박상규 (농촌진흥청 국립농업과학원)

한빛사논문

조회491

농촌진흥청 국립농업과학원

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Hortic. Res.-England, Dec 2023, 10 (12) | https://doi.org/10.1093/hr/uhad239 Gene editing of authentic Brassica rapa flavonol synthase 1 generates dihydroflavonol-accumulating Chinese cabbage

Sangkyu Park^1,†, Hyo Lee^1,†, Jaeeun Song¹, Chan Ju Lim², Jinpyo Oh², Sang Hoon Lee³, Saet Buyl Lee¹, Jong-Yeol Lee¹, Sunhyung Lim⁴, Jin A. Kim¹ and Beom-Gi Kim^1,*

¹Metabolic Engineering Division, National Institute of Agricultural Sciences, Rural Development Administration, Jeonju, 54874, South Korea

²Institute of Biotechnology and Breeding, Asiaseed Inc., Icheon, 17414, South Korea

³Food and Nutrition Division, National Institute of Agricultural Sciences, Rural Development Administration, Wanju, 55365, South Korea

⁴Department of Horticultural Biotechnology, School of Biotechnology, Hankyong National University, Anseong, 17579, South Korea

^†These authors contributed to this work equally.

^*Corresponding author : Beom-Gi Kim

Abstract

Flavonols are the major class of flavonoids of green Chinese cabbage ( Brassica rapa subsp. pekinensis). The B. rapa genome harbors seven flavonol synthase genes ( BrFLSs), but they have not been functionally characterized. Here, transcriptome analysis showed four BrFLSs mainly expressed in Chinese cabbage. Among them, only BrFLS1 showed major FLS activity and additional flavanone 3 β-hydroxylase (F3H) activity, while BrFLS2 and BrFLS3.1 exhibited only marginal F3H activities. We generated BrFLS1-knockout ( BrFLS1-KO) Chinese cabbages using CRISPR/Cas9-mediated genome editing and obtained transgene-free homozygous plants without off-target mutation in the T ₁ generation, which were further advanced to the T ₂ generation showing normal phenotype. UPLC-ESI-QTOF-MS analysis revealed that flavonol glycosides were dramatically decreased in the T ₂ plants, while dihydroflavonol glycosides accumulated concomitantly to levels corresponding to the reduced levels of flavonols. Quantitative PCR analysis revealed that the early steps of phenylpropanoid and flavonoid biosynthetic pathway were upregulated in the BrFLS1-KO plants. In accordance, total phenolic contents were slightly enhanced in the BrFLS1-KO plants, which suggests a negative role of flavonols in phenylpropanoid and flavonoid biosynthesis in Chinese cabbage. Phenotypic surveys revealed that the BrFLS1-KO Chinese cabbages showed normal head formation and reproductive phenotypes, but subtle morphological changes in their heads were observed. In addition, their seedlings were susceptible to osmotic stress compared to the controls, suggesting that flavonols play a positive role for osmotic stress tolerance in B.rapa seedling. In this study, we showed that CRISPR/Cas9-mediated BrFLS1-KO successfully generated a valuable breeding resource of Chinese cabbage with distinctive metabolic traits and that CRISPR/Cas9 can be efficiently applied in functional Chinese cabbage breeding.

논문정보

형식Research article
게재일2023년 12월 (BRIC 등록일 2023-12-15)
연구진국내 연구진
분야 생명과학 > 식량작물 및 원예작물

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