对正宗甘蓝型大白菜黄酮醇合成酶 1 进行基因编辑,可产生富含双氢黄酮醇的大白菜。

IF 7.6 Q1 GENETICS & HEREDITY
园艺研究(英文) Pub Date : 2023-11-14 eCollection Date: 2023-12-01 DOI:10.1093/hr/uhad239
Sangkyu Park, Hyo Lee, Jaeeun Song, Chan Ju Lim, Jinpyo Oh, Sang Hoon Lee, Saet Buyl Lee, Jong-Yeol Lee, Sunhyung Lim, Jin A Kim, Beom-Gi Kim
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引用次数: 0

摘要

黄酮醇是青江菜(Brassica rapa subsp.)B. rapa 基因组中含有七个黄酮醇合成酶基因(BrFLSs),但尚未对它们进行功能表征。在此,转录组分析显示了主要在大白菜中表达的四个 BrFLSs。其中,只有BrFLS1表现出主要的FLS活性和额外的黄烷酮3β-羟化酶(F3H)活性,而BrFLS2和BrFLS3.1仅表现出微弱的F3H活性。我们利用 CRISPR/Cas9 介导的基因组编辑技术生成了 BrFLS1 基因敲除(BrFLS1-KO)的大白菜,并在 T1 代获得了没有脱靶突变的无转基因同源植株,这些植株在 T2 代表现出正常的表型。UPLC-ESI-QTOF-MS分析表明,在T2代植株中,黄酮醇苷显著减少,而二氢黄酮醇苷同时积累到与黄酮醇减少水平相对应的水平。定量 PCR 分析表明,在 BrFLS1-KO 植株中,苯丙醇和类黄酮生物合成途径的早期步骤被上调。与此相对应,BrFLS1-KO植株的总酚含量略有增加,这表明黄酮类化合物在大白菜的苯丙类和黄酮类化合物生物合成中起负作用。表型调查显示,BrFLS1-KO 大白菜的头部形成和生殖表型正常,但头部出现了细微的形态变化。此外,与对照组相比,它们的幼苗易受渗透胁迫的影响,这表明黄酮醇对 B.rapa 幼苗的渗透胁迫耐受性起着积极的作用。本研究表明,CRISPR/Cas9介导的BrFLS1-KO成功产生了具有独特代谢性状的大白菜育种资源,CRISPR/Cas9可有效应用于大白菜功能育种。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Gene editing of authentic Brassica rapa flavonol synthase 1 generates dihydroflavonol-accumulating Chinese cabbage.

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 3β-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 T1 generation, which were further advanced to the T2 generation showing normal phenotype. UPLC-ESI-QTOF-MS analysis revealed that flavonol glycosides were dramatically decreased in the T2 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.

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