了番茄果实黄酮醇与黄烷酮含量，其中槲皮素3-O-芸香糖苷（芦丁）高达1 mg g^-1鲜重。转录组分析表明，slmyb32突变体中SlPAL6、Sl4CL3和Sl4CL4及五个候选SlUGTs的基因表达显著提高；双荧光素酶检测与EMSA分析发现SlMYB32可以与SlPAL6和Sl4CL3的启动子结合并抑制其转录活性；此外，slmyb32突变体中27个转录因子发生了显著差异表达，包括与已知类黄酮调控因子聚类在相同分支的2个SlMYBs、2个SlNACs、2个SlAP2s和1个SlWRKY，它们是今后研究SlMYB32调控网络的重要候选转录因子。本研究为果实生物活性物质的改良及黄酮醇和黄烷酮生物合成的负调控机制提供了新见解。

Abstract:

Flavonols and flavanones are important bioactive compounds with multiple pharmacological activities and health benefits. Transcriptional activation of flavonol and flavanone biosynthesis has been studied extensively, while little is known about the negative regulators. CRISPR/Cas9 gene-editing technology, with the advantage of precise genetic modification, is a desirable tool for breeding biofortified materials and exploring potential molecular mechanisms. In this study, a transcriptional repressor, SlMYB32, was characterized in tomato fruit. Phenotype and metabolomic analyses confirmed that knockout of SlMYB32 resulted in increased accumulation of flavonols and flavanones, especially about 1 mg g^–1 FW of quercetin 3-O-rutinoside (rutin). Transcriptome analysis indicated that expression of key genes SlPAL6, Sl4CL3 and Sl4CL4 as well as five candidate SlUGTs were significantly up-regulated in slmyb32 mutants. Dual-luciferase and EMSA assays indicated SlMYB32 could bind to and repress promoter activities of SlPAL6 and Sl4CL3. Expression of 27 transcription factors belonging to 12 families was significantly changed in slmyb32 mutants, among which two SlMYBs, two SlNACs, two SlAP2s and one SlWRKY were clustered with known flavonoid regulators. Our results provide new insights into improving bioactive compounds in fruit and understanding negative regulatory mechanisms in flavonol and flavanone biosynthesis.

Key words: flavonol , flavanone , CRISPR/Cas9 , repressor SlMYB32 , tomato , transcriptional regulation

Ruining Zhang, Yunlin Cao, Tong Zhang, Yingyue Ma, Jiajia Li, Kunsong Chen, Xian Li. CRISPR/Cas9 介导的转录抑制子SlMYB32突变诱导番茄果实黄酮醇和黄烷酮积累[J]. Journal of Integrative Agriculture, 2026, 25(4): 1463-1474.

Ruining Zhang, Yunlin Cao, Tong Zhang, Yingyue Ma, Jiajia Li, Kunsong Chen, Xian Li. CRISPR/Cas9-mediated mutagenesis of transcriptional repressor SlMYB32 improves flavonols and flavanones accumulation in tomato fruit[J]. Journal of Integrative Agriculture, 2026, 25(4): 1463-1474.

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CRISPR/Cas9 介导的转录抑制子SlMYB32突变诱导番茄果实黄酮醇和黄烷酮积累

CRISPR/Cas9-mediated mutagenesis of transcriptional repressor SlMYB32 improves flavonols and flavanones accumulation in tomato fruit

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