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