Preparation method and application of double auxotrophic engineering strain

CN119351437BActive Publication Date: 2026-08-28NANJING FORESTRY UNIV
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Patent Information

Application Number
CN202411431834.8
Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Filing Date
2024-10-14
Publication Date
2026-08-28
Estimated Expiration
2044-10-14

AI Technical Summary

Technical Problem

显而易见，与单营养缺陷型菌株相比，双营养缺陷型可进一步提高根癌农杆菌对补充营养成分的依赖，降低恢复突变的可能性，并且降低农杆菌在外植体上的过度生长，然而迄今未见通过基因删除创制双营养缺陷型根癌农杆菌的研究报道

Benefits of technology

[0066]1)本申请在宿主细胞中敲除leuA基因，构建营养缺陷型工程菌株EHA105leuA-；在EHA105leuA-菌株中进一步敲除hisD基因，构建双营养缺陷型工程菌株EHA105hisD-leuA-；本申请实施例结果表明：EHA105hisD-leuA-无法在不含有任何氨基酸的AB液体培养基中生长，也无法在单独添加100mg/L的亮氨酸或组氨酸的AB液体培养基中生长，而在同时添加100mg/L的亮氨酸和组氨酸的AB液体培养基中正常生长。在共培养的培养基中添加700mg/L的亮氨酸和200mg/L的组氨酸情况下，EHA105hisD-leuA-菌株具备与野生型EHA105菌株相同的遗传转化能力；在不添加抑菌抗生素的情况下，在筛选培养基上培养10天，叶盘上未见农杆菌过度生长，且在筛选培养基上培养40天，有表达外源基因的抗性愈伤出现。

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Abstract

The application discloses a preparation method and application of a double auxotrophic engineering strain, and belongs to the technical field of genetic engineering. The preparation method of the double auxotrophic engineering strain disclosed by the application knocks out a leuA gene in a host cell, constructs an auxotrophic engineering strain EHA105leuA-, further knocks out a hisD gene in the EHA105leuA- strain, and constructs a double auxotrophic engineering strain EHA105hisD-leuA-. The gene IDs of the leuA gene and the hisD gene are WP_010972185.1 and WP_004432479.1 respectively. The results of examples of the application show that the double auxotrophic engineering strain EHA105hisD-leuA- has a large reduction in survival cells in soil and plants, reduces the probability of agrobacterium escaping into a natural environment, and improves the biosafety in a plant genetic transformation process.
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Citation Information

Patent Citations

  • Acquisition method and application of double-auxotroph agrobacterium based on ilvC gene deletion

    CN118272430A