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WO-2026090993-A1 - CHIMERIC INSECTICIDAL PROTEIN AND USE THEREOF

WO2026090993A1WO 2026090993 A1WO2026090993 A1WO 2026090993A1WO-2026090993-A1

Abstract

The present invention relates to a chimeric insecticidal protein and a use thereof. The protein comprises domain I and domain II of Cry1Ac protein and domain III of Cry1Igg protein which are sequentially linked, and exhibits strong weight-inhibitory activity and lethal activity against various lepidopteran pests. The provided chimeric insecticidal protein broadens the insecticidal spectrum of the original protein, and also enhances toxicity against pests, effectively slowing the development of pest resistance in nature, and has good application prospects in agricultural production.

Inventors

  • WANG, Qinyang
  • PANG, Jie
  • XIE, Xiangting
  • ZHANG, Liangwei
  • Jia, Xiaowei

Assignees

  • 北京大北农生物技术有限公司

Dates

Publication Date
20260507
Application Date
20241031

Claims (16)

  1. A chimeric insecticidal protein, characterized in that the chimeric insecticidal protein comprises, in sequence, domain I and domain II of Cry1Ac protein and domain III of Cry1Ig protein.
  2. According to claim 1, the chimeric insecticidal protein is characterized in that the chimeric insecticidal protein comprises, in sequence, domains I and II of the Cry1Ac protein, domain III of the Cry1Ig protein, and the protoxin tail or a fragment thereof of the Cry1Ac protein.
  3. The chimeric insecticidal protein according to claim 1 or 2, characterized in that the amino acid sequence of the chimeric insecticidal protein comprises SEQ ID NO:1 or SEQ ID NO:2.
  4. A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the chimeric insecticidal protein according to any one of claims 1-3.
  5. The nucleic acid molecule according to claim 4, wherein the sequence of the nucleic acid molecule comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.
  6. An expression cassette, characterized in that the expression cassette comprises the nucleic acid molecule according to any one of claims 4 or 5.
  7. An expression vector, characterized in that the expression vector comprises the nucleic acid molecule according to any one of claims 4 or 5 or the expression cassette according to claim 6.
  8. A host cell, characterized in that the host cell comprises a chimeric protein according to any one of claims 1-3, a nucleic acid molecule according to any one of claims 4-5, an expression cassette according to claim 6, or an expression vector according to claim 7.
  9. A plant or plant part, characterized in that the plant or plant part comprises the chimeric insecticidal protein of any one of claims 1-3, the nucleic acid molecule of any one of claims 4-5, the expression cassette of claim 6, or the expression vector of claim 7.
  10. A method for preparing transgenic plants, the method comprising: 1) Transform the nucleic acid molecule of claims 4-5, the expression cassette of claim 6, or the expression vector of claim 7 into plant cells; 2) Select plant cells containing the nucleic acid molecule, expression cassette, or expression vector; 3) Regenerate plants from the selected plant cells.
  11. An insecticidal composition, characterized in that the insecticidal composition comprises the chimeric insecticidal protein according to any one of claims 1-3.
  12. A method for controlling lepidopteran pests, characterized in that it includes contacting the lepidopteran pests with the chimeric insecticidal protein of any one of claims 1-3, the host cell of claim 8, the plant or plant part of claim 9, or the insecticidal composition of claim 11, or introducing the nucleic acid molecule of any one of claims 4-5, the expression cassette of claim 6, or the expression vector of claim 7 into the plant, so that the lepidopteran pests come into contact with the plant.
  13. The method for controlling lepidopteran pests according to claim 12 is characterized in that the lepidopteran pests include noctuid moths or pyralid moths. Preferably, the lepidopteran pests include fall armyworm, beet armyworm, sugar beet armyworm, silver-striped armyworm, oriental armyworm, cotton bollworm and/or Asian corn borer.
  14. Use of the chimeric insecticidal protein of claims 1-3, the nucleic acid molecule of claims 4-5, the expression cassette of claim 6, the expression vector of claim 7, or the insecticidal composition of claim 11 in the control of lepidopteran pests.
  15. The use according to claim 14 is characterized in that it includes contacting lepidopteran pests with the insecticidal protein according to any one of claims 1-3 or the insecticidal composition according to claim 11 to control lepidopteran pests, or introducing the nucleic acid molecule according to claims 4-5, the expression cassette according to claim 6, or the recombinant expression vector according to claim 7 into a plant to allow lepidopteran pests to contact the plant and thereby control the lepidopteran pests. Preferably, the lepidopteran pests include noctuid moths or pyralid moths; More preferably, the lepidopteran pests include fall armyworm, beet armyworm, sugar beet armyworm, silver-striped armyworm, oriental armyworm, cotton bollworm and/or Asian corn borer.
  16. A product characterized in that the product is obtained from the plant or plant part of claim 9, or from the transgenic plant obtained by the method of preparing transgenic plants of claim 10, wherein the product is grain, starch, seed oil, syrup, flour, coarse flour, cereal or protein. Preferably, the product comprises the chimeric insecticidal protein of claims 1-3 or the nucleic acid molecule of claims 4-5.

Description

Chimeric insecticidal proteins and their uses Technical Field This invention relates to a chimeric insecticidal protein, a nucleic acid molecule encoding the protein, and methods and uses for controlling lepidopteran pests. Background Technology Currently, agricultural production faces both biotic stresses (such as diseases and pests) and abiotic stresses (such as drought, cold damage, and salinity damage), leading to weakened crop growth and reduced yields, posing a significant threat to global food security. Among these, pests are one of the main biotic stress factors affecting agricultural and forestry productivity. As the environmental problems caused by the use of chemical pesticides for pest control become increasingly serious, the use of biological pesticides is gradually coming into focus. Bacillus thuringiensis (Bt) is a Gram-positive bacterium widely distributed in nature and is also an entomopathogenic bacterium. The biggest difference between Bt and other Bacillus species is that Bt produces crystal proteins accompanying spore formation in the later stages of its growth; these are generally called parasporal crystal proteins (including Cry and Cyt proteins). These proteins are the main or decisive factors in producing insect pathogenicity. Numerous studies have reported the insecticidal activity of various Bt proteins against Lepidoptera, Coleoptera, Diptera, Hymenoptera, and Homoptera, such as Cry1Ab, Cry1Ac, Cry1F, Cry2Ab, and Cry3Bb. The commercial cultivation of Bt transgenic insect-resistant crops, prepared using plant genetic engineering, has become one of the main drivers of significantly improving agricultural productivity. These transgenic insect-resistant plants not only effectively control the occurrence and damage of target pests but also reduce the use of chemical pesticides, providing important guarantees for food and ecological security. However, with the widespread application of genetically modified crops, insects will evolve resistance to the Bt protein expressed in genetically modified plants under continuous selective pressure. If such resistance cannot be effectively controlled, it will limit the commercial value of genetically modified plant varieties containing Bt protein. At the same time, some Bt proteins also have strong cross-lethal activity against non-target organisms. These problems limit the further application of Bt protein. One approach to addressing the aforementioned problems is to rationally design functionally improved Bt chimeric proteins (or fusion proteins) based on the domains of Bt proteins. This method involves introducing or replacing domains of heterologous Bt proteins or other types of insecticidal proteins into a single Bt protein, or recombining domains of different Bt proteins, thereby obtaining Bt chimeric proteins with stronger insecticidal activity, a broader insecticidal spectrum, and even resistance to pesticide resistance in target pests. Currently, related research mainly focuses on domain recombination between Cry-type Bt proteins with relatively clear structures and functions. For example, Ballester V et al. reported that replacing domain III of Cry1Ab protein with domain III of Cry1C protein significantly improved the toxicity to diamondback moth compared to both Cry1Ab and Cry1C proteins (Applied and Environmental Microbiology, 1999, 65(5):1900-1903); SHAH J V et al. recombined domains of Cry9Aa... After replacing domain I with domain I of Cry1Ac, the lethal toxicity against cotton bollworm was increased by 4.9 times compared with Cry1Ac (Archives of Microbiology, 2017, 199(7):1069-1075); DAS A et al. spliced domain I of Cry1Aa with domain II of Cry1Ab and then spliced domain III of Cry1Ac, and the resulting hybrid had significantly higher insecticidal activity than the original protein (Frontiers in Plant Science, 2017, 8:1423). However, due to differences in structure, function, receptor binding sites, and insecticidal mechanisms among different Cry proteins, the reassembled chimeric proteins often exhibit incorrect structural forms, or fail to produce chimeric proteins that are identical to the parent protein or have enhanced insecticidal activity. Even with extensive analysis, design, screening, and validation, it is not always possible to construct chimeric insecticidal proteins that exhibit improved insecticidal activity compared to the parent protein. Therefore, designing more novel chimeric proteins with enhanced insecticidal activity and a broader insecticidal spectrum to provide more options for insecticidal proteins used in agricultural production remains a hot topic and a challenge in this field. Summary of the Invention To address the shortcomings of existing technologies, this invention provides a chimeric insecticidal protein capable of simultaneously targeting multiple lepidopteran pests. This chimeric insecticidal protein exhibits strong weight-inhibiting and lethal activity against fall armyworm, cotton bollworm, silve