Method for obtaining highly active cry1ai protein mutants and mutants thereof

Abstract

The invention relates to a method for acquiring high-activity Cry1Ai protein mutants and the mutants, and belongs to the biotechnology field. The method for acquiring the high-activity Cry1Ai protein mutants comprises the steps that continuous multi-point mutation is performed on an amino acid sequence in a loop 2 or / and a loop 3 of a Cry protein Domain II and comprises more than 10 continuous amino acid sequence changing. The insecticidal activity of the continuous multi-point mutant ModCry1Aid-loop 2 on cotton bollworm is improved by about 13 times, the activity of the continuous multi-point mutant ModCry1Aid-loop 2 on ostrinia nubilalis is improved by near 9 times compared with a Cry1Ai protein before improvement, and the activity of the continuous multi-point mutant ModCry1Aid-loop 2 and the continuous multi-point mutant ModCry1Aid-loop 3 on rice stem borer is improved by about 5-6 times compared with the Cry1Ai protein before improvement.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for obtaining a highly active Cry1Ai protein mutant and the highly active Cry1Ai protein mutant obtained by the method. Background technique [0002] The cry1Ai gene was cloned by the applicant in 2009 (patent number: ZL200910241558.8; Shu Changlong et al., 2013). suppressalis), American white moth (Hyphantria cunea) and silkworm (Bombyx mori) and other Lepidoptera insects have good insecticidal activity, but only have body weight inhibition effect on cotton bollworm (Helicoverpa armigera), which limits the The scope of application of the cry1Ai gene. cry1Ah1 (Patent No.: ZL200410009918.9; Zhang Jie et al., 2006), the Cry1Ah protein encoded by it has a high poisonous activity against Lepidoptera pests such as cotton bollworm, Asian corn borer and rice stem borer, and the Cry1Ah protein has a high toxicity to important The economic insect silkworm, however, exhibits low bio...

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Problems solved by technology

The above research results fully demonstrate that the loop region of the Cry protein is closely related to its insecticidal activity. However, there is no clear report on the specific amino acid region related to the insecticidal activity of the Cry protein against cotton bollworm, and most mutants are single point mutations with high activity. Most of them are lost, which is negatively speculated that the site may be related to insecticidal specificity, and there are few successful cases of changing insecticidal specificity and improving new activity

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