Insect-resistant protein and preparation method and application thereof

Abstract

The application discloses a new insect-resistant protein and application thereof, and belongs to the field of genetic engineering. The application provides a new insect-resistant protein, a coding gene of the insect-resistant protein, and application of the insect-resistant protein in cultivating insect-resistant crops.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to an insect-resistant protein, its preparation method and application, specifically a modified Cry1Ia protein. Background Technology

[0002] Currently, commercially available transgenic insect-resistant maize containing Cry1Ab protein (e.g., MON810, DBN9936) exhibits excellent control of corn borers. However, with the gradual expansion of the industrial planting scale of transgenic insect-resistant maize, there have been reports abroad of corn borers developing resistance to Cry1Ab maize. In order to manage corn borer resistance in advance, developing insect-resistant maize without Cry1A cross-resistance is one of the effective management methods. Existing studies have shown that Cry1I and Cry1Ab do not have cross-resistance, and substitution or superposition with this protein can delay the development of corn borer resistance (Xu L, Wang Z, Zhang J, et al. Cross-resistance of Cry1Ab-selected Asian corn borer to other Cry toxins[J]. Journal of Applied Entomology, 2010, 134(5):429-438.).

[0003] Among the identified Cry-type Bt insect-resistant genes (Bacillus thuringiensis genes, or Bt genes for short), Cry1I proteins are unique. While typically silent genes in Bacillus thuringiensis strains, they can be expressed in E. coli cultures as a protoxin of approximately 81 kDa, a molecular weight unique among Cry1 proteins. Furthermore, Cry1I proteins target insects from Lepidoptera and Coleoptera, and do not exhibit cross-resistance with Cry1A proteins. Therefore, research on Cry1I genes has significant theoretical and practical value, and will provide new gene options for addressing issues such as the narrow insecticidal spectrum of Bt toxins and the development of insecticide resistance in pests. However, there are currently few publications reporting on the insect-resistant performance and verification of Cry1I genes. Summary of the Invention

[0004] To overcome at least one problem in the existing technology, the inventors discovered in preliminary experiments that Cry1Ia1 has good activity against corn borers, and that modification of the toxic region of the insecticidal protein can further enhance the insecticidal activity of the protein. Therefore, this invention obtains better insecticidal activity against corn borers by modifying the Cry1Ia1 protein and applies it to the development of insect-resistant corn, which has good prospects for commercial application.

[0005] In previous research, the inventors mutated the nucleic acid molecule encoding the Cry1Ie1 protein using a sequential error-prone PCR method, expressed the mutant protein, and then screened for mutant proteins with higher insecticidal activity using a corn borer assay. Analysis of the nucleotide sequence of the encoded protein identified the mutation site. Since Cry1Ia and Cry1Ie both belong to the Cry1I class of proteins, based on the aforementioned previous research, this invention found a homologous active site in Cry1Ia and mutated it. Then, using a corn borer assay, three recombinant proteins superior to Cry1Ia1 were screened.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention is to provide an insect-resistant protein, which is a modified Cry1Ia protein; wherein the modified Cry1Ia protein contains the amino acid sequence shown in SEQ ID NO.1 and is mutated at at least one of the following sites: amino acids 82, 99, 113, 147, 182, 214, and 233. Further, at least one of the amino acids 113, 182, and 233 is mutated.

[0008] Furthermore, the modified Cry1Ia protein has mutation sites including: Y233N, E182K, N113S+E182V, V82N+N113S+E182V, N113S+E182V+Y233N, E182V, V82N+E182V, E182K+Y233N, E182V+Y233N, N99S+N113S+E182V, Y233N, or G147S+S214N+Y233N; even further, the modified Cry1Ia protein has mutation sites of Y233N, E182K, or N113S+E182V.

[0009] Furthermore, the amino acid sequence of the modified Cry1Ia protein is shown in SEQ ID No. 6 to SEQ ID No. 8; specifically, the amino acid sequence corresponding to the Y233N mutation is shown in SEQ ID No. 6, the amino acid sequence corresponding to the E182K mutation is shown in SEQ ID No. 7, and the amino acid sequence corresponding to the N113S+E182V mutation is shown in SEQ ID No. 8.

[0010] A second aspect of the present invention is to provide a nucleic acid molecule for encoding any of the above-described insect-resistant proteins, wherein the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO.1 is as shown in SEQ ID NO.2.

[0011] Further, the nucleic acid sequences encoding the amino acid sequences shown in SEQ ID No. 6 to SEQ ID No. 8 are shown in SEQ ID No. 3 to SEQ ID No. 5, respectively; specifically, the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID No. 6 is shown in SEQ ID No. 3, the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID No. 7 is shown in SEQ ID No. 4, and the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID No. 8 is shown in SEQ ID No. 5.

[0012] The amino acid and nucleic acid sequences involved are shown in Table 1 below:

[0013] Table 1 – Sequence information related to insect-resistant proteins

[0014]

[0015]

[0016]

[0017]

[0018]

[0019] A third aspect of the present invention is to provide a biological material associated with any of the above-described insect-resistant proteins or with any of the above-described nucleic acid molecules, comprising a recombinant vector, recombinant microorganism, or recombinant cell line containing any of the above-described nucleic acid molecules.

[0020] Furthermore, the recombinant vector is the pET28a expression vector, which can also be replaced by other suitable expression vectors commonly used in the art.

[0021] Furthermore, the recombinant cell line is the Escherichia coli BL21 cell line, but other suitable strains of Escherichia coli cell lines may also be used.

[0022] A fourth aspect of the present invention is to provide a method for preparing any of the above-mentioned insect-resistant proteins, comprising the steps of: modifying the nucleic acid sequence encoding the Cry1Ia protein as described in SEQ ID NO.2 according to the corresponding mutation site by homologous recombination (e.g., continuous error-prone PCR strategy), constructing the mutated nucleic acid molecule into the pET28a expression vector, and transforming it into the Escherichia coli BL21 cell line for expression of the modified Cry1Ia protein.

[0023] Furthermore, when designing the nucleic acid sequence encoding the Cry1Ia protein, its codons were set to be E. coli (K12 strain) preferred and to avoid XhoI and HindIII restriction sites; the mutated nucleic acid molecule was cloned into the pET28a expression vector between the restriction endonuclease XhoI and HindIII sites to obtain the protein expression vector.

[0024] Further, the specific steps for protein expression in the *E. coli* BL21 cell line include: inoculating a single colony into LB liquid medium and culturing until the medium becomes turbid; adding IPTG (Isopropyl-β-D-thiogalactoside) to the bacterial culture and continuing culturing; adding loading buffer to the obtained bacterial culture for electrophoresis; comparing the results of the negative control and the IPTG-induced culture to determine whether expression has occurred; inoculating the expressed bacterial culture into LB liquid medium to obtain a seed culture; inoculating the seed culture into LB liquid medium again and culturing; adding IPTG to the obtained culture medium to precipitate *E. coli* cells; discarding the supernatant and collecting the precipitate; adding buffer to the precipitate; sonicating and centrifuging; and detecting whether the supernatant contains recombinant protein.

[0025] A fifth aspect of the present invention is to provide an application of any of the above-mentioned insect-resistant proteins, any of the above-mentioned nucleic acid molecules or any of the above-mentioned biological materials, selected from at least one of the following applications: application in insect resistance, application in the preparation of insect-resistant agents, application in the cultivation of insect-resistant crops.

[0026] Furthermore, the insect-resistant crop is a genetically modified crop resistant to the corn borer.

[0027] Furthermore, the insect-resistant substance has insecticidal activity against the corn borer.

[0028] Furthermore, the genetically modified crop is corn.

[0029] Compared with the prior art, the present invention, by adopting the above technical solution, has the following beneficial effects:

[0030] This invention obtains a novel insecticidal protein (mutated Cry1Ia protein) with high insecticidal activity through random mutation and insecticidal activity screening. By modifying the toxic region of the insecticidal protein, the insecticidal activity of the protein is further enhanced. It can be used to cultivate insect-resistant crops. Experiments have verified that the above recombinant protein can be expressed in crops such as corn, so it can be used to cultivate transgenic crops resistant to corn borer, and can also be used to prepare novel insecticides to control corn borer. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify the source are all commercially available raw materials. The equipment used in each step of the following embodiments are all conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0032] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to specific embodiments, but this is not intended to limit the scope of the invention.

[0033] The following definitions and methods are provided in this embodiment of the invention to better define this application and to guide those skilled in the art in its practice. Unless otherwise stated, the terms are to be understood in accordance with their conventional usage by those skilled in the art. All patent documents, academic papers, industry standards, and other publicly available publications cited herein are incorporated herein by reference in their entirety.

[0034] As used herein, “plant” means any plant, including the whole plant, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which the plant can regenerate, plant callus, and intact plant cells in a plant or plant part, such as embryo, pollen, ovule, seed, leaf, flower, branch, fruit, stem, root, root tip, anther, etc. Unless otherwise indicated, nucleic acids are written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in an amino to carboxyl direction. Amino acids may be represented herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature. Similarly, nucleotides may be represented by commonly accepted single-letter codes. Numerical ranges include numbers that define the range. As used herein, “nucleic acid” includes deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form, and unless otherwise limited, includes known analogs (e.g., peptide nucleic acids) that have the basic properties of natural nucleotides and hybridize with single-stranded nucleic acids in a manner similar to that of naturally occurring nucleotides. As used herein, the term “encoding” or “encoded” in the context of a particular nucleic acid means that the nucleic acid contains the essential information that guides the translation of the nucleotide sequence into a particular protein. Codons are used to represent the information encoding the protein. As used herein, “full-length sequence” referring to a particular polynucleotide or the protein it encodes means the entire nucleic acid sequence or the entire amino acid sequence having a natural (non-synthetic) endogenous sequence. The full-length polynucleotide encodes the full-length, catalytically active form of the particular protein. The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. This term is used for amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. This term is also used for naturally occurring amino acid polymers. The terms “residue,” “amino acid residue,” or “amino acid” are used interchangeably herein to refer to an amino acid incorporated into a protein, polypeptide, or peptide (collectively, “protein”). Amino acids can be naturally occurring amino acids and, unless otherwise limited, may include known analogs of natural amino acids that can function in a similar manner to naturally occurring amino acids.

[0035] The term "trait" refers to the physiological, morphological, biochemical, or physical characteristics of a plant or a particular plant material or cell. In some cases, this trait is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch, or oil content of seeds or leaves, or by observing metabolic or physiological processes, such as by measuring tolerance to water deprivation or specific salt, sugar, or nitrogen concentrations, or by observing the expression levels of one or more genes, or by agronomic observations such as tolerance to osmotic stress or yield.

[0036] “Transgenic” means any cell, cell line, callus, tissue, plant part, or plant whose genome has been altered by the presence of a heterologous nucleic acid, such as a recombinant DNA construct. As used herein, the term “transgenic” includes those initial transgenic events and those resulting from those events through sexual hybridization or asexual reproduction, and does not cover genomic (chromosomal or extrachromosomal) alterations made through conventional plant breeding methods or through naturally occurring events such as random cross-fertilization, infection with a non-recombinant virus, transformation by a non-recombinant bacteria, non-recombinant transposition, or spontaneous mutation.

[0037] In this application, the terms "comprising," "including," or variations thereof should be understood to include other elements, numbers, or steps besides those described. "Test plant" or "test plant cell" refers to a plant or plant cell in which genetic modification has taken effect, or a progeny cell of such a modified plant or cell containing the modification. "Control" or "control plant" provides a reference point for measuring phenotypic changes in the test plant.

[0038] Negative or control plants may include, for example: (a) wild-type plants or cells, i.e., plants or cells with the same genotype as the genetic modification starting material, the genetic modification producing the test plants or cells; (b) plants or plant cells with the same genotype as the starting material but transformed with an empty construct (i.e., a construct with no known effect on the target trait, such as a construct containing the target gene); (c) plants or plant cells that are non-transformed isomers of the test plants or plant cells; (d) plants or plant cells that are genetically identical to the test plants or plant cells but not exposed to conditions or stimuli that would induce the expression of the target gene; or (e) the test plants or plant cells themselves, which are under conditions where the target gene is not expressed.

[0039] Those skilled in the art will readily recognize that advances in molecular biology, such as site-specific and random mutagenesis, polymerase chain reaction methods, and protein engineering techniques, have provided a wide range of appropriate tools and procedures for modifying or engineering the amino acid sequences and potential gene sequences of proteins of interest in agriculture.

[0040] In some embodiments, the nucleotide sequence of this application may be modified to perform conserved amino acid substitutions. Principles and examples of conserved amino acid substitutions are further described below. In some embodiments, the nucleotide sequence of this application may be substituted without altering the amino acid sequence according to disclosed monocotyledonous codon preferences; for example, a codon encoding the same amino acid sequence may be replaced with a codon preferred by monocotyledons without changing the amino acid sequence encoded by the nucleotide sequence. In some embodiments, a portion of the nucleotide sequence in this application may be replaced with a different codon encoding the same amino acid sequence, thereby changing the nucleotide sequence without altering the encoded amino acid sequence. Conserved variants include those sequences that encode an amino acid sequence of one of the proteins of the embodiments due to genetic codon degeneracy. In some embodiments, a portion of the nucleotide sequence in this application may be replaced according to a codon preferred by monocotyledons. Those skilled in the art will recognize that amino acid additions and / or substitutions are generally based on the relative similarity of amino acid side-chain substituents, such as the hydrophobicity, charge, size, etc., of the substituents. Exemplary amino acid substituents having the various properties considered above are well known to those skilled in the art and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the target protein can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC) (incorporated herein by reference). Conserved substitutions, such as replacing one amino acid with another amino acid having similar properties, can be performed. Sequence identity verification includes hybridization techniques. For example, a known nucleotide sequence, in whole or in part, can be used as a probe for selective hybridization with other corresponding nucleotide sequences present in cloned genomic DNA fragments or cDNA fragment groups (i.e., genomic libraries or cDNA libraries) from a selected organism. The hybridization probe may be a genomic DNA fragment, cDNA fragment, RNA fragment, or other oligonucleotide, and may be labeled with a detectable group such as 32P or other detectable markers. Thus, for example, hybridization probes can be prepared by labeling synthetic oligonucleotides based on sequences from the embodiment. Methods for preparing hybridization probes and constructing cDNA and genomic libraries are generally known in the art. Hybridization of the sequences can be performed under stringent conditions. As used herein, the terms "stringent conditions" or "stringent hybridization conditions" refer to conditions under which the probe will hybridize with its target sequence to a detectable extent (e.g., at least 2, 5, or 10 times the background) relative to hybridization with other sequences.Harsh conditions are sequence-dependent and vary across different environments. By controlling hybridization harshness and / or washing conditions, target sequences 100% complementary to the probe can be identified (homologous probe method). Alternatively, harsh conditions can be adjusted to allow for some sequence mismatches in order to detect lower similarities (heterologous probe method). Typically, probe lengths are less than about 1000 or 500 nucleotides. Typically, harsh conditions are those where the salt concentration is less than about 1.5 M Na ions at pH 7.0 to 8.3, typically about 0.01 M to 1.0 M Na ion concentration (or other salts), and the temperature conditions are: at least about 30 °C for short probes (e.g., 10 to 50 nucleotides) and at least about 60 °C for long probes (e.g., greater than 50 nucleotides). Harsh conditions can also be achieved by adding a destabilizing agent such as formamide. Exemplary low-threshold conditions include hybridization at 37°C using 30% to 35% formamide buffer, 1M NaCl, and 1% SDS (sodium dodecyl sulfate), followed by washing at 50°C to 55°C in 1× to 2× SSC (20× SSC = 3.0M NaCl / 0.3M trisodium citrate). Exemplary medium-threshold conditions include hybridization at 37°C using 40% to 45% formamide, 1.0M NaCl, and 1% SDS, followed by washing at 55°C to 60°C in 0.5× to 1× SSC. Exemplary high-threshold conditions include hybridization at 37°C using 50% formamide, 1M NaCl, and 1% SDS, followed by a final wash at 60°C to 65°C in 0.1× SSC for at least about 20 minutes. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. Hybridization duration is typically less than about 24 hours, typically from about 4 hours to about 12 hours. Specificity typically depends on post-hybridization washing, with key factors being the ionic strength and temperature of the final washing solution. The Tm (thermodynamic melting point) of DNA-DNA hybrids can be approximated by the formula from Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm = 81.5℃ + 16.6 (logM) + 0.41 (%GC) - 0.61 (%formamide) - 500 / L; where M is the molar concentration of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, "formamide%" is the percentage of formamide in the hybridization solution, and L is the base pair length of the hybrid. Tm is the temperature at which 50% of the complementary target sequence hybridizes with a perfectly matched probe (at a given ionic strength and pH). Washing is typically performed at least until equilibration is reached and a low hybridization background level is achieved, such as for 2 hours, 1 hour, or 30 minutes. Each 1% mispairing should lower Tm by approximately 1°C; therefore, Tm, hybridization, and / or washing conditions can be adjusted to hybridize with the desired sequence of homology. For example, if a sequence with ≥90% homology is required, Tm can be lowered by 10°C.Typically, the stringency conditions are selected to be approximately 5°C lower than the Tm of the specific sequence and its complementary sequence at the defined ionic strength and pH. However, under very stringent conditions, hybridization and / or washing can be performed at 4°C lower than the Tm; under moderately stringent conditions, hybridization and / or washing can be performed at 6°C lower than the Tm; and under low stringency conditions, hybridization and / or washing can be performed at 11°C lower than the Tm.

[0041] In some embodiments, a fragment of a nucleotide sequence and the amino acid sequence it encodes is also included. As used herein, the term "fragment" refers to a portion of the nucleotide sequence of a polynucleotide of an embodiment or a portion of the amino acid sequence of a polypeptide. A fragment of the nucleotide sequence may encode a protein fragment that retains the biological activity of the native or corresponding full-length protein and thus has protein activity. Mutant proteins include biologically active fragments of native proteins containing consecutive amino acid residues that retain the biological activity of the native protein. Some embodiments also include transformed plant cells or transgenic plants containing a nucleotide sequence of at least one embodiment. In some embodiments, plants are transformed using an expression vector containing a nucleotide sequence of at least one embodiment and a promoter operatively linked thereto that drives expression in plant cells. Transformed plant cells and transgenic plants represent plant cells or plants whose genome contains a heteropolynucleotide. Generally, the heteropolynucleotide is stably integrated into the genome of the transformed plant cell or transgenic plant to pass the polynucleotide to offspring. The heteropolynucleotide may be integrated into the genome alone or as part of an expression vector. In some embodiments, the plants involved in this application include plant cells, plant protoplasts, plant cell tissue cultures capable of regenerating plants, plant callus, plant masses, and plant cells that are whole plants or parts of plants, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, kernels, ears, rachis, husks, straw, roots, root tips, anthers, etc. This application also includes plant cells, protoplasts, tissues, callus, embryos, flowers, stems, fruits, leaves, and roots derived from transgenic plants of this application or their progeny, and thus at least partially containing the nucleotide sequences of this application.

[0042] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of this application. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al.'s *Molecular Cloning: A Laboratory Manual* (Sambrook J & Russell DW, 2001), or according to the conditions recommended in the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are all commercially available conventional reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0043] Example 1 - Screening for protein mutation sites

[0044] In this embodiment, Cry1Ie1 protein was used as a template for amino acid mutation modification. It is generally believed that domain I of Bt protein is involved in the formation of the midgut canal in insects and determines toxicity, while domains II and III determine the specific binding of the protein to the receptor. Amino acids 1-648 of Cry1Ie1 protein (sequence shown in SEQ ID NO.9) constitute its core insecticidal region, including domains I-III. To improve the insecticidal activity of Cry1Ie1 protein, the modified region was mainly concentrated in domain I, i.e., amino acids 58-285.

[0045] Based on the sequence shown in SEQ ID NO.9, a nucleic acid sequence encoding this sequence was designed (using the online tool http: / / www.friendbio.com / codon.html), with codons set to be E. coli (K12 strain) preferred and avoiding XhoI and HindIII restriction sites. A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.9 was obtained, and the sequence of the nucleic acid molecule is shown in SEQ ID NO.10. Further, the sequence shown in SEQ ID NO.10 was mutated using a sequential error-prone PCR strategy to obtain the mutated nucleic acid molecule.

[0046] A total of 158 mutant nucleic acid molecules were obtained. These, along with unmutated nucleic acid molecules, were cloned into the pET28a expression vector between the restriction endonuclease XhoI and HindIII sites to obtain a protein expression vector. This vector was then transformed into the *E. coli* BL21 cell line for protein expression. Specific steps:

[0047] A single colony was inoculated into 0.5 mL of LB liquid medium and cultured at 37°C for 4 h until the medium became turbid. 100 μL of the bacterial culture was then inoculated with IPTG (Isopropyl-β-D-thiogalactoside) to a final concentration of 0.8 mM. 100 μL of the bacterial culture was also used as a negative control. The culture was continued for another 4 h. 25 μL of loading buffer was added to the 100 μL of bacterial culture for electrophoresis. The results of the negative control and the IPTG-induced colony were compared to determine whether expression was present. For colonies showing expression, the remaining 20 μL was inoculated into 2 mL of LB liquid medium and cultured at 37°C for 12-16 h as a seed culture. This seed culture was then inoculated into 250 mL of LB liquid medium until OD600 = 0.5-0.6. IPTG (Isopropyl-β-D-thiogalactoside) was then added to a concentration of 0.8 mM, and the culture was continued under the same conditions for 4 h. The culture was centrifuged at 5000 g for 10 minutes to precipitate the *E. coli* cells. The supernatant was discarded, and the precipitate was collected. Add 30 mL of 20 mM Tris-50 mM NaCl buffer to the precipitate and sonicate. After centrifugation, check the supernatant for recombinant proteins. A total of 141 recombinant proteins were obtained. Some expression vectors could not yield soluble proteins, possibly due to mutations affecting normal protein expression or folding.

[0048] The 141 recombinant proteins obtained were subjected to insecticidal activity testing. 50 μL of each insecticidal protein was plated onto the surface of a 24-well plate containing approximately 1 mL of artificial insect feed. Newly hatched first-instar larvae of the corn borer (Ostrinia furnacalis) were then fed the plates for insecticidal activity determination. The insecticidal rate was calculated after 7 days of rearing. Tris-HCl buffer was used as a blank control, the pET28a empty vector expression product as a negative control, and Cry1Ab protein as a positive control.

[0049] The results showed that 132 of the 141 recombinant proteins had insecticidal activity comparable to or weaker than that of the unmutated Cry1Ie1, while 9 recombinant proteins showed significantly improved insecticidal activity compared to the unmutated Cry1Ie1. Further testing yielded 6 recombinant proteins with higher activity. The testing method was as follows:

[0050] Bioassays were performed using a surface coating method. Approximately 1 mL of unconsolidated artificial feed (about 0.5 g) was first added to a 24-well plate, gently shaken to spread the feed evenly across the bottom of the wells, and allowed to solidify. Then, protein solutions of different concentrations (25 μL / well) were added, gently shaken to evenly spread the solution on the feed surface, and allowed to air dry in a fume hood for 1 hour. Five concentration gradients (0, 0.5, 10, 25, 50 μg / g) and a blank control (buffered buffer) were included. Each treatment contained 24 newly hatched larvae (hatching time 2–12 hours), with three replicates. Larvae were cultured in a rearing room at 25 ± 2℃, a photoperiod of 14:10 (L:D) h, and a relative humidity of 50–70%. Mortality was assessed after 7 days. Larvae were considered dead if they did not move when their tails were lightly touched with a brush; larvae not reaching the second instar were also considered dead.

[0051] Calculate the mortality rate and adjusted mortality rate using the following formulas, and calculate the LC50 value using GraphPad.

[0052]

[0053]

[0054] The six Cry1Ie1 recombinant proteins with high activity are: 1) I82V+S99N+L111I+K147G+N214S; 2) D233N; 3) E182K; 4) D113S+E182V; 5) I82S+S99D; 6) D233Y. Their insecticidal activities are shown in Table 2.

[0055] Table 2 - Insecticidal activity of Cry1Ie1 mutant protein

[0056]

[0057] "*" indicates a significant difference compared to the non-mutated protein control (α = 0.05). 1: Unit: μg / g.

[0058] Example 2 - Mutation of the target protein Cry1Ia

[0059] Because Cry1Ia1 and Cry1Ie1 are similar, this embodiment further compared the sequences of Cry1Ia1 and Cry1Ie1 to identify the seven mutation sites (I82, S99, D113, K147, E182, N214, D233) in Cry1Ia that correspond to Cry1Ie1. These sites were modified using homologous recombination on the nucleic acid sequence of Cry1Ia1 (as shown in SEQ ID NO.2). The mutated nucleic acid molecules were then constructed into the pET28a expression vector according to the method in Example 1, and transformed into the *E. coli* BL21 cell line for protein expression. Ultimately, a total of 12 recombinant proteins were obtained. Some expression vectors could not yield soluble proteins, possibly due to mutations affecting normal protein expression or folding.

[0060] The 12 recombinant proteins were tested for insecticidal activity. Specifically, the insecticidal proteins were purified and quantified to 0.02 mg / mL. 50 μL of each protein was plated into a 24-well plate containing approximately 1 mL of artificial insect feed. The plate was then stored overnight at 10°C to allow the protein to infiltrate the feed. The next day, newly hatched larvae of a susceptible corn borer (Ostrinia furnacalis) were introduced for insecticidal activity determination. Each protein was tested in duplicate, with 10 insects per replicate. The insecticidal rate of each protein was calculated after 7 days of rearing. PBS buffer was used as a blank control, the pET28a empty vector expression product as a negative control, and Cry1Ia protein as a parallel control.

[0061] The results showed that the insecticidal activity of 3 out of 12 recombinant proteins was significantly improved compared with that of the unmutated Cry1Ia (Table 3). These 3 Cry1Ia recombinant proteins contained the following mutations: 1) Y233N; 2) E182K; 3) N113S+E182V.

[0062] Table 3 - Insecticidal activity of Cry1Ia mutant protein

[0063]

[0064] Example 3 - Insecticidal activity test of mutant protein Cry1Ia

[0065] This embodiment further determines the half-lethal concentration (LC50) of the three recombinant Cry1Ia proteins (Y233N, E182K, and N113S+E182V) obtained in Example 2 and compares them with Cry1Ab and Cry1Ia. The test method is as follows:

[0066] Bioassays were performed using a surface coating method. Approximately 1 mL of unconsolidated artificial feed (about 1 g) was first added to a 24-well plate. After the feed solidified, protein solutions of different concentrations (50 μL / well) were added. The plate was gently shaken to evenly spread the solution on the feed surface. It was then stored overnight at 10°C to allow the protein to penetrate the feed. The next day, the plate was air-dried in a fume hood for 1 hour. After the surface moisture was dried, newly hatched corn borer larvae of a sensitive strain were inoculated for insecticidal activity assays. Five concentration gradients (0.01, 0.1, 0.5, 1, 2 μg / g) and a blank control (buffered buffer) were set up. Each treatment was inoculated with 24 newly hatched larvae (hatching time 2–12 hours), with three replicates. The larvae were cultured in a rearing room at 25±2°C, a photoperiod of 14:10 (L:D) h, and a relative humidity of 50–70%. Mortality was assessed after 7 days. If you gently touch the tail of the larva with a brush and the larva does not move, it is considered dead. Larvae that have not developed to the second instar are also considered dead.

[0067] Calculate the mortality rate and adjusted mortality rate using the following formulas, and use SSPS to calculate the LC50 value.

[0068]

[0069]

[0070] The insecticidal activity results of the mutant protein Cry1Ia are shown in Table 4, which indicates that the three recombinant proteins exhibit good insecticidal activity.

[0071] Table 4 - Insecticidal activity of Cry1Ia mutant protein

[0072]

[0073] * indicates a significant difference compared to the non-mutated protein control (α = 0.05). 1: Unit: μg / g.

[0074] The three Cry1Ia recombinant proteins (Y233N, E182K, N113S+E182V) in the above embodiments can be expressed in crops such as corn, thereby being used to breed transgenic crops resistant to corn borers, or to prepare novel insecticides to control corn borers.

[0075] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. An insect-resistant protein, characterized in that, The insect-resistant protein is a modified Cry1Ia protein; wherein the modified Cry1Ia protein is based on the amino acid sequence shown in SEQ ID NO. 1 and undergoes the following mutations: Y233N, E182K or N113S+E182V.

2. A nucleic acid molecule for encoding the insect-resistant protein as described in claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that, The amino acid sequence of the nucleic acid molecule is as shown in any one of SEQ ID No. 3, SEQ ID No. 4, or SEQ ID No.

5.

4. A biomaterial related to the insect-resistant protein of claim 1 or the nucleic acid molecule of any one of claims 2-3, characterized in that, The biomaterial is a recombinant vector or recombinant cell line containing nucleic acid molecules as described in any one of claims 2 to 3.

5. The biomaterial according to claim 4, characterized in that, The recombinant vector is the pET28a expression vector, and the recombinant cell line is the Escherichia coli BL21 cell line.

6. A method for preparing the insect-resistant protein as described in claim 1, characterized in that, The steps include: modifying the nucleic acid sequence of Cry1Ia, as described in SEQ ID NO. 2, by homologous recombination at the mutation site described in claim 1; constructing the mutated nucleic acid molecule into the pET28a expression vector; and transforming it into the Escherichia coli BL21 cell line for expression of the modified Cry1Ia protein.

7. The application of an insect-resistant protein as described in claim 1, a nucleic acid molecule as described in any one of claims 2-3, or a biomaterial as described in any one of claims 4-5, characterized in that, The application is selected from at least one of the following applications: application in insect resistance, application in the preparation of insect-resistant agents, and application in the cultivation of insect-resistant crops; The insect-resistant crop is a genetically modified crop resistant to corn borer, and the insect resistance means that it has insecticidal activity against corn borer; the crop is corn.

Images