Establishment and application of highly efficient regeneration, genetic transformation and genome editing system for cucurbitacins biosynthesis in Hemsleya chinensis.

IF 4.3 2区 生物学 Q1 PLANT SCIENCES
Juan Wang, Chao-Hui Li, Chun-Fan Xiang, Pin-Han Zhou, Le-Song Li, Xia Li, Sheng-Chao Yang, Guang-Hui Zhang, Yan Zhao
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引用次数: 0

Abstract

Background: Hemsleya Chinensis is a perennial plant in the Cucurbitaceae family containing antibacterial and anti-inflammatory compounds. The lack of genetic transformation systems makes it difficult to verify the functions of genes controlling important traits and conduct molecular breeding in H. chinensis.

Results: Highly efficient calli were induced on MS medium added 1.5 mg·L- 1 6-benzylaminopurine (6-BA) and 0.02 mg·L- 1 1-naphthylacetic acid (NAA) with high efficiency (> 95%). The frequency of shoot induction was increased to 90% with a plant growth regulator combination of 1.5 mg·L- 1 6-BA and 0.1 mg·L- 1 NAA. Our results also showed that 100% of shoot regeneration was achieved in a shoot regeneration medium. Additionally, more than 92% of kanamycin-resistant plants were confirmed. Furthermore, we achieved 42% genome editing efficiency by applying this transformation method to HcOSC6, a gene that catalyzes the formation of cucurbitadienol. HPLC analysis showed OE-HcOSC6 lines exhibited significantly higher cucurbitadienol levels than the genome-edited lines. Transcriptomic analysis revealed that some downstream genes related to cucurbitadienol biosynthesis, such as HcCYP87D20, HcCYP81Q58, and HcSDR34, were up-regulated in OE lines and down-regulated in mutants.

Conclusions: Here, we established a process for regeneration, transformation, and genome editing of H. chinensis using stem segments. This provides valuable insight into the underlying molecular mechanisms of medicinal compound production. By combining high-efficiency tissue culture, transformation, and genome editing systems, we provide a powerful platform that supports functional research on molecular mechanisms of secondary metabolism.

用于葫芦素生物合成的高效再生、遗传转化和基因组编辑系统的建立与应用。
背景:鹤望兰(Hemsleya Chinensis)是葫芦科多年生植物,含有抗菌和消炎化合物。由于缺乏遗传转化系统,很难验证控制其重要性状的基因的功能,也很难进行分子育种:结果:在添加了 1.5 mg-L- 1 6-苄基氨基嘌呤(6-BA)和 0.02 mg-L- 1 1-萘乙酸(NAA)的 MS 培养基上诱导出高效胼胝体,诱导效率高(> 95%)。使用 1.5 mg-L- 1 6-BA 和 0.1 mg-L- 1 NAA 的植物生长调节剂组合后,新芽诱导率提高到 90%。我们的结果还显示,在芽再生培养基中,芽的再生率达到了 100%。此外,超过 92% 的抗卡那霉素植株得到了确认。此外,通过对催化葫芦二烯醇形成的基因 HcOSC6 应用这种转化方法,我们实现了 42% 的基因组编辑效率。高效液相色谱分析显示,OE-HcOSC6 株系的葫芦烯醇含量明显高于基因组编辑株系。转录组分析表明,与葫芦二烯醇生物合成相关的一些下游基因,如 HcCYP87D20、HcCYP81Q58 和 HcSDR34,在 OE 株系中上调,而在突变体中下调:结论:在此,我们建立了一种利用茎段再生、转化和基因组编辑 H. chinensis 的方法。结论:在此,我们建立了利用茎段进行再生、转化和基因组编辑的过程,这为深入了解药用化合物生产的分子机制提供了宝贵的视角。通过结合高效的组织培养、转化和基因组编辑系统,我们提供了一个强大的平台,支持对次生代谢分子机制的功能研究。
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来源期刊
BMC Plant Biology
BMC Plant Biology 生物-植物科学
CiteScore
8.40
自引率
3.80%
发文量
539
审稿时长
3.8 months
期刊介绍: BMC Plant Biology is an open access, peer-reviewed journal that considers articles on all aspects of plant biology, including molecular, cellular, tissue, organ and whole organism research.
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