CN103739683B - Insecticidal protein, and encoding gene and use thereof - Google Patents

Abstract

The present invention relates to an insecticidal protein, its coding gene and its use. The insecticidal protein includes: (a) a protein composed of the amino acid sequence shown in SEQ ID NO: 2; or (b) the amino acid sequence in (a) A protein derived from (a) that has been substituted and/or deleted and/or added one or several amino acids and has insecticidal activity. The insecticidal gene of the present invention is particularly suitable for expression in monocotyledonous plants, especially rice, which not only significantly improves the expression level and stability of the PIC10-01 insecticidal protein, but also significantly enhances the effect of the PIC10-01 insecticidal protein on insect pests. Virulence, especially for insect pests of the order Lepidoptera.

Description

Insecticidal protein, its coding gene and application

technical field

The present invention relates to an insecticidal protein, its encoding gene and application, in particular to a modified PIC10-01 insecticidal protein, its encoding gene and application.

Background technique

Plant pests are the main factor leading to the loss of crops, causing significant economic losses to farmers, and even affecting the living conditions of the local population. In order to control plant pests, people usually use broad-spectrum chemical insecticides and biological insecticides, but both have limitations in practical application: chemical insecticides will cause environmental pollution problems and lead to the emergence of resistant insects. However, biopesticides are easily degraded in the environment and require repeated application in production, which greatly increases production costs.

In order to solve the limitations in the practical application of chemical insecticides and biological insecticides, scientists have discovered through research that by transferring insect-resistant genes encoding insecticidal proteins into plants, some insect-resistant transgenic plants can be obtained to control plant pests. PIC10 insecticidal protein is one of many insecticidal proteins, and it is an insoluble parasporal crystal protein.

The PIC10 protein is ingested into the midgut by insects, and the protoxin is dissolved in the alkaline pH environment of the insect midgut. The N- and C-terminals of the protein are digested by alkaline protease, and the protoxin is converted into an active fragment; the active fragment binds to the receptor on the upper surface of the insect midgut epithelial cell membrane, and inserts into the intestinal membrane, resulting in perforated lesions in the cell membrane, destroying the inner and outer membranes. Changes in osmotic pressure and pH balance, etc., disturb the digestive process of insects and eventually lead to their death.

Rice is the main food crop in China, and every year there is a huge loss of food due to plant pests, such as the rice stem borer, the rice stem borer or the large borer. There is no report on the expression level and toxicity of PIC10 insecticidal protein in plants.

Contents of the invention

The object of the present invention is to provide an insecticidal protein, its coding gene and its use. The PIC10-01 insecticidal protein has a relatively high expression level and toxicity in plants (especially rice).

To achieve the above object, the invention provides a kind of insecticidal protein, comprising:

(a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2; or

(b) The amino acid sequence in (a) has been substituted and/or deleted and/or one or several amino acids have been added, and the protein derived from (a) has insecticidal activity.

To achieve the above object, the invention provides a kind of insecticidal gene, comprising:

(a) a nucleotide sequence encoding said pesticidal protein; or

(b) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence defined in (a) and that encodes a protein having pesticidal activity; or

(c) have the nucleotide sequence shown in SEQ ID NO:1.

The stringent conditions can be hybridized at 65°C in 6×SSC (sodium citrate), 0.5% SDS (sodium dodecyl sulfate) solution, and then mixed with 2×SSC, 0.1% SDS and 1×SSC, Wash each membrane once with 0.1% SDS.

To achieve the above object, the present invention also provides an expression cassette, comprising the insecticidal gene under the control of an operably linked regulatory sequence.

To achieve the above purpose, the present invention also provides a recombinant vector comprising the insecticidal gene or the expression cassette.

To achieve the above object, the present invention also provides a method for producing an insecticidal protein, comprising:

obtaining cells of a transgenic host organism comprising said pesticidal gene or said expression cassette;

culturing the cells of said transgenic host organism under conditions that permit production of the pesticidal protein;

The pesticidal protein is recovered.

Further, the cells of the transgenic host organism include plant cells, animal cells, bacteria, yeast, baculovirus, nematodes or algae.

Preferably, the plant is corn, soybean, cotton, rice or wheat.

To achieve the above object, the present invention also provides a method for increasing the range of insect targets, comprising: combining the insecticidal protein or the insecticidal protein encoded by the expression cassette in plants with at least one The pesticidal protein or the second pesticidal nucleotide of the pesticidal protein encoded by the expression cassette is expressed together.

Further, the second insecticidal nucleotide may encode Cry-like insecticidal protein, Vip-like insecticidal protein, protease inhibitor, lectin, α-amylase or peroxidase.

Optionally, the second insecticidal nucleotide is dsRNA that suppresses important genes in target insect pests.

In the present invention, the expression of PIC10-01 insecticidal protein in a transgenic plant may be accompanied by the expression of one or more Cry-like insecticidal proteins and/or Vip-like insecticidal proteins. Such co-expression of more than one insecticidal toxin in the same transgenic plant can be achieved by genetically engineering the plant to contain and express the desired gene. In addition, one plant (the first parent) can express PIC10-01 insecticidal protein through genetic engineering, and the second plant (second parent) can express Cry insecticidal protein and/or Vip insecticidal protein through genetic engineering protein. Progeny plants expressing all the genes introduced into the first parent and the second parent are obtained by crossing the first parent and the second parent.

RNA interference (RNA interference, RNAi) refers to the phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (double-stranded RNA, dsRNA), which is highly conserved during evolution. Therefore, RNAi technology can be used to specifically knock out or turn off the expression of specific genes.

To achieve the above object, the present invention also provides a method for producing insect-resistant plants, comprising: introducing the insecticidal gene or the expression cassette or the recombinant vector into plants.

Preferably, the plant is corn, soybean, cotton, rice or wheat.

To achieve the above object, the present invention also provides a method for protecting plants from damage caused by insect pests, comprising: introducing the insecticidal gene or the expression cassette or the recombinant vector into the plant , causing the introduced plant to produce an amount of the insecticidal protein sufficient to protect it from insect pests.

Preferably, the plant is corn, soybean, cotton, rice or wheat.

Introduce the insecticidal gene or the expression cassette or the recombinant vector into the plant. In the present invention, the exogenous DNA is introduced into the plant cell. Conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, Microshot bombardment, direct DNA uptake into protoplasts, electroporation, or silicon whisker-mediated DNA introduction.

To achieve the above object, the present invention also provides a method for controlling insect pests, comprising: contacting insect pests with an inhibitory amount of the insecticidal protein or the insect inhibitory protein encoded by the insecticidal gene.

Preferably, the insect pests are insect pests of the order Lepidoptera.

To achieve the above object, the present invention also provides a use of the insecticidal protein or the insect-inhibiting protein encoded by the insecticidal gene to control insect pests.

The genome of a plant, plant tissue or plant cell in the present invention refers to any genetic material in a plant, plant tissue or plant cell, and includes nucleus, plastid and mitochondrial genome.

The polynucleotides and/or nucleotides described in the present invention form an entire "gene" that encodes a protein or polypeptide in a desired host cell. Those skilled in the art will readily recognize that the polynucleotides and/or nucleotides of the present invention can be placed under the control of regulatory sequences in the intended host.

As is well known to those skilled in the art, DNA typically exists in double-stranded form. In this arrangement, one strand is complementary to the other and vice versa. As DNA replicates in plants other complementary strands of DNA are produced. Thus, the present invention includes the use of the polynucleotides exemplified in the Sequence Listing and their complements. "Coding strand" as commonly used in the art refers to the strand combined with the antisense strand. To express a protein in vivo, one strand of DNA is typically transcribed into a complementary strand of mRNA, which serves as a template for translation of the protein. mRNA is actually transcribed from the "antisense" strand of DNA. The "sense" or "coding" strand has a series of codons (codons are three nucleotides, read three at a time to yield a specific amino acid) that can be read as an open reading frame (ORF) to form the protein or peptide of interest. The present invention also includes RNA and PNA (peptide nucleic acid) that are functionally equivalent to the exemplified DNA.

In the present invention, the nucleic acid molecules or fragments thereof are hybridized with the insecticidal gene of the present invention under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the pesticidal gene of the present invention. Nucleic acid molecules or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. In the present invention, if two nucleic acid molecules can form an antiparallel double-stranded nucleic acid structure, it can be said that the two nucleic acid molecules can specifically hybridize to each other. A nucleic acid molecule is said to be the "complement" of another nucleic acid molecule if two nucleic acid molecules exhibit perfect complementarity. In the present invention, two nucleic acid molecules are said to exhibit "complete complementarity" when every nucleotide of one nucleic acid molecule is complementary to the corresponding nucleotide of the other nucleic acid molecule. Two nucleic acid molecules are said to be "minimally complementary" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under at least conventional "low stringency" conditions. Similarly, two nucleic acid molecules are said to be "complementary" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under conventional "high stringency" conditions. Deviations from perfect complementarity are permissible as long as the deviation does not completely prevent the two molecules from forming a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe, it only needs to be sufficiently complementary in sequence to form a stable double-stranded structure under the particular solvent and salt concentration employed.

In the present invention, a substantially homologous sequence is a nucleic acid molecule that can specifically hybridize to a complementary strand of another matched nucleic acid molecule under highly stringent conditions. Appropriate stringent conditions to promote DNA hybridization, for example, treatment with 6.0× sodium chloride/sodium citrate (SSC) at approximately 45°C, followed by washing with 2.0×SSC at 50°C, are known to those skilled in the art. is well known. For example, the salt concentration in the washing step can be selected from about 2.0×SSC, 50°C for low stringency conditions to about 0.2×SSC, 50°C for high stringency conditions. In addition, the temperature conditions in the washing step can be increased from about 22°C at room temperature for low stringency conditions to about 65°C for high stringency conditions. Both the temperature condition and the salt concentration can be changed, or one can be kept constant while the other variable is changed. Preferably, the stringent conditions of the present invention can be in 6 × SSC, 0.5% SDS solution, at 65 ° C with SEQ ID NO: 1 specific hybridization, and then use 2 × SSC, 0.1% SDS and 1 × SSC , 0.1% SDS each washed once.

Therefore, a sequence having insect-resistant activity and hybridizing to sequence 1 of the present invention under stringent conditions is included in the present invention. These sequences are at least about 40%-50% homologous, about 60%, 65% or 70% homologous, even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% homologous to the sequences of the invention %, 94%, 95%, 96%, 97%, 98%, 99% or more homology. That is, the sequence identity ranges from at least about 40%-50%, about 60%, 65% or 70% homology, even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% %, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity.

The genes and proteins described in the present invention not only include specific example sequences, but also include parts and/or fragments (compared with the full-length protein and/or terminal parts) that preserve the insecticidal activity characteristics of the specific example proteins. deletions), variants, mutants, substitutions (proteins with substituted amino acids), chimeras and fusion proteins. The "variant" or "variation" refers to the nucleotide sequence encoding the same protein or encoding an equivalent protein with insecticidal activity. The "equivalent protein" refers to a protein having the same or substantially the same biological activity against lepidopteran insect pests as the claimed protein.

The "fragment" or "truncation" of a DNA molecule or protein sequence in the present invention refers to a part of the original DNA or protein sequence (nucleotide or amino acid) or its artificially modified form (such as a sequence suitable for plant expression) ), including adjacent fragments and internal and/or terminal deletions compared to the full-length molecule, the aforementioned sequences may vary in length but are long enough to ensure that the (encoded) protein is an insect toxin. In some cases (especially expression in plants) it may be advantageous to use truncated genes encoding truncated proteins. Preferred truncated genes generally encode 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 of the full-length protein , 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 , 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.

Due to the redundancy of the genetic code, many different DNA sequences can encode the same amino acid sequence. It is well within the level of skill in the art to generate such alternative DNA sequences encoding identical or substantially identical proteins. These different DNA sequences are included within the scope of the present invention. The "substantially identical" sequence refers to a sequence that has amino acid substitutions, deletions, additions or insertions but does not substantially affect the insecticidal activity, and also includes fragments that retain insecticidal activity.

The substitution, deletion or addition of the amino acid sequence in the present invention is a routine technique in the art, and such amino acid changes are preferably: small characteristic changes, that is, conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, Typically deletions of about 1-30 amino acids; small amino- or carboxy-terminal extensions, eg, amino-terminal extensions of a methionine residue; small linker peptides, eg, about 20-25 residues in length.

Examples of conservative substitutions are those that occur within the following groups of amino acids: basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids ( such as glutamine, asparagine), hydrophobic amino acids (such as leucine, isoleucine, and valine), aromatic amino acids (such as phenylalanine, tryptophan, and tyrosine), and small molecules Amino acids (such as glycine, alanine, serine, threonine, and methionine). Those amino acid substitutions that generally do not alter a particular activity are well known in the art and have been described, for example, by N. Neurath and R.L. Hill in Protein, Academic Press, New York, 1979. The most common interchanges are Ala/Ser, Val/Ile, Asp/Glu, Thu/Ser, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, and their opposite interchanges.

It will be apparent to those skilled in the art that such substitutions can be made outside of regions important to the function of the molecule and still result in an active polypeptide. Amino acid residues essential for the activity of the polypeptides of the present invention and thus selected not to be substituted can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (see, for example, Cunningham and Wells , 1989, Science 244: 1081-1085). The latter technique introduces mutations at every positively charged residue in the molecule, and detects the anti-insect activity of the resulting mutant molecules, so as to determine the amino acid residues that are important for the activity of the molecule. Substrate-enzyme interaction sites can also be determined by analysis of their three-dimensional structure by techniques such as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (see, e.g., de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol 224:899-904; Wlodaver et al., 1992, FEBS Letters 309:59-64).

Therefore, amino acid sequences having certain homology with the amino acid sequence shown in Sequence 2 are also included in the present invention. The similarity/identity of these sequences to the sequences of the present invention is typically greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and may be greater than 95%. Preferred polynucleotides and proteins of the invention can also be defined in terms of more specific identity and/or similarity ranges. For example, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% identical and/or similar.

The regulatory sequences in the present invention include but not limited to promoters, transit peptides, terminators, enhancers, leader sequences, introns and other regulatory sequences operably linked to the insecticidal gene.

The promoter is a promoter that can be expressed in plants, and the "promoter that can be expressed in plants" refers to a promoter that ensures the expression of the coding sequence linked to it in plant cells. A promoter expressible in plants may be a constitutive promoter. Examples of promoters directing constitutive expression in plants include, but are not limited to, 35S promoter derived from cauliflower mosaic virus, maize ubi promoter, rice GOS2 gene promoter and the like. Alternatively, the promoter expressible in plants may be a tissue-specific promoter, i.e., the promoter directs expression of the coding sequence at higher levels in some tissues of the plant, such as in green tissues, than in other tissues of the plant (which can be determined by routine RNA assays), such as the PEP carboxylase promoter. Alternatively, the promoter expressible in plants may be a wound-inducible promoter. A wound-inducible promoter or a promoter directing a wound-induced expression pattern means that when a plant is subjected to mechanical or insect-induced wounds, the expression of the coding sequence under the regulation of the promoter is significantly increased compared with normal growth conditions. Examples of wound-inducible promoters include, but are not limited to, the promoters of the potato and tomato protease inhibitors (pinI and pinII) and the maize proteinase inhibitor (MPI).

The transit peptide (also called secretory signal sequence or targeting sequence) directs the transgene product to a specific organelle or cellular compartment. The transit peptide can be heterologous to the receptor protein, for example, using a gene encoding a chloroplast transport The peptide sequences target the chloroplast, or the endoplasmic reticulum using the 'KDEL' retention sequence, or the vacuole using the CTPP of the barley lectin gene.

The leader sequence includes, but is not limited to, a picornavirus leader sequence, such as an EMCV leader sequence (5' non-coding region of encephalomyocarditis virus); a potyvirus leader sequence, such as a MDMV (maize dwarf mosaic virus) leader sequence; Human immunoglobulin heavy chain binding protein (BiP); untranslated leader of coat protein mRNA of alfalfa mosaic virus (AMV RNA4); tobacco mosaic virus (TMV) leader.

The enhancers include, but are not limited to, cauliflower mosaic virus (CaMV) enhancers, Scrophulariaceae mosaic virus (FMV) enhancers, carnation weathering ring virus (CERV) enhancers, cassava vein mosaic virus (CsVMV) enhancers , Mirabilis Mosaic Virus (MMV) Enhancer, Night Scent Yellow Leaf Curl Virus (CmYLCV) Enhancer, Multan Cotton Leaf Curl Virus (CLCuMV), Commelina Yellow Mottle Virus (CoYMV) and Peanut Chlorotic Streak Flower Leaf virus (PCLSV) enhancer.

For monocot applications, the introns include, but are not limited to, the maize hsp70 intron, the maize ubiquitin intron, the Adh intron 1, the sucrose synthase intron, or the rice Act1 intron. For dicot applications, such introns include, but are not limited to, the CAT-1 intron, the pKANNIBAL intron, the PIV2 intron, and the "super ubiquitin" intron.

The terminator may be a suitable polyadenylation signal sequence that functions in plants, including but not limited to, the polyadenylation signal sequence derived from the nopaline synthase (NOS) gene of Agrobacterium tumefaciens , the polyadenylation signal sequence from the protease inhibitor Ⅱ (pinⅡ) gene, the polyadenylation signal sequence from the pea ssRUBISCO E9 gene and the polyadenylation signal sequence from the α-tubulin gene Polyadenylation signal sequence.

The "operably linked" in the present invention refers to the linkage of nucleic acid sequences, which allows one sequence to provide the required function for the linked sequence. The "operably linked" in the present invention can be linking a promoter with a sequence of interest, so that the transcription of the sequence of interest is controlled and regulated by the promoter. "Operably linked" when a sequence of interest encodes a protein and expression of that protein is desired means that a promoter is linked to said sequence in such a way that the resulting transcript is efficiently translated. If the junction of the promoter and coding sequence is a transcript fusion and expression of the encoded protein is desired, the junction is made such that the first translation initiation codon in the resulting transcript is that of the coding sequence. Alternatively, if the junction of the promoter and coding sequence is a translational fusion and expression of the encoded protein is to be achieved, the junction is made such that the first translation initiation codon contained in the 5' untranslated sequence is fused with the promoter linked in such a way that the resulting translation product is in-frame with the translational open reading frame encoding the desired protein. Nucleic acid sequences that may be "operably linked" include, but are not limited to: sequences that provide gene expression function (i.e., gene expression elements such as promoters, 5' untranslated regions, introns, protein coding regions, 3' untranslated regions, polynucleotides adenylation sites and/or transcription terminators), sequences that provide DNA transfer and/or integration functions (i.e. T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), provide selection Sequences for sexual function (i.e., antibiotic resistance markers, biosynthetic genes), sequences that provide scoreable marker function, sequences that facilitate sequence manipulation in vitro or in vivo (i.e., polylinker sequences, site-specific recombination sequences), and sequences that provide Sequences that are functional for replication (i.e., bacterial origins of replication, autonomously replicating sequences, centromere sequences).

The term "insecticide" in the present invention means that it is toxic to crop pests. More specifically, the target insects are pests, such as, but not limited to, most of the Lepidoptera pests, such as corn borer, rice stem borer, oriental armyworm, rice stem borer or rice borer, and the like.

In the present invention, the insecticidal protein is the amino acid sequence of PIC10-01, as shown in SEQ ID NO:2 in the sequence listing. The insecticidal gene is a PIC10-01 nucleotide sequence, as shown in SEQ ID NO:1 in the sequence listing. The insecticidal gene is a DNA sequence used for transformation of plants, especially rice. In addition to the coding region comprising the protein encoded by the PIC10-01 nucleotide sequence, it may also contain other elements, such as the coding region encoding the transit peptide, The coding region of a protein encoding a selectable marker or a protein that confers herbicide resistance.

The PIC10-01 insecticidal protein of the present invention is toxic to most Lepidoptera pests. The plants of the present invention, especially rice, contain exogenous DNA in their genome, said exogenous DNA includes PIC10-01 nucleotide sequence, and protect it from the threat of pests by expressing an inhibitory amount of this protein. An inhibitory amount refers to a lethal or sublethal dose. At the same time, the plants should be normal in morphology and can be cultivated for consumption and/or production of the product under conventional methods. Furthermore, the plants can substantially eliminate the need for chemical or biological insecticides against insects targeted by the protein encoded by the PIC10-01 nucleotide sequence.

Expression levels of insecticidal crystal proteins (ICPs) in plant material can be detected by a variety of methods described in the art, such as by application of specific primers to quantify mRNA encoding insecticidal proteins produced in tissues, or by direct specificity. The amount of pesticidal protein produced was measured.

Different assays can be used to determine the insecticidal efficacy of ICPs in plants. In the present invention, the target insects are mainly Lepidoptera pests, more specifically Spores borer or Chilorrhizae etc.

In addition, the expression cassette comprising the insecticidal protein (PIC10-01 amino acid sequence) of the present invention can also be expressed in plants together with at least one protein encoding a herbicide resistance gene, and the herbicide resistance gene includes but not limited to, Glufosinate resistance genes (e.g. bar gene, pat gene), bendichlor resistance gene (e.g. pmph gene), glyphosate resistance gene (e.g. EPSPS gene), bromoxynil (bromoxynil) resistance gene, sulfonate ureide resistance gene, resistance gene to herbicide palapat, resistance gene to cyanamide or resistance gene to glutamine synthetase inhibitors (such as PPT), so as to obtain both high insecticidal activity and Genetically modified plants for herbicide resistance.

The invention provides an insecticidal protein, its coding gene and its use, which have the following advantages:

1. Strong toxicity. The insecticidal protein PIC10-01 of the present invention has strong insecticidal toxicity, especially for Lepidoptera pests that harm rice.

2. High expression level. The insecticidal gene PIC10-01 of the present invention optimizes the insecticidal protein PIC10-01 according to the preferred codons of rice, and simultaneously removes the sequence that makes the mRNA unstable, the PolyA tailing signal and the similar site of intron splicing, Moreover, the GC content is increased, which fully conforms to the characteristics of rice genes, so that the insecticidal gene of the present invention is particularly suitable for expression in monocotyledonous plants, especially rice, which has a high expression level and good stability.

3. Broad insecticidal spectrum. The insecticidal protein PIC10-01 protein of the present invention not only exhibits high resistance to Chilo suppressalis, but also has high activity against S. chinensis, so it has broad application prospects in plants.

The technical solutions of the present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

Description of drawings

Fig. 1 is the construction flowchart of the recombinant cloning vector DBN01-T containing the PIC10-01 nucleotide sequence of the insecticidal protein of the present invention, its encoding gene and its application;

Fig. 2 is a construction flow diagram of the recombinant expression vector DBN100323 containing the PIC10-01 nucleotide sequence of the insecticidal protein of the present invention, its encoding gene and its application;

Fig. 3 is the anti-insect effect diagram of the transgenic rice plant inoculated with the insecticidal protein of the present invention, its coding gene and application;

Fig. 4 is a graph showing the insecticidal effect of the insecticidal protein of the present invention, its coding gene and its application when the transgenic rice plant is inoculated with the large borer.

Detailed ways

The technical scheme of the insecticidal protein, its coding gene and application of the present invention will be further illustrated below through specific examples.

The first embodiment, the acquisition and synthesis of PIC10-01 gene sequence

The amino acid sequence (630 amino acids) of the PIC10-01 insecticidal protein is shown in SEQ ID NO: 2 in the sequence listing; the amino acid sequence (630 amino acids) corresponding to the PIC10-01 insecticidal protein encoding is obtained according to rice preference codons amino acid) nucleotide sequence (1896 nucleotides), as shown in SEQID NO:1 in the sequence listing. The PIC10-01 nucleotide sequence (shown as SEQ ID NO: 1 in the sequence listing) was synthesized by Nanjing KingScript Biotechnology Co., Ltd.; the synthesized PIC10-01 nucleotide sequence (SEQ ID NO: 1) The 5' end of the PIC10-01 nucleotide sequence (SEQ ID NO: 1) is also connected with a PvuI restriction site at the 3' end.

The second embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium

1. Construction of recombinant cloning vector DBN01-T containing PIC10-01 nucleotide sequence

The synthesized PIC10-01 nucleotide sequence was ligated into the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the operation steps were carried out according to the instructions of the pGEM-T vector produced by Promega Company to obtain the recombinant cloning vector DBN01-T , its construction process is shown in Figure 1 (among them, Amp represents the ampicillin resistance gene; f1 represents the replication origin of phage f1; LacZ is the LacZ start codon; SP6 is the SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter sub; PIC10-01 is the nucleotide sequence of PIC10-01 (SEQ ID NO: 1); MCS is the multiple cloning site).

Then, the recombinant cloning vector DBN01-T was transformed into Escherichia coli T1 competent cells (Transgen, Beijing, China, CAT: CD501) by heat shock method. The heat shock conditions were: 50 μl E. coli T1 competent cells, 10 μl plasmid DNA (recombinant Cloning vector DBN01-T), in 42°C water bath for 30 seconds; ice bath cells for 1-2 minutes; L, adjust the pH to 7.5 with NaOH) for 1 hour (shaking on a shaker at 100 rpm), and coat the surface with IPTG (isopropylthio-β-D-galactoside) and X-gal (5- Bromo-4-chloro-3-indole-β-D-galactoside) ampicillin (100 mg/L) on LB solid plates (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L , agar 15g/L, adjust the pH to 7.5 with NaOH) and grow overnight. White colonies were picked and cultured overnight in LB liquid medium at a temperature of 37°C. Extract the plasmid by alkaline method: centrifuge the bacterial solution at 12000rpm for 1min, remove the supernatant, and use 100μl ice-precooled solution I (25mM Tris-HCl, 10mM EDTA (ethylenediaminetetraacetic acid), 50mM glucose , pH8.0) suspension; add 200 μl freshly prepared solution II (0.2M NaOH, 1% SDS (sodium dodecyl sulfate)), invert the tube 5 times, mix, put on ice for 3-5min; add 150 μl ice-cold Solution III (3M potassium acetate, 5M acetic acid), mix thoroughly immediately, place on ice for 5-10min; centrifuge at 4°C and 12000rpm for 5min, take an appropriate amount of supernatant, add 2 times the volume of water and ethanol, mix well and place at room temperature for 5 min; centrifuge at 4 °C and 12000 rpm for 5 min, discard the supernatant, wash the precipitate with ethanol with a concentration (V/V) of 70% and dry it; add 30 μl of RNase ( 20μg/ml) TE (10mM Tris-HCl, 1mM EDTA, pH8.0) to dissolve the precipitate; in a water bath at 37°C for 30min to digest RNA; store at -20°C for later use.

After the extracted plasmid was digested and identified by EcoRV and SmaI, the positive clones were sequenced and verified, and the results showed that the PIC10-01 nucleotide sequence inserted in the recombinant cloning vector DBN01-T was shown in SEQ ID NO: 1 in the sequence table The nucleotide sequence, that is, the PIC10-01 nucleotide sequence was correctly inserted.

2. Construction of recombinant expression vector DBN100323 containing PIC10-01 nucleotide sequence

Recombinant cloning vector DBN01-T and expression vector DBNBC-01 (vector backbone: pCAMBIA2301 (available from CAMBIA institutions)) were digested with restriction enzymes SpeI and PvuI respectively, and the excised PIC10-01 nucleotide sequence fragment was inserted into Between the SpeI and PvuI sites of the expression vector DBNBC-01, it is well known to those skilled in the art to construct the vector by using the conventional enzyme digestion method, and construct the recombinant expression vector DBN100323, and its construction process is shown in Figure 2 (Kan: Kanamycin gene; RB: right border; Ubi: corn Ubiquitin (ubiquitin) gene promoter (SEQ ID NO: 3); PIC10-01: PIC10-01 nucleotide sequence (SEQ ID NO: 1); Nos : terminator of nopaline synthase gene (SEQ ID NO:4); PMI: phosphomannose isomerase gene (SEQ ID NO:5); LB: left border).

The recombinant expression vector DBN100323 was transformed into Escherichia coli T1 competent cells by heat shock method, and the heat shock conditions were: 50 μl Escherichia coli T1 competent cells, 10 μl plasmid DNA (recombinant expression vector DBN100323), 42 ° C water bath for 30 seconds; ice bath cells 1-2 minutes; shake culture in LB liquid medium at 37°C for 1 hour (shaking on a shaker at 100rpm); Cultivate for 12 hours, pick white colonies, and put them in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, kanamycin 50mg/L, adjust pH to 7.5 with NaOH) at temperature Incubate overnight at 37°C. The plasmid was extracted by alkaline method. The extracted plasmid was identified after digestion with restriction endonucleases EcoRV and SmaI, and the positive clones were sequenced and identified. The results showed that the nucleotide sequence between the SpeI and PvuI sites of the recombinant expression vector DBN100323 was SEQ ID in the sequence table The nucleotide sequence shown in NO:1 is the PIC10-01 nucleotide sequence.

3. Construction of a recombinant expression vector DBN100323N containing a known sequence

According to the method of constructing the recombinant cloning vector DBN01-T containing the PIC10-01 nucleotide sequence described in 1 of the second embodiment of the present invention, a known sequence (SEQ ID NO: 6) was used to construct a recombinant clone containing a known sequence Vector DBN01R-T. The positive clones were sequenced and verified, and the results showed that the known sequence inserted into the recombinant cloning vector DBN01R-T was the nucleotide sequence shown in SEQ ID NO: 6 in the sequence listing, that is, the known sequence was inserted correctly.

According to the method for constructing the recombinant expression vector DBN100323 containing the PIC10-01 nucleotide sequence described in 2 of the second embodiment of the present invention, the known sequence was used to construct the recombinant expression vector DBN100323N containing the known sequence. The positive clones were sequenced and verified, and the results showed that the known sequence inserted into the recombinant expression vector DBN100323N was the nucleotide sequence shown in SEQ ID NO: 6 in the sequence listing, that is, the known sequence was inserted correctly.

4. Transformation of recombinant expression vector into Agrobacterium

Transform the correctly constructed recombinant expression vectors DBN100323 and DBN100323N (known sequence) into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) with liquid nitrogen method, and the transformation conditions are: 100 μL Agrobacterium LBA4404 , 3 μL of plasmid DNA (recombinant expression vector); put in liquid nitrogen for 10 minutes, and warm water bath at 37°C for 10 minutes; inoculate the transformed Agrobacterium LBA4404 in LB test tubes and incubate for 2 hours at a temperature of 28°C and a rotation speed of 200 rpm , spread on the LB solid plate containing 50mg/L rifampicin (Rifampicin) and 100mg/L kanamycin (Kanamycin) until a positive monoclonal grows, pick the monoclonal culture and extract its plasmid, use restriction The recombinant expression vectors DBN100323 and DBN100323N (known sequence) were digested with the endonuclease AhdI and EcoRV, and the results showed that the structures of the recombinant expression vectors DBN100323 and DBN100323N (known sequence) were completely correct.

The third embodiment, the acquisition and verification of rice plants transferred to the PIC10-01 nucleotide sequence

1. Obtain rice plants transferred to the PIC10-01 nucleotide sequence

According to the routinely used Agrobacterium infection method, the callus of the aseptically cultured japonica rice variety Nipponbare was co-cultured with the Agrobacterium described in 4 in the second embodiment, so that the recombinant strains constructed in 2 and 3 in the second embodiment The T-DNA (including the promoter sequence of maize Ubiquitin gene, PIC10-01 nucleotide sequence, known sequence, PMI gene and Nos terminator sequence) in the expression vectors DBN100323 and DBN100323N (known sequence) was transferred into rice chromosome In the group, the rice plants transformed with the PIC10-01 nucleotide sequence and the rice plants transformed with the known sequence were obtained; at the same time, wild-type rice plants were used as controls.

For Agrobacterium-mediated transformation of rice, briefly, rice seeds were inoculated in induction medium (N6 salts, N6 vitamins, casein 300 mg/L, sucrose 30 g/L, 2,4-dichlorophenoxyacetic acid (2, 4-D) On 2mg/L, Phytogel 3g/L, pH 5.8), callus was induced from mature rice embryos (step 1: callus induction step), after which, preferably callus, was treated with Agrobacterium The suspension is contacted with callus, wherein the Agrobacterium is capable of delivering the PIC10-01 nucleotide sequence and/or known sequences to at least one cell on the callus (step 2: infection step). In this step, the callus is preferably immersed in Agrobacterium suspension (OD660=0.3, infection medium (N6 salt, N6 vitamin, casein 300mg/L, sucrose 30g/L, glucose 10g/L, acetosyringone (AS) 40mg/L, 2,4-dichlorophenoxyacetic acid (2,4-D) 2mg/L, pH5.4)) to initiate infection. The callus was co-cultured with Agrobacterium for a period of time (3 days) (step 3: co-cultivation step). Preferably, the callus is cultured in solid medium (N6 salt, N6 vitamin, casein 300mg/L, sucrose 30g/L, glucose 10g/L, acetosyringone (AS) 40mg/L, 2, 4-dichlorophenoxyacetic acid (2,4-D) 2mg/L, plant gel 3g/L, pH5.8). After this co-culture phase, there is a "recovery" step. In the "recovery" step, recovery medium (N6 salt, N6 vitamin, casein 300mg/L, sucrose 30g/L, 2,4-dichlorophenoxyacetic acid (2,4-D) 2mg/L, phytocondensate Glue 3 g/L, pH 5.8) in the presence of at least one antibiotic (cephalosporin) known to inhibit the growth of Agrobacterium, without addition of a selection agent for plant transformants (step 4: recovery step). Preferably, calli are grown on solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for infected cells. Next, the inoculated calli are cultured on a medium containing a selection agent (mannose) and growing transformed calli are selected (step 5: selection step). Preferably, the callus is screened in a selective agent solid medium (N6 salt, N6 vitamins, casein 300mg/L, sucrose 10g/L, mannose 10g/L, 2,4-dichlorophenoxyacetic acid (2 , 4-D) 2 mg/L, Phytogel 3 g/L, pH 5.8), resulting in selective growth of transformed cells. Then, the callus regenerates into plants (step 6: regeneration step), preferably, calli grown on media containing selection agents are cultured on solid media (N6 differentiation medium and MS rooting medium) to regenerated plants.

The screened resistant callus was transferred to the N6 differentiation medium (N6 salt, N6 vitamin, casein 300mg/L, sucrose 20g/L, 6-benzylaminoadenine 2mg/L, Naacetic acid 1mg/L, Plant gel 3g/L, pH5.8), cultured and differentiated at 25°C. The differentiated seedlings were transferred to the MS rooting medium (MS salt, MS vitamin, casein 300mg/L, sucrose 15g/L, plant gel 3g/L, pH5.8), and cultivated at 25°C to about 10cm High, moved to the greenhouse to cultivate until firm. In the greenhouse, culture was carried out at 30°C every day.

2. Use TaqMan to verify the rice plants transferred to the PIC10-01 nucleotide sequence

About 100 mg of leaves of rice plants with PIC10-01 nucleotide sequence and rice plants with known sequences were taken as samples, and the genomic DNA was extracted with Qiagen's DNeasy Plant Maxi Kit, and then tested by Taqman probe fluorescence quantitative PCR method Detect the copy number of PIC10 gene. At the same time, wild-type rice plants were used as a control, and the detection and analysis were carried out according to the above method. The experiment was repeated 3 times, and the average value was taken.

The specific method for detecting the copy number of PIC10 gene is as follows:

Step 11. Take 100 mg of leaves of rice plants transferred to PIC10-01 nucleotide sequence, rice plants transferred to known sequences, and wild-type rice plants, respectively, and grind them into homogenate with liquid nitrogen in a mortar, Take 3 repetitions for each sample;

Step 12, use Qiagen's DNeasy Plant Mini Kit to extract the genomic DNA of the above sample, and refer to its product manual for specific methods;

Step 13, using NanoDrop2000 (Thermo Scientific) to measure the genomic DNA concentration of the above sample;

Step 14, adjusting the genomic DNA concentration of the above samples to the same concentration value, the concentration value ranges from 80-100ng/μl;

Step 15, using the Taqman probe fluorescent quantitative PCR method to identify the copy number of the sample, using the sample with known copy number as the standard, and using the sample of the wild-type rice plant as the control, each sample was repeated 3 times, and the average Value; Fluorescence quantitative PCR primer and probe sequences are respectively:

The following primers and probes were used to detect the PIC10-01 nucleotide sequence:

Primer 1 (CF1): CAGGACTGGATCACCTATAATCGG as shown in SEQ ID NO: 7 in the sequence listing;

Primer 2 (CR1): AAAGAACGCGGCAATGTCC as shown in SEQ ID NO: 8 in the sequence listing;

Probe 1 (CP1): CAGGCGCGATCTTACTTTGACGGTCC as shown in SEQ ID NO:9 in the sequence listing;

The following primers and probes were used to detect known sequences:

Primer 3 (CF2): CAAGGAATGGGAAGAAGATCCTAAC as shown in SEQ ID NO: 10 in the sequence listing;

Primer 4 (CR2): TTCAAGAAGTCCATCAAGGATACG as shown in SEQ ID NO: 11 in the sequence listing;

Probe 2 (CP2): CCAGCAACCAGGACCAGAGTGATCGATAG as shown in SEQ ID NO: 12 in the sequence listing;

The PCR reaction system is:

The 50X primer/probe mix contained 45 μl of each primer at a concentration of 1 mM, 50 μl of probe at a concentration of 100 μM and 860 μl of 1X TE buffer, and was stored in amber tubes at 4°C.

The PCR reaction conditions are:

Data were analyzed using SDS2.3 software (Applied Biosystems).

The experimental results showed that both the PIC10-01 nucleotide sequence and the known sequence had been integrated into the genome of the rice plants tested, and the rice plants transferred with the PIC10-01 nucleotide sequence and the rice plants transferred with the known sequence Transgenic rice plants containing a single copy of the PIC10 gene were obtained from the plants.

2. Detection of insect resistance effect of transgenic rice plants

The rice plants transferred to the PIC10-01 nucleotide sequence, the rice plants transferred to the known sequence, the wild-type rice plants and the rice plants identified as non-transgenic by Taqman were tested for their resistance to Sarmaea and Chilo suppressalis.

(1) Chilo suppressalis: rice plants transferred to PIC10-01 nucleotide sequence, rice plants transferred to known sequences, wild-type rice plants, and non-transgenic rice plants (tillering stage) identified by Taqman Rinse the fresh leaves with sterile water and dry the water on the leaves with gauze, then remove the veins from the rice leaves, and cut them into strips of about 1cm×4cm, and put 1 long strip of leaves into On the filter paper at the bottom of a circular plastic petri dish, the filter paper is moistened with distilled water, and 10 artificially reared Chilo suppressalis (newly hatched larvae) are placed in each petri dish. After being placed for 3 days at 28°C, relative humidity 70%-80%, and photoperiod (light/dark) 16:8, the total resistance was obtained according to the three indicators of the developmental progress, mortality rate and leaf damage rate of Chilo borer larvae. Score: total score = 100×mortality+[100×mortality 90×(number of newly hatched worms/total number of inoculated worms)+60×(number of newly hatched-negative control worms/total number of inoculated worms)+10×(negative control worms number/total number of inoculated insects)]+100×(1-leaf damage rate). A total of 3 strains (S1, S2 and S3) transferred with PIC10-01 nucleotide sequence, a total of 3 strains transferred with known sequence (S4, S5 and S6), were identified as non-transgenic by Taqman ( NGM) and 1 wild-type (CK) strain; 3 strains were selected from each strain for testing, and each strain was repeated 6 times. The results are shown in Table 1 and Figure 3.

Table 1. Results of insect resistance experiment of transgenic rice plants inoculated with Chilo borer

The results in Table 2 show that: both the rice plants transferred with the PIC10-01 nucleotide sequence and the rice plants transferred with known sequences can be selected to have certain resistance to Chilo suppressalis, but the PIC10-01 nuclear The total bioassay score of rice plants with nucleotide sequences was significantly higher than that of rice plants with known sequences. The total biometric score of the rice plants transferred with the PIC10-01 nucleotide sequence was about 240 points, while that of the rice plants transferred with the known sequence was about 200 points. The results in Fig. 3 show that: compared with the rice plants transferred with the known sequence, the rice plants transferred with the PIC10-01 nucleotide sequence can cause a large number of deaths of newly hatched larvae, and greatly inhibit the developmental progress of the larvae, After 3 days, the larvae were basically still in the newly hatched state or between the newly hatched and negative control states, and the leaf damage rate was relatively small, about 10%.

(2) Scutellaria spp.: fresh rice plants with PIC10-01 nucleotide sequence, rice plants with known sequence, wild-type rice plants, and non-transgenic rice plants (tiller stage) identified by Taqman were taken respectively. Rinse the leaves with sterile water and dry the water on the leaves with gauze, then remove the veins from the rice leaves, and cut them into strips of about 1cm×4cm. On the filter paper at the bottom of a shaped plastic petri dish, the filter paper is moistened with distilled water, and 10 artificially reared larvae (newly hatched larvae) are placed in each petri dish. , relative humidity 70%-80%, and photoperiod (light/dark) 16:8, after 3 days, the total resistance score was obtained according to the three indicators of the developmental progress of the larvae, mortality and leaf damage rate: total Score=100×mortality+[100×mortality 90×(number of newly hatched worms/total number of inoculated worms)+60×(number of newly hatched-negative control worms/total number of inoculated worms)+10×(number of negative control worms/total number of inoculated worms) total number of insects)]+100×(1-leaf damage rate). A total of 3 strains (S1, S2 and S3) transferred with PIC10-01 nucleotide sequence, a total of 3 strains transferred with known sequence (S4, S5 and S6), were identified as non-transgenic by Taqman ( NGM) and 1 wild-type (CK) strain; 3 strains were selected from each strain for testing, and each strain was repeated 6 times. The results are shown in Table 2 and Figure 4.

Table 2. Insect resistance experiment results of transgenic rice plants inoculated with S.

The results in Table 2 show that plants that have a certain inhibitory effect on S. mori can be selected from the rice plants that have been transferred to the PIC10-01 nucleotide sequence and the rice plants that have been transferred to the known sequence, but the PIC10-01 nucleotide sequence The total bioassay score of rice plants with acid sequence was significantly higher than that of rice plants with known sequence. The total biometric score of the rice plants transferred with the PIC10-01 nucleotide sequence was about 230 points, while that of the rice plants transferred with the known sequence was about 160 points. The results in Fig. 4 show that: compared with the rice plants transferred with the known sequence, the rice plants transferred with the PIC10-01 nucleotide sequence can cause a large number of deaths of newly hatched larvae, and greatly inhibit the developmental progress of the larvae. After 3 days, the larvae were basically still in the newly hatched state or between the newly hatched and negative control states, and the leaf damage rate was also controlled below 15%.

This proves that the rice plants transferred to the PIC10-01 nucleotide sequence have higher insect resistance, that is, the rice plants transferred to the PIC10-01 nucleotide sequence that express a high level of PIC10-01 protein also have higher virulence Therefore, the PIC10-01 nucleotide sequence optimized according to the preferred codons of rice significantly increases the virulence of the PIC10-01 protein expressed in rice.

In summary, the PIC10-01 insecticidal gene of the present invention adopts the preferred codons of rice, making the insecticidal gene of the present invention particularly suitable for expression in monocotyledonous plants, especially rice. The PIC10-01 insecticidal protein of the present invention not only expresses High and stable, strong toxicity to insect pests, especially Lepidoptera insect pests.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be The scheme shall be modified or equivalently replaced without departing from the spirit and scope of the technical scheme of the present invention.

Claims (16)

1. An insecticidal protein, characterized in that, the protein composed of the amino acid sequence shown in SEQ ID NO:2. 2. A kind of insecticidal gene, is characterized in that, is selected from: (a) the nucleotide sequence encoding the insecticidal protein of claim 1; or (b) a nucleotide sequence of a protein that hybridizes to the nucleotide sequence defined in (a) under stringent conditions and encodes an amino acid sequence as shown in SEQ ID NO: 2; or (c) Nucleotide sequence as shown in SEQ ID NO:1. 3. An expression cassette, characterized in that it comprises the insecticidal gene of claim 2 under the regulation of an operably linked regulatory sequence. 4. A recombinant vector comprising the insecticidal gene of claim 2 or the expression cassette of claim 3. 5. A method for producing an insecticidal protein, comprising: obtaining the cells of the transgenic host organism comprising the insecticidal gene of claim 2 or the expression cassette of claim 3; culturing the cells of said transgenic host organism under conditions that permit production of the pesticidal protein; The pesticidal protein is recovered. 6. The method for producing an insecticidal protein according to claim 5, wherein the cells of the transgenic host organism include plant cells, animal cells, bacteria, yeast, baculovirus or algae. 7. The method for producing an insecticidal protein according to claim 6, wherein the plant is corn, soybean, cotton, rice or wheat. 8. A method for increasing the range of insect targets, comprising: using the insecticidal gene according to claim 2 or the expression cassette according to claim 3 in plants with at least one gene different from that described in claim 2 The insecticidal gene or the second insecticidal nucleotide sequence of the expression cassette according to claim 3 are expressed together. 9. The method for increasing the insect target range according to claim 8, characterized in that, the second insecticidal nucleotide sequence encodes Cry class insecticidal protein, Vip class insecticidal protein, protease inhibitor, agglutination α-amylase or peroxidase. 10. The method for increasing the target range of insects according to claim 8, characterized in that, the second insecticidal nucleotide sequence is a dsRNA that suppresses important genes in target insect pests. 11. A method for producing insect-resistant plants, characterized by comprising: introducing the insecticidal gene of claim 2 or the expression cassette of claim 3 or the recombinant vector of claim 4 into plants. 12. The method for producing insect-resistant plants according to claim 11, wherein said plants are corn, soybean, cotton, rice or wheat. 13. A method for protecting plants from damage caused by Lepidoptera insect pests, comprising: applying the insecticidal gene according to claim 2 or the expression cassette according to claim 3 or the expression cassette according to claim 4 The recombinant vector is introduced into plants, so that the introduced plants can produce enough insecticidal protein to protect them from lepidopteran insect pests. 14. The method for protecting plants from damage caused by Lepidoptera insect pests according to claim 13, characterized in that said plants are corn, soybean, cotton, rice or wheat. 15. A method for controlling lepidopteran insect pests, characterized in that, comprising: making the lepidopteran insect pests and the insecticidal protein described in claim 1 or the insects encoded by the insecticidal gene described in claim 2 Inhibitory protein contacts. 16. The use of the insecticidal protein as claimed in claim 1 or the insect-inhibiting protein encoded by the insecticidal gene as claimed in claim 2 in controlling Lepidoptera insect pests.