CN116410277A - Novel insect-resistant gene and application thereof - Google Patents

Abstract

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

Description

Novel insect-resistant gene and application thereof

Technical Field

The invention relates to a novel insect-resistant gene and application thereof, belonging to the field of genetic engineering.

Background

Among the Cry Bt insect-resistant genes that have been identified, cry1I proteins are unique, e.g., they are typically silent genes in Bacillus thuringiensis strains, but can be expressed in E.coli cultures as a protoxin of about 81 kDa, a unique molecular mass in Cry1 proteins. In addition, different Cry1 type I proteins have been identified as active against both lepidopteran and coleopteran pests. More importantly, insect resistance to Cry1A proteins that develop resistance does not exhibit cross resistance to Cry1I proteins (Zhao C, jurat-Funtes J L, abdelgaffar H M, et al identification of a New Cry1I-Type Gene as a Candidate for Gene Pyramiding in Corn To Control Ostrinia Species Larvae [ J ]. Appl Environ Microbiol,2015,81 (11): 3699-705.).

The Cry1Ab has good corn borer insecticidal activity, and a plurality of transgenic insect-resistant corn transformants are also developed by using the Cry1Ab (such as MON810 and DBN 9936). However, with the gradual expansion of the industrialized planting of transgenic insect-resistant corn in China, the drug resistance of corn borers to Cry1Ab proteins appears sooner or later. Superimposing new insect-resistant proteins with different insecticidal mechanisms is a better solution in a comprehensive sense. Studies have shown that Cry1Ie has no cross resistance to Cry1Ab, can be superimposed on this protein, and delays 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)

However, the insecticidal activity of Cry1Ie on corn borers is still poorer than that of Cry1Ab, so that transgenic corn developed by utilizing Cry1Ie has a certain insecticidal activity, but the resistance effect is not ideal, and the requirement of commercial planting is difficult to be met. The transformation of the toxic region of the insecticidal protein can enhance the insecticidal activity of the protein, so that the insecticidal activity of the corn borer is better as long as the Cry1Ie protein is transformed, and the novel protein which has no interaction resistance with Cry1Ab is applied to the development of insect-resistant corn, and has good commercial application prospect.

In order to solve the problems, the invention carries out mutation on a nucleic acid molecule encoding Cry1Ie1 protein by a continuous error-prone PCR method, expresses the mutant into protein, then screens the mutant protein with higher insecticidal activity by a corn borer biological test, analyzes the nucleotide sequence of the encoded protein, and finds out the mutation site.

Disclosure of Invention

The invention provides an amino acid variation site of a protein, which is characterized in that the site is positioned at any one or more of the following sites of a sequence shown in SEQ ID NO. 1: 1) I82; 2) S99; 3) D113; 4) K147; 5) E182; 6) N214; 7) And D233.

The invention also provides a protein which is characterized by comprising an amino acid sequence shown in SEQ ID NO.1 and having the following mutation: 1) I82v+s99n+l111i+k147g+n214S; 2) D233N; 3) E182K; 4) D113s+e182V; 5) I680s+s99d; 6) D233Y.

The invention also provides a nucleic acid, which is characterized in that the nucleic acid codes for the protein.

In some embodiments, the nucleotide sequence of the nucleic acid is set forth in any one of SEQ ID NO.3-SEQ ID NO. 8.

The invention also provides application of the protein and the nucleic acid in cultivation of insect-resistant crops.

In some embodiments, the crop described in the above application is corn.

The invention has the beneficial effects that: through random mutation and insecticidal activity screening, the invention obtains 6 novel insecticidal proteins with high insecticidal activity, and can be used for cultivating insect-resistant crops.

Detailed Description

The following definitions and methods are provided 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 organelles, plant protoplasts, plant cell tissue cultures from which plants can regenerate, plant callus tissue, 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 the 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 salts 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 alteration 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 may be substituted according to the disclosed monocot codon bias without altering the amino acid sequence, e.g., codons encoding the same amino acid sequence may be substituted 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 replaced 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 threonic acid; 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) than 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 probe 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 of an embodiment or a portion of the amino acid sequence of a polypeptide. 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 contiguous 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 at least partially comprising 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 mutagenesis of target protein

The invention uses Cry1Ie1 protein as a template to carry out amino acid mutation transformation. 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. 1) 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.

The sequence shown in SEQ ID No.1 was designed to encode a nucleic acid sequence of this type (http:// www.friendbio.com/codon. Html. In the following in-line tool) in which codons were set to E.coli (K12 strain) preference and XhoI and HindIII cleavage sites were avoided. A nucleic acid molecule encoding the sequence shown as SEQ ID NO.1 (the sequence is shown as SEQ ID NO. 2) is obtained. Further, the sequence shown in SEQ ID NO.2 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 in the vector pET28a expression vector, respectively, 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 0.5mL of LB liquid culture medium, culturing at 37 ℃ for 4 hours until the culture medium is turbid, adding IPTG (Isopropyl-beta-D-thiohinging) into 100uL of bacterial liquid to a final concentration of 0.8mM, simultaneously taking 100uL of bacterial liquid as negative control, continuously culturing for 4 hours, adding 25uL of loading buffer solution into 100uL of 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 20uL of the expression was inoculated into 2mL of LB liquid medium and cultured at 37℃for 12 to 16 hours as a seed liquid, the seed liquid was inoculated into 250mL of LB liquid medium again to OD600 = 0.5 to 0.6, then IPTG (isopopyl-beta-D-thiogaside) was added to a concentration of 0.8mM, and the culture was continued under the same conditions for 4 hours. The culture medium was centrifuged at 5000g 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 examined 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.

EXAMPLE 2 insecticidal Activity test of muteins

141 recombinant proteins obtained in example 1 were tested for insecticidal activity. The method comprises the following steps:

50. Mu.L of each insecticidal protein was applied to the surface of a 24-well plate to which about 1mL of artificial feed had been added, and corn borer (Ostrinia furnacalis) newborn first-instar larvae were raised for insecticidal activity determination. The insecticidal rate was counted after 7 days of feeding. 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 the 141 recombinant proteins had insecticidal activity comparable to or weaker than that of unmutated Cry1Ie1, and that 9 recombinant proteins had significantly improved insecticidal activity compared to unmutated Cry1Ie 1.

These 9 recombinant proteins were further subjected to a detailed insecticidal activity test. The testing method comprises the following steps:

the biological assay is carried out by adopting a surface smearing method, firstly, about 1mL 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, protein solutions (25 mu L/hole) with different concentrations are added after the feed is solidified, the liquid medicine is evenly paved on the surface of the feed by slight shaking after the addition, and the feed is naturally dried in a fume hood for 1h. 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 corrective mortality were calculated according to the following formulas, and LC50 values were calculated using graphpad.

Mortality (%) = (number of insects-number of surviving insects+number of insects whose age is still below 2 years)/number of insects×100 (formula 1)

The test results show that 6 recombinant proteins have better insecticidal activity. Sequencing the inserted nucleic acid molecular region on the expression vector for expressing the 6 proteins to obtain nucleotide sequences (shown as SEQ ID NO. 3-8), determining the protein amino acid sequence according to the nucleotide sequences, and comparing the protein amino acid sequence with the amino acid sequence before mutation to obtain mutation sites. The mutation sites of the above 6 mutations and the insecticidal activity after the mutation are shown in Table 1.

Table 1 insecticidal Activity of muteins

". Times." indicates a significant difference (α=0.05) relative to the unmutated protein control. 1: in μg/g.

The 6 recombinant proteins can be expressed in crops such as corn, and 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.

While the invention has been described in detail in the general context and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Sequence listing

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gttcgtatcc gttacgcttc taccaccgac ctgcagttcc acacctctat caacggtaaa 1740

gctatcaacc agggtaactt ctctgctacc atgaaccgtg gtgaagacct ggactacaaa 1800

accttccgta ccgttggttt caccaccccg ttctctttct ctgacgttca gtctaccttc 1860

accatcggtg cttggaactt ctcttctggt aacgaagttt acatcgaccg tatcgaattc 1920

gttccggttg aagttaccta cgaa 1944

<210> 3

<211> 1944

<212> DNA

<213> Synthesis (unown)

<400> 3

atgaaactga aaaacccgga caaacaccag tctctgtctt ctaacgctaa agttgacaaa 60

atcgctaccg actctctgaa aaacgaaacc gacatcgaac tgaaaaacat caaccacgaa 120

gacttcctgc gtatgtctga acacgaatct atcgacccgt tcgtttctgc ttctaccatc 180

cagaccggta tcggtatcgc tggtaaaatc ctgggtaccc tgggtgttcc gttcgctggt 240

caggtcgctt ctctgtactc tttcatcctg ggtgaactgt ggccgaaagg taaaaatcag 300

tgggaaatct tcatggaaca cgttgaagaa atcatcgacc agaaaatctc tacctacgct 360

cgtaacatcg ctctggctga cctgaaaggt ctgggtgacg ctctggctgt ttaccacgaa 420

tctctggaat cttggatcgg aaaccgtaac aacgctcgtg ctacctctgt tgttaaatct 480

cagtacatcg ctctggaact gctgttcgtt cagaaactgc cgtctttcgc tgtttctggt 540

gaagaagttc cgctgctgcc gatctacgct caggctgcta acctgcacct gctgctgctg 600

cgtgacgctt ctgttttcgg taaagaatgg ggtctgtcta gctctcagat ctctaccttc 660

tacaaccgtc aggttgaacg tacctctgac tactctgacc actgcgttaa atggtactct 720

accggtctga acaacctgcg tggtaccaac gctgaatctt gggttcgtta caaccagttc 780

cgtaaagaca tgaccctgat ggttctggac ctgatcgctc tgttcccgtc ttacgacacc 840

ctggtttacc cgatcaaaac cacctctcag ctgacccgtg aagtttacac cgacgctatc 900

ggtaccgttc acccaaatgc tagcttcgct tctaccactt ggtacaacaa caacgctccg 960

tctttctctg ctatcgaatc tgctgttgtt cgtaacccgc acctgctgga cttcctggaa 1020

caggttacca tctactctct gctgtctcgt tggtctaaca cccagtacat gaacatgtgg 1080

ggtggtcacc gtctggaatt ccgtaccatc ggtggtgttc tgaacacctc tacccagggt 1140

tctaccaaca cctctatcaa cccggttacc ctgccgttca cctctcgtga cgtttaccgt 1200

accgaatctc tggctggtct gaacctgttc ctgacccagc cggttaacgg tgttccgcgt 1260

gttgacttcc actggaaatt cgctaccctg ccgatcgctt ctgacaactt ctactacctg 1320

ggttacgctg gtgttggtac ccagctgcag gactctgaaa acgaactgcc gccggaaacc 1380

accggtcagc cgaactacga atcttactct caccgtctgt ctcacatcgg tctgatctct 1440

gcttctcacg ttaaagctct ggtttactct tggacccacc gttctgctga ccgtaccaac 1500

accatcgaac cgaactctat cacccagatc ccgctggtta aagcgttcaa cctgtcttct 1560

ggtgctgctg ttgttcgtgg tccgggtttc accggtggtg acatcctgcg tcgtaccaac 1620

accggtacct tcggtgacat ccgtgttaac atcaacccgc cgttcgctca gcgttaccgt 1680

gttcgtatcc gttacgcttc taccaccgac ctgcagttcc acacctctat caacggtaaa 1740

gctatcaacc agggtaactt ctctgctacc atgaaccgtg gtgaagacct ggactacaaa 1800

accttccgta ccgttggttt caccaccccg ttctctttct ctgacgttca gtctaccttc 1860

accatcggtg cttggaactt ctcttctggt aacgaagttt acatcgaccg tatcgaattc 1920

gttccggttg aagttaccta cgaa 1944

<210> 4

<211> 1944

<212> DNA

<213> Synthesis (unown)

<400> 4

atgaaactga aaaacccgga caaacaccag tctctgtctt ctaacgctaa agttgacaaa 60

atcgctaccg actctctgaa aaacgaaacc gacatcgaac tgaaaaacat caaccacgaa 120

gacttcctgc gtatgtctga acacgaatct atcgacccgt tcgtttctgc ttctaccatc 180

cagaccggta tcggtatcgc tggtaaaatc ctgggtaccc tgggtgttcc gttcgctggt 240

cagatcgctt ctctgtactc tttcatcctg ggtgaactgt ggccgaaagg taaatctcag 300

tgggaaatct tcatggaaca cgttgaagaa ctcatcgacc agaaaatctc tacctacgct 360

cgtaacatcg ctctggctga cctgaaaggt ctgggtgacg ctctggctgt ttaccacgaa 420

tctctggaat cttggatcaa aaaccgtaac aacgctcgtg ctacctctgt tgttaaatct 480

cagtacatcg ctctggaact gctgttcgtt cagaaactgc cgtctttcgc tgtttctggt 540

gaagaagttc cgctgctgcc gatctacgct caggctgcta acctgcacct gctgctgctg 600

cgtgacgctt ctgttttcgg taaagaatgg ggtctgtcta actctcagat ctctaccttc 660

tacaaccgtc aggttgaacg tacctctgac tactctaacc actgcgttaa atggtactct 720

accggtctga acaacctgcg tggtaccaac gctgaatctt gggttcgtta caaccagttc 780

cgtaaagaca tgaccctgat ggttctggac ctgatcgctc tgttcccgtc ttacgacacc 840

ctggtttacc cgatcaaaac cacctctcag ctgacccgtg aagtttacac cgacgctatc 900

ggtaccgttc acccaaatgc tagcttcgct tctaccactt ggtacaacaa caacgctccg 960

tctttctctg ctatcgaatc tgctgttgtt cgtaacccgc acctgctgga cttcctggaa 1020

caggttacca tctactctct gctgtctcgt tggtctaaca cccagtacat gaacatgtgg 1080

ggtggtcacc gtctggaatt ccgtaccatc ggtggtgttc tgaacacctc tacccagggt 1140

tctaccaaca cctctatcaa cccggttacc ctgccgttca cctctcgtga cgtttaccgt 1200

accgaatctc tggctggtct gaacctgttc ctgacccagc cggttaacgg tgttccgcgt 1260

gttgacttcc actggaaatt cgctaccctg ccgatcgctt ctgacaactt ctactacctg 1320

ggttacgctg gtgttggtac ccagctgcag gactctgaaa acgaactgcc gccggaaacc 1380

accggtcagc cgaactacga atcttactct caccgtctgt ctcacatcgg tctgatctct 1440

gcttctcacg ttaaagctct ggtttactct tggacccacc gttctgctga ccgtaccaac 1500

accatcgaac cgaactctat cacccagatc ccgctggtta aagcgttcaa cctgtcttct 1560

ggtgctgctg ttgttcgtgg tccgggtttc accggtggtg acatcctgcg tcgtaccaac 1620

accggtacct tcggtgacat ccgtgttaac atcaacccgc cgttcgctca gcgttaccgt 1680

gttcgtatcc gttacgcttc taccaccgac ctgcagttcc acacctctat caacggtaaa 1740

gctatcaacc agggtaactt ctctgctacc atgaaccgtg gtgaagacct ggactacaaa 1800

accttccgta ccgttggttt caccaccccg ttctctttct ctgacgttca gtctaccttc 1860

accatcggtg cttggaactt ctcttctggt aacgaagttt acatcgaccg tatcgaattc 1920

gttccggttg aagttaccta cgaa 1944

<210> 5

<211> 1944

<212> DNA

<213> Synthesis (unown)

<400> 5

atgaaactga aaaacccgga caaacaccag tctctgtctt ctaacgctaa agttgacaaa 60

atcgctaccg actctctgaa aaacgaaacc gacatcgaac tgaaaaacat caaccacgaa 120

gacttcctgc gtatgtctga acacgaatct atcgacccgt tcgtttctgc ttctaccatc 180

cagaccggta tcggtatcgc tggtaaaatc ctgggtaccc tgggtgttcc gttcgctggt 240

cagatcgctt ctctgtactc tttcatcctg ggtgaactgt ggccgaaagg taaatctcag 300

tgggaaatct tcatggaaca cgttgaagaa ctcatcgacc agaaaatctc tacctacgct 360

cgtaacatcg ctctggctga cctgaaaggt ctgggtgacg ctctggctgt ttaccacgaa 420

tctctggaat cttggatcaa aaaccgtaac aacgctcgtg ctacctctgt tgttaaatct 480

cagtacatcg ctctggaact gctgttcgtt cagaaactgc cgtctttcgc tgtttctggt 540

gaaaaagttc cgctgctgcc gatctacgct caggctgcta acctgcacct gctgctgctg 600

cgtgacgctt ctgttttcgg taaagaatgg ggtctgtcta actctcagat ctctaccttc 660

tacaaccgtc aggttgaacg tacctctgac tactctgacc actgcgttaa atggtactct 720

accggtctga acaacctgcg tggtaccaac gctgaatctt gggttcgtta caaccagttc 780

cgtaaagaca tgaccctgat ggttctggac ctgatcgctc tgttcccgtc ttacgacacc 840

ctggtttacc cgatcaaaac cacctctcag ctgacccgtg aagtttacac cgacgctatc 900

ggtaccgttc acccaaatgc tagcttcgct tctaccactt ggtacaacaa caacgctccg 960

tctttctctg ctatcgaatc tgctgttgtt cgtaacccgc acctgctgga cttcctggaa 1020

caggttacca tctactctct gctgtctcgt tggtctaaca cccagtacat gaacatgtgg 1080

ggtggtcacc gtctggaatt ccgtaccatc ggtggtgttc tgaacacctc tacccagggt 1140

tctaccaaca cctctatcaa cccggttacc ctgccgttca cctctcgtga cgtttaccgt 1200

accgaatctc tggctggtct gaacctgttc ctgacccagc cggttaacgg tgttccgcgt 1260

gttgacttcc actggaaatt cgctaccctg ccgatcgctt ctgacaactt ctactacctg 1320

ggttacgctg gtgttggtac ccagctgcag gactctgaaa acgaactgcc gccggaaacc 1380

accggtcagc cgaactacga atcttactct caccgtctgt ctcacatcgg tctgatctct 1440

gcttctcacg ttaaagctct ggtttactct tggacccacc gttctgctga ccgtaccaac 1500

accatcgaac cgaactctat cacccagatc ccgctggtta aagcgttcaa cctgtcttct 1560

ggtgctgctg ttgttcgtgg tccgggtttc accggtggtg acatcctgcg tcgtaccaac 1620

accggtacct tcggtgacat ccgtgttaac atcaacccgc cgttcgctca gcgttaccgt 1680

gttcgtatcc gttacgcttc taccaccgac ctgcagttcc acacctctat caacggtaaa 1740

gctatcaacc agggtaactt ctctgctacc atgaaccgtg gtgaagacct ggactacaaa 1800

accttccgta ccgttggttt caccaccccg ttctctttct ctgacgttca gtctaccttc 1860

accatcggtg cttggaactt ctcttctggt aacgaagttt acatcgaccg tatcgaattc 1920

gttccggttg aagttaccta cgaa 1944

<210> 6

<211> 1944

<212> DNA

<213> Synthesis (unown)

<400> 6

atgaaactga aaaacccgga caaacaccag tctctgtctt ctaacgctaa agttgacaaa 60

atcgctaccg actctctgaa aaacgaaacc gacatcgaac tgaaaaacat caaccacgaa 120

gacttcctgc gtatgtctga acacgaatct atcgacccgt tcgtttctgc ttctaccatc 180

cagaccggta tcggtatcgc tggtaaaatc ctgggtaccc tgggtgttcc gttcgctggt 240

cagatcgctt ctctgtactc tttcatcctg ggtgaactgt ggccgaaagg taaatctcag 300

tgggaaatct tcatggaaca cgttgaagaa ctcatcagcc agaaaatctc tacctacgct 360

cgtaacatcg ctctggctga cctgaaaggt ctgggtgacg ctctggctgt ttaccacgaa 420

tctctggaat cttggatcaa aaaccgtaac aacgctcgtg ctacctctgt tgttaaatct 480

cagtacatcg ctctggaact gctgttcgtt cagaaactgc cgtctttcgc tgtttctggt 540

gaagtagttc cgctgctgcc gatctacgct caggctgcta acctgcacct gctgctgctg 600

cgtgacgctt ctgttttcgg taaagaatgg ggtctgtcta actctcagat ctctaccttc 660

tacaaccgtc aggttgaacg tacctctgac tactctgacc actgcgttaa atggtactct 720

accggtctga acaacctgcg tggtaccaac gctgaatctt gggttcgtta caaccagttc 780

cgtaaagaca tgaccctgat ggttctggac ctgatcgctc tgttcccgtc ttacgacacc 840

ctggtttacc cgatcaaaac cacctctcag ctgacccgtg aagtttacac cgacgctatc 900

ggtaccgttc acccaaatgc tagcttcgct tctaccactt ggtacaacaa caacgctccg 960

tctttctctg ctatcgaatc tgctgttgtt cgtaacccgc acctgctgga cttcctggaa 1020

caggttacca tctactctct gctgtctcgt tggtctaaca cccagtacat gaacatgtgg 1080

ggtggtcacc gtctggaatt ccgtaccatc ggtggtgttc tgaacacctc tacccagggt 1140

tctaccaaca cctctatcaa cccggttacc ctgccgttca cctctcgtga cgtttaccgt 1200

accgaatctc tggctggtct gaacctgttc ctgacccagc cggttaacgg tgttccgcgt 1260

gttgacttcc actggaaatt cgctaccctg ccgatcgctt ctgacaactt ctactacctg 1320

ggttacgctg gtgttggtac ccagctgcag gactctgaaa acgaactgcc gccggaaacc 1380

accggtcagc cgaactacga atcttactct caccgtctgt ctcacatcgg tctgatctct 1440

gcttctcacg ttaaagctct ggtttactct tggacccacc gttctgctga ccgtaccaac 1500

accatcgaac cgaactctat cacccagatc ccgctggtta aagcgttcaa cctgtcttct 1560

ggtgctgctg ttgttcgtgg tccgggtttc accggtggtg acatcctgcg tcgtaccaac 1620

accggtacct tcggtgacat ccgtgttaac atcaacccgc cgttcgctca gcgttaccgt 1680

gttcgtatcc gttacgcttc taccaccgac ctgcagttcc acacctctat caacggtaaa 1740

gctatcaacc agggtaactt ctctgctacc atgaaccgtg gtgaagacct ggactacaaa 1800

accttccgta ccgttggttt caccaccccg ttctctttct ctgacgttca gtctaccttc 1860

accatcggtg cttggaactt ctcttctggt aacgaagttt acatcgaccg tatcgaattc 1920

gttccggttg aagttaccta cgaa 1944

<210> 7

<211> 1944

<212> DNA

<213> Synthesis (unown)

<400> 7

atgaaactga aaaacccgga caaacaccag tctctgtctt ctaacgctaa agttgacaaa 60

atcgctaccg actctctgaa aaacgaaacc gacatcgaac tgaaaaacat caaccacgaa 120

gacttcctgc gtatgtctga acacgaatct atcgacccgt tcgtttctgc ttctaccatc 180

cagaccggta tcggtatcgc tggtaaaatc ctgggtaccc tgggtgttcc gttcgctggt 240

cagagcgctt ctctgtactc tttcatcctg ggtgaactgt ggccgaaagg taaagatcag 300

tgggaaatct tcatggaaca cgttgaagaa ctcatcgacc agaaaatctc tacctacgct 360

cgtaacatcg ctctggctga cctgaaaggt ctgggtgacg ctctggctgt ttaccacgaa 420

tctctggaat cttggatcaa aaaccgtaac aacgctcgtg ctacctctgt tgttaaatct 480

cagtacatcg ctctggaact gctgttcgtt cagaaactgc cgtctttcgc tgtttctggt 540

gaagaagttc cgctgctgcc gatctacgct caggctgcta acctgcacct gctgctgctg 600

cgtgacgctt ctgttttcgg taaagaatgg ggtctgtcta actctcagat ctctaccttc 660

tacaaccgtc aggttgaacg tacctctgac tactctgacc actgcgttaa atggtactct 720

accggtctga acaacctgcg tggtaccaac gctgaatctt gggttcgtta caaccagttc 780

cgtaaagaca tgaccctgat ggttctggac ctgatcgctc tgttcccgtc ttacgacacc 840

ctggtttacc cgatcaaaac cacctctcag ctgacccgtg aagtttacac cgacgctatc 900

ggtaccgttc acccaaatgc tagcttcgct tctaccactt ggtacaacaa caacgctccg 960

tctttctctg ctatcgaatc tgctgttgtt cgtaacccgc acctgctgga cttcctggaa 1020

caggttacca tctactctct gctgtctcgt tggtctaaca cccagtacat gaacatgtgg 1080

ggtggtcacc gtctggaatt ccgtaccatc ggtggtgttc tgaacacctc tacccagggt 1140

tctaccaaca cctctatcaa cccggttacc ctgccgttca cctctcgtga cgtttaccgt 1200

accgaatctc tggctggtct gaacctgttc ctgacccagc cggttaacgg tgttccgcgt 1260

gttgacttcc actggaaatt cgctaccctg ccgatcgctt ctgacaactt ctactacctg 1320

ggttacgctg gtgttggtac ccagctgcag gactctgaaa acgaactgcc gccggaaacc 1380

accggtcagc cgaactacga atcttactct caccgtctgt ctcacatcgg tctgatctct 1440

gcttctcacg ttaaagctct ggtttactct tggacccacc gttctgctga ccgtaccaac 1500

accatcgaac cgaactctat cacccagatc ccgctggtta aagcgttcaa cctgtcttct 1560

ggtgctgctg ttgttcgtgg tccgggtttc accggtggtg acatcctgcg tcgtaccaac 1620

accggtacct tcggtgacat ccgtgttaac atcaacccgc cgttcgctca gcgttaccgt 1680

gttcgtatcc gttacgcttc taccaccgac ctgcagttcc acacctctat caacggtaaa 1740

gctatcaacc agggtaactt ctctgctacc atgaaccgtg gtgaagacct ggactacaaa 1800

accttccgta ccgttggttt caccaccccg ttctctttct ctgacgttca gtctaccttc 1860

accatcggtg cttggaactt ctcttctggt aacgaagttt acatcgaccg tatcgaattc 1920

gttccggttg aagttaccta cgaa 1944

<210> 8

<211> 1944

<212> DNA

<213> Synthesis (unown)

<400> 8

atgaaactga aaaacccgga caaacaccag tctctgtctt ctaacgctaa agttgacaaa 60

atcgctaccg actctctgaa aaacgaaacc gacatcgaac tgaaaaacat caaccacgaa 120

gacttcctgc gtatgtctga acacgaatct atcgacccgt tcgtttctgc ttctaccatc 180

cagaccggta tcggtatcgc tggtaaaatc ctgggtaccc tgggtgttcc gttcgctggt 240

cagatcgctt ctctgtactc tttcatcctg ggtgaactgt ggccgaaagg taaatctcag 300

tgggaaatct tcatggaaca cgttgaagaa ctcatcgacc agaaaatctc tacctacgct 360

cgtaacatcg ctctggctga cctgaaaggt ctgggtgacg ctctggctgt ttaccacgaa 420

tctctggaat cttggatcaa aaaccgtaac aacgctcgtg ctacctctgt tgttaaatct 480

cagtacatcg ctctggaact gctgttcgtt cagaaactgc cgtctttcgc tgtttctggt 540

gaagaagttc cgctgctgcc gatctacgct caggctgcta acctgcacct gctgctgctg 600

cgtgacgctt ctgttttcgg taaagaatgg ggtctgtcta actctcagat ctctaccttc 660

tacaaccgtc aggttgaacg tacctctgac tactctyacc actgcgttaa atggtactct 720

accggtctga acaacctgcg tggtaccaac gctgaatctt gggttcgtta caaccagttc 780

cgtaaagaca tgaccctgat ggttctggac ctgatcgctc tgttcccgtc ttacgacacc 840

ctggtttacc cgatcaaaac cacctctcag ctgacccgtg aagtttacac cgacgctatc 900

ggtaccgttc acccaaatgc tagcttcgct tctaccactt ggtacaacaa caacgctccg 960

tctttctctg ctatcgaatc tgctgttgtt cgtaacccgc acctgctgga cttcctggaa 1020

caggttacca tctactctct gctgtctcgt tggtctaaca cccagtacat gaacatgtgg 1080

ggtggtcacc gtctggaatt ccgtaccatc ggtggtgttc tgaacacctc tacccagggt 1140

tctaccaaca cctctatcaa cccggttacc ctgccgttca cctctcgtga cgtttaccgt 1200

accgaatctc tggctggtct gaacctgttc ctgacccagc cggttaacgg tgttccgcgt 1260

gttgacttcc actggaaatt cgctaccctg ccgatcgctt ctgacaactt ctactacctg 1320

ggttacgctg gtgttggtac ccagctgcag gactctgaaa acgaactgcc gccggaaacc 1380

accggtcagc cgaactacga atcttactct caccgtctgt ctcacatcgg tctgatctct 1440

gcttctcacg ttaaagctct ggtttactct tggacccacc gttctgctga ccgtaccaac 1500

accatcgaac cgaactctat cacccagatc ccgctggtta aagcgttcaa cctgtcttct 1560

ggtgctgctg ttgttcgtgg tccgggtttc accggtggtg acatcctgcg tcgtaccaac 1620

accggtacct tcggtgacat ccgtgttaac atcaacccgc cgttcgctca gcgttaccgt 1680

gttcgtatcc gttacgcttc taccaccgac ctgcagttcc acacctctat caacggtaaa 1740

gctatcaacc agggtaactt ctctgctacc atgaaccgtg gtgaagacct ggactacaaa 1800

accttccgta ccgttggttt caccaccccg ttctctttct ctgacgttca gtctaccttc 1860

accatcggtg cttggaactt ctcttctggt aacgaagttt acatcgaccg tatcgaattc 1920

gttccggttg aagttaccta cgaa 1944

Claims (6)

1. An amino acid variation site of a protein, characterized in that the site is located at any one or more of the following sites in the sequence shown in SEQ ID NO. 1:

1)I82；

2)S99；

3)D113；

4)K147；

5)E182；

6)N214；

7)D233。

2. a protein comprising the amino acid sequence of SEQ ID No.1 and having the following mutations:

1)I82V+S99N+L111I+K147G+N214S；

2)D233N；

3)E182K；

4)D113S+E182V；

5)I82S+S99D

6)D233Y。

3. a nucleic acid encoding the protein of claim 2.

4. A nucleic acid according to claim 3, wherein the nucleotide sequence of the nucleic acid is as set forth in any one of SEQ ID No.3 to SEQ ID No. 8.

5. Use of a protein according to claim 2 and a nucleic acid according to claim 3 to claim 4 for the cultivation of insect-resistant crops.

6. The use of claim 5, wherein the crop is corn.