Random recombinant mutant protein of Bacillus thuringiensis sip1aa protein

Abstract

The invention relates to a random recombination mutant protein of Bacillus thuringiensis Sip1Aa protein, belonging to the field of biotechnology. The present invention obtains Sip1Aa highly efficient soluble insecticidal protein mutants M6 and M8 proteins, respectively changing one amino acid, the insecticidal activity of the obtained mutant protein M8 is increased by 4.02 times, and the insecticidal activity of M6 is increased by 2.19 times, effectively To overcome the problem of finding a gene expressed by Bacillus thuringiensis that is highly virulent to the great ape leaf beetle, and to delay the development of insecticide resistance of pests to engineering bacteria and transgenic plants.

Description

technical field [0001] The invention belongs to the technical field of biological control, and in particular relates to a random recombination mutant protein of a Bt insecticidal gene with high toxicity to coleopteran agricultural pests. Background technique [0002] Bacillus thuringiensis (Bt) is a Gram-positive bacterium that widely exists in nature, and its shape is generally short rod-shaped, solitary or in short chains. In 1911, German biologist Bernai (Berliner) isolated the same bacterium from the diseased larva of a Mediterranean mealworm in Thuringiensis, Germany, and named it Bacillus thuringiensis. Bacillus thuringiensis has a research history of more than 100 years. The reports on this bacterium involve biomolecules, cytology, taxonomic nomenclature, active components of Bt, and its insecticidal mechanism. There are tens of thousands of research reports. [0003] At present, there are few reports on Bt Sip protein at home and abroad. In the process of studying ...

AI Technical Summary

Problems solved by technology

In 2012, our laboratory cloned and identified the sip gene containing 1038bp and encoding 345 amino acid sequences from the Bt strain QZL26, but its insecticidal activity has not been reported (Liu Yanjie, Li Haitao, Liu Rongmei, etc. Cloning of the new Bt gene sip , expression and bioinformatics analysis[J]. Biotechnology Bulletin, 2012(12):101-105)

In 2013, Murawska et al. applied genome sequencing technology and found that the IS5056 strain contained the sip gene, but did not conduct in-depth research

Even so, when the number of mutated bases in each target gene exceeds 6, it will often cause the protein to lose its original function

[0007] In addition, in the previous research on the Cry protein, a series of mutants were constructed through random mutation, among which the mutants with improved insecticidal activity were all at low mutation frequency, and when the mutation frequency was too high, most of the mutations were Harmful, it is almost impossible to screen for beneficial mutations (Vartanian JP, Henry M, Wain-Hobson S. Hypermutagenic PCR involving all four transitions and a sizeable proportion of transformations. Nucleic Acids Res, 1996, 24(14): 2627-2631.)

At the same time, if the mutation frequency is too low, the wild-type protein will occupy the advantage of the mutant library, and the ideal mutant cannot be screened.

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