CN116284280B

CN116284280B - Insect-resistant protein and preparation method and application thereof

Info

Publication number: CN116284280B
Application number: CN202310581628.4A
Authority: CN
Inventors: 张玉静; 旷乐; 王晖; 王营; 左丹; 李晨
Original assignee: Wuhan Laiken Boao Technology Co ltd; Laiken Biotechnology Hainan Co ltd
Current assignee: Wuhan Laiken Boao Technology Co ltd; Laiken Biotechnology Hainan Co ltd
Priority date: 2023-05-23
Filing date: 2023-05-23
Publication date: 2023-08-08
Anticipated expiration: 2043-05-23
Also published as: CN116284280A

Abstract

The invention discloses a preparation method and application of a novel insect-resistant protein, and belongs to the field of genetic engineering. The invention provides a novel insect-resistant protein and a coding gene thereof, wherein the novel protein is an insect-resistant mutant protein, and the insect-resistant mutant protein is a protein in which a Cry1Ie2 protein is mutated at 233 th position. The application is the application of the coding gene in cultivating insect-resistant transgenic corn plants, the application in preparing insect-resistant mutant proteins and the application of the insect-resistant mutant proteins in preparing pesticides. The invention improves the activity of Cry1Ie2 protein by artificial modification.

Description

Insect-resistant protein and preparation method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an insect-resistant protein, a preparation method and application thereof, and specifically relates to a modified Cry1Ie2 protein.

Background

Currently, among the commercialized transgenic insect-resistant corn, corn containing Cry1Ab proteins (e.g., MON810, DBN 9936) has good corn borer control effects. However, with the gradual expansion of the industrialized planting scale of transgenic insect-resistant corn, the development of resistance of corn borers to Cry1Ab corn has been reported abroad, and the policy of commercialization of transgenic corn in China is also gradually opened. In order to make resistance management of corn borers in advance, development of insect-resistant corn without Cry1A cross resistance is one of effective management means. Studies have shown that Cry1I has no Cross resistance with Cry1Ab, and can delay the development of corn borer resistance by replacing or overlapping with the protein (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.).

In the case of the Cry Bt insect-resistant gene (Bacillus thuringiensis)bacillus thuringiensisGenes, abbreviated Bt genes), cry1 class I proteins are unique in that they are typically silent genes in bacillus thuringiensis strains, but can be expressed in the form of a protoxin of about 81 kDa in e. Furthermore, target insect species for Cry1 class I proteins include lepidopteran and coleopteran, and do not develop cross-resistance with Cry1 class a proteins. Therefore, the research of Cry1I genes has more important theoretical significance and practical value, and provides a new gene selection for solving the problems of narrow insecticidal spectrum of Bt toxins, drug resistance of pests and the like. However, researches and performance verification of Cry1I genes in insect resistance are reported in the prior literature.

However, the insecticidal activity of Cry1Ie2 against corn borer is still poorer than that of Cry1Ab, so that transgenic corn developed by utilizing Cry1Ie2 has a certain insecticidal activity, but the resistance effect is not ideal, and the planting requirement is difficult to reach. The transformation of the toxic region of the insecticidal protein can enhance the insecticidal activity of the protein, so that if the insecticidal activity of corn borer is better and the novel protein which has no cross resistance with Cry1Ab is obtained by transforming Cry1Ie2 protein, the novel protein has good application prospect in development of insect-resistant corn.

Disclosure of Invention

In order to overcome at least one problem in the prior art, the inventor of the application improves the insecticidal activity of the Cry1Ie2 protein through a great amount of experiments and repeated fumbling, and expands the application range of the Cry1Ie2 protein.

The inventors found that Cry1Ie2 was not significant in activity against corn borers, whereas modification of the toxic region of the insecticidal protein could enhance the insecticidal activity of the protein. Therefore, the invention obtains better corn borer insecticidal activity by modifying Cry1Ie2 protein and is applied to the development of insect-resistant corn, and has good application prospect.

The inventor carries out mutation on nucleic acid molecules encoding Cry1Ie1 protein by a continuous error-prone PCR method, expresses the mutant protein, screens out mutant protein with higher insecticidal activity by a corn borer biological test, analyzes the nucleotide sequence of the encoded protein, and finds out mutation sites. Because the proteins belong to Cry1Ie class, on the basis of the research, the invention finds homologous active sites in Cry1Ie2 and makes mutation, and then 5 recombinant proteins superior to Cry1Ie2 are screened out through corn borer bioassay.

In order to achieve the above purpose, the invention adopts the following technical scheme:

a first aspect of the present invention provides an insect-resistant protein that is an engineered Cry1Ie2 protein; wherein the engineered Cry1Ie2 protein contains the amino acid sequence shown in SEQ ID NO. 1 and is mutated at least one of the following sites: 233 th bit.

In one embodiment, the amino acid at position 233 is mutated to a non-D amino acid, e.g., a, V, G, L, E, F, W, Y, N, S, Q, K, M, T, C, P, H, R, I; preferably, the mutation is Y or N.

Further, the engineered Cry1Ie2 protein has a mutation site that further comprises: 182 th bit; still further, the engineered Cry1Ie2 proteins have a mutein combination of: 1) D233Y; 2) D233N; 3) E182K+D233N; 4) E182N+D233Y;

further, the amino acid sequence of the modified Cry1Ie2 protein D233Y is shown in the SEQ ID No. 3.

In a second aspect, the invention provides a nucleic acid molecule for encoding any of the above 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.

Further, the nucleic acid sequences encoding the amino acid sequences shown in SEQ ID No.3 are shown in SEQ ID No. 4 respectively;

the amino acid and nucleic acid sequences referred to above are shown in Table 1 below:

TABLE 1 sequence information relating to insect-resistant proteins

In a third aspect, the invention provides a biological material associated with any of the above insect-resistant proteins or with any of the above nucleic acid molecules, comprising a recombinant vector, recombinant microorganism, recombinant cell line comprising a nucleic acid molecule as described in any of the above.

Further, the recombinant vector is a pET28a expression vector, which may be replaced with other suitable expression vectors conventionally used in the art.

Further, the recombinant cell line is an E.coli BL21 cell line, and suitable prokaryotic expression cell lines of other strain types can also be used.

The fourth aspect of the present invention provides a method for preparing any one of the above insect-resistant proteins, comprising the steps of: on a nucleic acid sequence of the coded Cry1Ie2 protein shown in SEQ ID NO. 2, modifying the nucleic acid sequence according to a corresponding mutation site by a homologous recombination method, constructing a mutated nucleic acid molecule into a pET28a expression vector, and transferring into an escherichia coli BL21 cell line to express the modified Cry1Ie2 protein.

Further, when designing a nucleic acid sequence encoding a Cry1Ie2 protein, its codon was set to e.coli (K12 strain) preference and XhoI and HindIII cleavage sites were avoided; cloning the mutated nucleic acid molecule between the sites of restriction enzymes XhoI and HindIII in the pET28a expression vector to obtain the protein expression vector.

Further, the specific steps of protein expression in the E.coli BL21 cell line include: inoculating a single colony to an LB liquid culture medium, culturing until the culture medium is turbid, taking bacterial liquid, adding IPTG (isopopyl-beta-D-thiogalactide), continuously culturing, adding a sample buffer solution into the bacterial liquid, preparing samples, electrophoresis, and judging whether the expression exists according to the comparison of a negative control and the result induced by adding IPTG; inoculating the expressed bacterial liquid into an LB liquid culture medium for culture to obtain seed liquid, inoculating the seed liquid into the LB liquid culture medium for culture, and then adding IPTG for continuous culture; the obtained culture solution is subjected to centrifugal precipitation of escherichia coli cells, and then the supernatant is discarded to collect the precipitate; adding buffer solution into the precipitate, ultrasonic crushing, centrifuging, and detecting whether the supernatant contains recombinant protein.

In a fifth aspect, the present invention provides the use of any one of the above-described insect-resistant proteins, any one of the above-described nucleic acid molecules or any one of the above-described biological materials, selected from at least one of the following: the application of the plant extract in insect resistance, the application in preparing insect-resistant preparations and the application in cultivating insect-resistant crops.

Further, the insect-resistant crop is a transgenic crop resistant to corn borers.

Further, the insect-resistant agent has insecticidal activity to corn borers.

Further, the transgenic crop is maize.

Compared with the prior art, the invention has the following beneficial effects by adopting the technical scheme:

according to the invention, through random mutation and insecticidal activity screening, a novel insecticidal protein (mutated Cry1Ie2 protein) with high insecticidal activity is obtained, the insecticidal activity of the protein is further enhanced through modification of a toxic region of the insecticidal protein, the insecticidal protein can be used for cultivating insect-resistant crops, and experimental verification proves that: the recombinant protein can be expressed in crops such as corn, so that the recombinant protein can be used for cultivating transgenic crops resistant to corn borers, and can also be used for preparing novel pesticides to prevent and treat corn borers.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The experimental procedures, which are not specified in the following examples, are generally determined according to national standards. The experimental materials not shown in the examples below are all commercially available. The equipment used in each step in the following examples is conventional equipment. If the corresponding national standard does not exist, the method is carried out according to the general international standard, the conventional condition or the condition recommended by the manufacturer. Unless otherwise indicated, all parts are parts by weight and all percentages are percentages by mass. Unless defined or otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any method and material similar or equivalent to those described may be used in the methods of the present invention.

It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The invention will be further illustrated, but is not limited, by the following examples.

The following definitions and methods are provided in the embodiments of the present invention to better define the present application and to guide those of ordinary skill in the art in the practice of the present application. Unless otherwise indicated, terms are to be construed according to conventional usage by those of ordinary skill in the relevant art. All patent documents, academic papers, industry standards, and other publications cited herein are incorporated by reference in their entirety.

As used herein, a "plant" is any plant, including whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, whole plant cells in plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, and the like. Unless otherwise indicated, nucleic acids are written in the 5 'to 3' direction from left to right; the amino acid sequence is written in the amino to carboxyl direction from left to right. Amino acids may be represented herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB biochemical nomenclature committee. Likewise, nucleotides may be referred to by commonly accepted single letter codes. The numerical range includes the numbers defining the range. As used herein, "nucleic acid" includes reference to deoxyribonucleotide or ribonucleotide polymers in either single-or double-stranded form, and unless otherwise limited, includes known analogs (e.g., peptide nucleic acids) having the basic properties of natural nucleotides that hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides. As used herein, the term "encode" or "encoded" when used in the context of a particular nucleic acid, means that the nucleic acid contains the necessary information to direct translation of the nucleotide sequence into a particular protein. The information encoding the protein is represented using codons. As used herein, reference to a "full-length sequence" of a particular polynucleotide or protein encoded thereby refers to an entire nucleic acid sequence or an 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 "polypeptide", "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The 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 acid. The term is also used for naturally occurring amino acid polymers. The terms "residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively, "protein"). Amino acids may be naturally occurring amino acids, and unless otherwise limited, may include known analogs of natural amino acids, which analogs may function in a similar manner to naturally occurring amino acids.

The term "trait" refers to a physiological, morphological, biochemical or physical characteristic of a plant or a particular plant material or cell. In some cases, this property 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 the seed or leaf, or by observing metabolic or physiological processes, for example by measuring tolerance to water deprivation or specific salt or sugar or nitrogen concentrations, or by observing the expression level of one or more genes, or by agronomic observations such as osmotic stress tolerance or yield.

"transgenic" refers to 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. The term "transgene" as used herein includes those initial transgenic events as well as those produced from the initial transgenic events by sexual hybridization or asexual reproduction, and does not encompass genomic (chromosomal or extrachromosomal) changes by conventional plant breeding methods or by naturally occurring events such as random fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

In this application, the terms "comprises," "comprising," or variations thereof, are to be understood to encompass other elements, numbers, or steps in addition to those described. "subject plant" or "subject plant cell" refers to a plant or plant cell in which genetic engineering has been effected, or a progeny cell of a plant or cell so engineered, which progeny cell comprises the engineering. "control" or "control plants" provide a reference point for measuring phenotypic changes in a subject plant.

Negative or control plants can include, for example: (a) Wild-type plants or cells, i.e., plants or cells having the same genotype as the genetically engineered starting material, which genetic engineering produces the subject plant or cell; (b) A plant or plant cell having the same genotype as the starting material but which has been transformed with an empty construct (i.e., with a construct that has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) A plant or plant cell that is a non-transformed isolate of the subject plant or plant cell; (d) A plant or plant cell genetically identical to the test plant or plant cell but not exposed to conditions or stimuli that induce expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed.

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, provide a wide range of suitable tools and procedures for engineering or engineering amino acid sequences and potentially genetic sequences of proteins of agricultural interest.

In some embodiments, the nucleotide sequences of the present application may be altered to make conservative amino acid substitutions. The principles and examples of conservative amino acid substitutions are described further below. In certain embodiments, the nucleotide sequences of the present application can be subjected to substitutions in accordance with the disclosed monocot codon preferences that do not alter the amino acid sequence, e.g., codons encoding the same amino acid sequence can be replaced with monocot-preferred codons without altering the amino acid sequence encoded by the nucleotide sequence. In some embodiments, a portion of the nucleotide sequence herein is replaced with a different codon encoding the same amino acid sequence, such that the amino acid sequence encoded thereby is not changed while the nucleotide sequence is changed. Conservative variants include those sequences that encode the amino acid sequence of one of the proteins of an embodiment due to the degeneracy of the genetic code. In some embodiments, a portion of the nucleotide sequences herein are substituted according to monocot preference codons. 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, e.g., hydrophobicity, charge, size, etc., of the substituents. Exemplary amino acid substituents having various of the aforementioned contemplated properties 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. Guidelines for suitable amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al (1978) Atlas of Protein Sequence and Structure (protein sequence and structure atlas) (Natl. Biomed. Res. Foundation, washington, D.C.), incorporated herein by reference. Conservative substitutions, such as substitution of one amino acid for another with similar properties, may be made. Identification of sequence identity includes hybridization techniques. For example, all or part of a known nucleotide sequence is used as a probe for selective hybridization with other corresponding nucleotide sequences present in a cloned genomic DNA fragment or population of cDNA fragments (i.e., a genomic library or cDNA library) from a selected organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32P or other detectable marker. Thus, for example, hybridization probes can be prepared by labeling synthetic oligonucleotides based on the sequences of the embodiments. Methods for preparing hybridization probes and constructing cDNA and genomic libraries are generally known in the art. Hybridization of the sequences may be performed under stringent conditions. As used herein, the term "stringent conditions" or "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target sequence to a detectably greater extent (e.g., at least 2-fold, 5-fold, or 10-fold over background) relative to hybridization to other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the hybridization stringency and/or controlling the washing conditions, target sequences 100% complementary to the probes can be identified (homologous probe method). Alternatively, stringent conditions can be adjusted to allow for some sequence mismatches in order to detect lower similarity (heterologous probe method). Typically, the probe is less than about 1000 or 500 nucleotides in length. Typically, stringent conditions are those in which the salt concentration is less than about 1.5M Na ion, typically about 0.01M to 1.0M Na ion concentration (or other salt) at a pH of 7.0 to 8.3, and the temperature conditions are: when used with short probes (e.g., 10 to 50 nucleotides), at least about 30 ℃; when used with long probes (e.g., greater than 50 nucleotides), at least about 60 ℃. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization at 37 ℃ with 30% to 35% formamide buffer, 1M NaCl, 1% sds (sodium dodecyl sulfate), washing in 1 x to 2 x SSC (20 x SSC = 3.0M NaCl/0.3M trisodium citrate) at 50 ℃ to 55 ℃. Exemplary moderately stringent conditions include hybridization in 40% to 45% formamide, 1.0M NaCl, 1% SDS at 37℃and washing in 0.5 XSSC to 1 XSSC at 55℃to 60 ℃. Exemplary high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37 ℃ and a final wash in 0.1 x SSC at 60 ℃ to 65 ℃ for at least about 20 minutes. Optionally, the wash buffer may comprise about 0.1% to about 1% sds. The duration of hybridization is typically less than about 24 hours, typically from about 4 hours to about 12 hours. Specificity generally depends on post-hybridization washing, the key factors being the ionic strength and temperature of the final wash solution. The Tm (thermodynamic melting point) of DNA-DNA hybrids can be approximated from the formula Meinkoth and Wahl (1984) Anal biochem. 138:267-284: tm=81.5 ℃ +16.6 (log) +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 a defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. Washing is typically performed at least until equilibrium is reached and a low hybridization background level is reached, such as 2 hours, 1 hour, or 30 minutes. Each 1% mismatch corresponds to a decrease in Tm of about 1 ℃; thus, tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of desired identity. For example, if sequences with ≡90% identity are desired, the Tm can be reduced by 10 ℃. Typically, stringent conditions are selected to be about 5 ℃ lower than the Tm for the specific sequence and its complement at a defined ionic strength and pH. However, under very stringent conditions, hybridization and/or washing may be performed at 4℃below the Tm; hybridization and/or washing may be performed at 6 ℃ below the Tm under moderately stringent conditions; hybridization and/or washing can be performed at 11℃below the Tm under low stringency conditions.

In some embodiments, fragments of the nucleotide sequence and the amino acid sequence encoded thereby are also included. As used herein, the term "fragment" refers to a portion of the nucleotide sequence of a polynucleotide or a portion of the amino acid sequence of a polypeptide of an embodiment. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native or corresponding full-length protein and thus have protein activity. Mutant proteins include biologically active fragments of a native protein that comprise consecutive amino acid residues that retain the biological activity of the native protein. Some embodiments also include a transformed plant cell or transgenic plant comprising the nucleotide sequence of at least one embodiment. In some embodiments, the plant is transformed with an expression vector comprising the nucleotide sequence of at least one embodiment and operably linked thereto a promoter that drives expression in a plant cell. Transformed plant cells and transgenic plants refer to plant cells or plants comprising a heterologous polynucleotide within the genome. In general, the heterologous polynucleotide is stably integrated within the genome of the transformed plant cell or transgenic plant, such that the polynucleotide is delivered to the offspring. The heterologous polynucleotide may be integrated into the genome, either alone or as part of an expression vector. In some embodiments, the plants contemplated herein include plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells, which are whole plants or parts of plants, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, nuts, ears, cobs, hulls, stalks, roots, root tips, anthers, and the like. The present application also includes plant cells, protoplasts, tissues, calli, embryos and flowers, stems, fruits, leaves and roots derived from the transgenic plants of the present application or progeny thereof, and thus comprising at least in part the nucleotide sequences of the present application.

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present application. Examples follow conventional experimental conditions, such as the MolecuLar cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecuLar cloning: a laboratory manual, 2001), or conditions recommended by the manufacturer's instructions, unless otherwise indicated. Unless otherwise indicated, all chemical reagents used in the examples were conventional commercial reagents, and the technical means used in the examples were conventional means well known to those skilled in the art.

EXAMPLE 1 screening of protein mutation sites

In this example, the Cry1Ie1 protein was used as a template for amino acid mutagenesis. It is believed that domain I of Bt proteins is involved in the formation of intestinal tracts in insects and determines toxicity, and domains II and III determine specific binding of proteins to receptors. The amino acids 1-648 of Cry1Ie1 protein (the sequence is shown as SEQ ID NO. 5) are the core insecticidal region comprising structural domains I-III, and in order to improve the insecticidal activity of Cry1Ie1 protein, the modified region is mainly concentrated in the structural domain I region, namely the amino acids 58-285.

According to the sequence shown in SEQ ID No. 5, the nucleic acid sequence encoding this sequence was designed (http:// www.friendbio.com/codon. Html. In the following in-line tool) with codons set to E.coli (K12 strain) preference and avoiding XhoI and HindIII cleavage sites. Obtaining the nucleic acid molecule of the amino acid sequence shown in SEQ ID NO. 5, wherein the sequence of the nucleic acid molecule is shown in SEQ ID NO. 6. Further, the sequence shown in SEQ ID NO. 6 was mutated using a sequential error-prone PCR (sequential error-prone PCR) strategy to obtain a mutated nucleic acid molecule.

A total of 158 mutant nucleic acid molecules were obtained, which together with the unmutated nucleic acid molecules were cloned between the sites of restriction enzymes XhoI and HindIII, respectively, in the vector pET28a expression vector, to obtain a protein expression vector. The vector is transferred into an escherichia coli BL21 cell line and protein expression is carried out. The method comprises the following specific steps:

inoculating a single colony to 0.5 mL of LB liquid medium, culturing at 37 ℃ until the culture medium is turbid, adding IPTG (isopopyl-beta-D-thiohinging) into 100 uL bacterial liquid to a final concentration of 0.8 mM, simultaneously taking 100 uL bacterial liquid as negative control, continuously culturing 4 h, adding 25 uL loading buffer into 100 uL bacterial liquid for sample preparation electrophoresis, and comparing according to the negative control and the result induced by adding IPTG, and judging whether the expression exists. The remaining 20. 20 uL with expression was inoculated into 2 mL of LB liquid medium and cultured at 37 ℃ for 12 to 16 h as seed liquid, the seed liquid was inoculated into 250 mL of LB liquid medium again to an OD 600=0.5 to 0.6, IPTG (isopopyl- β -D-thiogaside) was then added to a concentration of 0.8 mM, and the culture was continued under the same conditions for 4 hours. The culture medium was centrifuged at 5000 g for 10 minutes to pellet E.coli cells, and the supernatant was discarded to collect the pellet. The precipitate was sonicated with 30mL of 20mM Tris-50mM NaCl buffer. After centrifugation, the supernatant was checked for the presence of recombinant proteins. A total of 141 recombinant proteins were obtained, and part of the expression vector failed to obtain soluble proteins, possibly due to the effects of normal expression or folding of the proteins after mutation.

The 141 recombinant proteins obtained were tested for insecticidal activity. 50 μl of each insecticidal protein was applied to the surface of a 24-well plate to which about 1 mL insect artificial feed had been added, and corn borer (Ostrinia furnacalis) newborn first instar larvae were raised for insecticidal activity determination. After 7 days of feeding, the insecticidal rate was counted. Tris-HCl buffer was used as a blank control, pET28a empty vector product as a negative control, cry1Ab protein as a positive control.

The results show that 132 of 141 recombinant proteins have the insecticidal activity equivalent to or weaker than that of unmutated Cry1Ie1, 9 recombinant proteins have the insecticidal activity remarkably improved compared with that of unmutated Cry1Ie1, and 6 recombinant proteins with higher activity are obtained through further tests. The testing method comprises the following steps:

the biological assay is carried out by adopting a surface smearing method, firstly, about 1 mL of non-solidified artificial feed (about 0.5 g) is added into a 24-hole plate, the feed is paved on the bottom of the hole plate by slight shaking, after the feed is solidified, protein solutions (25 mu L/hole) with different concentrations are added, after the addition, the liquid medicine is evenly paved on the surface of the feed by slight shaking, and then the feed is naturally dried in a fume hood for 1 h. The experiment was set up with 5 gradient concentrations (0, 0.5, 10, 25, 50. Mu.g/g) and a blank (buffer), each treatment was inoculated with 24 first larvae (incubation time 2-12 h), 3 replicates were set up, placed at 25.+ -. 2 ℃ and photoperiod 14:10 (L: D) h, incubated in a chamber with 50-70% relative humidity, and mortality was investigated after 7 days. The tail of the larva is touched by a writing brush, the larva is regarded as dead, and the larva which does not develop 2 years is also regarded as dead.

Mortality and corrected mortality were calculated according to the following formulas, and LC50 values were calculated using graphpad.

The 6 Cry1Ie1 recombinant proteins with higher activity are respectively: 1) I82v+s99n+l111i+k147g+n214S; 2) D233N; 3) E182K; 4) D113s+e182V; 5) I680s+s99d; 6) D233Y. The insecticidal activity is shown in Table 2.

TABLE 2 insecticidal Activity of Cry1Ie1 muteins

EXAMPLE 2 mutagenesis of the target protein Cry1Ie2

Since Cry1Ie2 and Cry1Ie1 have similarity, this example further aligned the Cry1Ie2 and Cry1Ie1 sequences to find the 8 mutation sites (I82, S99, L111, D113, K147, E182, N214, D233) in Cry1Ie2 that correspond to Cry1Ie 1. On the nucleic acid sequence of Cry1Ie2 (shown as SEQ ID NO. 2), these sites were modified by homologous recombination, and the mutated nucleic acid molecule was constructed into a pET28a expression vector according to the method of example 1, and transformed into the E.coli BL21 cell line, and protein expression was performed. Finally, a total of 32 recombinant proteins were obtained, and a part of the expression vector could not obtain soluble proteins, possibly due to the effect of normal expression or folding of proteins after mutation.

The above 32 recombinant proteins were tested for insecticidal activity. The method comprises the following steps: after purification of insecticidal proteins, the insecticidal proteins were quantified to 0.02mg/mL, 50 μl of the insecticidal proteins were applied to 24-well plates to which about 1 mL insect artificial feed had been added, placed in a refrigerator at 10 ℃ for overnight storage, the proteins were allowed to soak into the feed, and the next day corn borer (Ostrinia furnacalis) initially hatched larvae of sensitive strain were inoculated for insecticidal activity determination, 2 replicates per protein test, and 24 replicates per test. After 7 days of feeding, the insecticidal rate of each protein was counted. PBS buffer was used as a blank control, pET28a empty vector product as a negative control, and Cry1Ie protein as a parallel control. 12 recombinant proteins with better activity are screened out.

The 12 Cry1Ie2 recombinant proteins with higher activity are respectively: 1) D233Y; 2) D233N; 3) E182K; 4) E182N; 5) D113s+e182V; 6) I680s+s99n; 7) I82v+s99n+k147g+n214S; 8) I680n+e182 v+d233n; 9) E182K+D233N;10 E182n+d233y;11 I680v+s99n; 12 I82s+s99n+g147 s+n214S.

The insecticidal activity is shown in Table 3.

TABLE 3 insecticidal Activity of Cry1Ie2 muteins

EXAMPLE 3 insecticidal Activity test of mutein Cry1Ie2

This example further carried out a semi-lethal concentration determination of the 12 Cry1Ie2 recombinant proteins obtained in example 2 and compared with Cry1Ab and Cry1Ie 2. The testing method comprises the following steps:

the biological assay is carried out by adopting a surface smearing method, firstly, about 1 mL of non-coagulated artificial feed (about 1 g) is added into a 24-hole plate, after the feed is coagulated, protein solutions (50 mu L/hole) with different concentrations are added, the mixture is gently shaken after the addition to uniformly spread the liquid medicine on the surface of the feed, the mixture is placed into a refrigerator with the temperature of 10 ℃ for overnight storage, the protein is immersed into the feed, the next day of natural air drying is carried out in a fume hood for 1 h, and after the moisture on the surface of the feed is dried, the corn borer initially hatched larvae of sensitive strains are inoculated for the determination of insecticidal activity. The experiment is provided with 6 gradient concentrations (0.01, 0.1, 0.5, 1, 2 and 5 mug/g) and a blank control (buffer solution), each treatment is connected with 24 first hatched larvae (hatching time is 2-12 h), 3 times of repetition are set, the treatment is placed in a pest-culturing room with the temperature of 25+/-2 ℃ and the photoperiod of 14:10 (L: D) h, the relative humidity of 50-70% for culturing, and the death rate is investigated after 7 days. The tail of the larva is touched by a writing brush, the larva is regarded as dead, and the larva which does not develop 2 years is also regarded as dead.

Mortality and corrected mortality were calculated according to the following formulas, and LC50 values were calculated using SSPS.

The insecticidal activity results of the mutant proteins Cry1Ie are shown in Table 4, which shows that 4 recombinant proteins exhibit better insecticidal activity.

Table 4 insecticidal Activity of Cry1ie2 muteins

As can be seen from table 4, the combination comprising the 233 st site mutation in the Cry1Ie2 mutein has significantly better activity than the original protein. Among them, the mutant protein Cry1Ie2-1 with 233 th amino acid mutated from aspartic acid to tyrosine has the best activity of killing corn borer.

The 4 Cry1Ie2 recombinant proteins (Cry 1Ie2-1, cry1Ie2-2, cry1Ie2-9 and Cry1Ie 2-10) in the above embodiments can be expressed in crops such as corn, so that the novel recombinant protein can be used for cultivating transgenic crops resistant to corn borers, and can also be used for preparing novel pesticides for preventing and controlling corn borers.

The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for the present invention will occur to those skilled in the art, and are also within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.

Claims

1. An insect-resistant mutein characterized in that the insect-resistant mutein comprises a mutation in the amino acid position corresponding to the amino acid sequence shown in SEQ ID No. 1 at the following amino acid position compared to the amino acid sequence of the parent insect-resistant protein:

1) D at position 233 of SEQ ID NO. 1 is mutated to Y;

2) D at position 233 of SEQ ID NO. 1 is mutated to N;

3) E at position 182 of SEQ ID NO. 1 is mutated to K and D at position 233 is mutated to N;

4) E at position 182 of SEQ ID NO. 1 is mutated to N and D at position 233 is mutated to Y.

2. A nucleic acid molecule encoding the insect-resistant mutein of any one of claims 1.

3. A vector comprising the nucleic acid molecule of claim 2 and operably linked thereto a regulatory element.

4. A recombinant cell of a prokaryote, wherein said recombinant cell comprises the nucleic acid molecule of claim 2 or the vector of claim 3.

5. Use of the insect-resistant mutein of any one of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, the prokaryotic recombinant cell of claim 4 for the preparation of an anti-corn borer preparation or cultivation of an anti-corn borer crop.