Insecticidal use of proteins

Abstract

The invention relates to an insecticidal use of a protein which can be used for controlling diplodia diplosa, and a method for controlling the diplodia diplosa comprises the step of delivering at least GRIC23 protein to the diplodia diplosa. The GRIC23 protein disclosed by the invention has a good effect of controlling the diplodia diplosa, and an insect-resistant mechanism can be increased.

Description

Technical Field

[0001] The present invention relates to insecticidal use of a protein, and in particular to the use of a GRIC23 protein for controlling the two-spotted leaf beetle. Background Art

[0002] Coleopteran pests are extremely harmful to agriculture and forestry. Most Coleopteran pests are soil-dwelling pests that live mainly underground and feed on the underground parts of crops, including germinating seeds, rhizomes of seedlings and other tissues. Their feeding behavior often causes a large number of seedling shortages and broken ridges in crop planting areas, affecting the yield and quality of crops in the middle and late stages of crop development. Traditional pesticide prevention of Coleopteran pests needs to be combined with deep plowing and soil turning, which requires a lot of manpower and material resources.

[0003] Monolepta hieroglyphica (Motschulsky) is also called long-tarsed leaf beetle. It belongs to the family Chrysomelidae in the order Coleoptera. It is a new type of holometabolous insect of the family Chrysomelidae that harms many crops. The two-spotted leaf beetle harms corn, mainly from July to September. When the adults harm corn leaves, the leaves become gauze-like in mild cases, and the entire leaves dry up in severe cases. The insect has the habit of gathering in groups and tending to harm tender leaves. It often concentrates on a plant to feed on the corn mesophyll from top to bottom. The tender leaves are bitten into holes. After the middle and lower leaves are eaten, the reticular veins or epidermis remain, which appear as small irregular white spots from a distance, which has a great impact on photosynthesis. After the corn tassels and silks, the insect likes to feed on anthers and filaments, which seriously affects the normal flowering and pollination of corn, and easily causes ear rot.

[0004] Crystallized and secreted soluble insecticidal toxins are highly specific to their hosts and have been recognized worldwide as an alternative to chemical insecticides. For example, insecticidal proteins (such as delta-endotoxins produced by Bt strains) have been used in various agricultural applications to protect agriculturally important plants from insect infestations, reduce the need for chemical insecticide applications, and increase yields. Insecticidal proteins control agriculturally relevant pests of crops by mechanical methods such as spraying to disperse microbial preparations containing various bacterial strains onto plant surfaces, and by using genetic transformation techniques to produce transgenic plants and seeds expressing insecticidal proteins.

[0005] The global use of transgenic plants expressing insecticidal proteins and the limited number of insecticidal proteins used in these crops creates selection pressure on existing insect alleles that confer resistance to currently utilized insecticidal proteins.

[0006] The development of resistance to insecticidal proteins in target insect pests creates a continuing need to discover and develop new forms of insecticidal proteins that can be used to manage the increase in insect resistance in transgenic crops expressing insecticidal proteins. New insecticidal proteins with improved efficacy and that exhibit control over a wider range of susceptible insect species will reduce the number of surviving insects that can develop resistance alleles. In addition, the use of two or more transgenic insecticidal proteins in one plant that are toxic to the same insect pest and exhibit different modes of action can reduce the likelihood of resistance development in any single target insect species.

[0007] To meet this need, the present invention discloses a novel insecticidal protein GRIC23 that is completely different from traditional Bt proteins and exhibits good activity against the coleopteran leaf beetle. The GRIC23 protein of the present invention addresses the continuing need in the art to engineer other insecticidal proteins to have improved insecticidal properties, such as increased efficacy against a wider range of target pest species and different modes of action. Summary of the invention

[0008] The purpose of the present invention is to provide an insecticidal use of a protein, to provide for the first time a method for controlling the two-spotted firefly leaf beetle by expressing the GRIC23 protein, and to effectively supplement an insecticidal protein having a control effect on the two-spotted firefly leaf beetle.

[0009] To achieve the above object, the present invention provides a method for controlling the two-spotted firefly leaf beetle, comprising delivering at least a GRIC23 protein to the two-spotted firefly leaf beetle, wherein the GRIC23 protein has an amino acid sequence shown in SEQ ID NO:1.

[0010] Alternatively, the delivery method is that the two-spotted firefly leaf beetle contacts the GRIC23 protein by ingesting a formulated protein composition containing at least the GRIC23 protein, and the growth of the two-spotted firefly leaf beetle is inhibited and / or the beetle dies after contact.

[0011] Optionally, the delivery method is: the GRIC23 protein is present in at least the bacteria that produce the GRIC23 protein on the surface of the plant part, the two-spotted firefly leaf beetle contacts the GRIC23 protein by feeding on the plant part to which the bacteria are attached, and after contact, the growth of the two-spotted firefly leaf beetle is inhibited and / or it dies.

[0012] Alternatively, the delivery method is: the two-spotted firefly leaf beetle contacts the GRIC23 protein by ingesting at least the transgenic plant part that produces the GRIC23 protein, and after contact, the growth of the two-spotted firefly leaf beetle is inhibited and / or the beetle dies.

[0013] Furthermore, the transgenic plant also produces at least one protein selected from the following group: Cry insecticidal proteins, Vip insecticidal proteins, protease inhibitors, lectins, α-amylases, peroxidases and herbicide tolerance proteins.

[0014] Optionally, the transgenic plants also produce dsRNA that inhibits important genes in the two-spotted leaf beetle.

[0015] Based on the above technical solution, the nucleic acid molecule encoding the GRIC23 protein has a nucleotide sequence shown in SEQ ID NO:2.

[0016] To achieve the above object, the present invention also provides a use of a GRIC23 protein for controlling the two-spotted leaf beetle, wherein the GRIC23 protein has an amino acid sequence shown in SEQ ID NO:1.

[0017] To achieve the above objectives, the present invention also provides a method for producing a plant for controlling the two-spotted leaf beetle, comprising introducing a nucleic acid molecule encoding the GRIC23 protein into the genome of the plant, wherein the GRIC23 protein has an amino acid sequence shown in SEQ ID NO: 1.

[0018] To achieve the above objectives, the present invention also provides a method for producing plant seeds for controlling the two-spotted firefly leaf beetle, comprising selfing the plant obtained by the method, or hybridizing the plant obtained by the method as a first plant with a second plant, thereby producing plant seeds containing a nucleic acid molecule encoding the GRIC23 protein.

[0019] The following description and definition are provided to better define the present invention and guide those skilled in the art to practice the present invention. Unless otherwise specified, terms should be understood according to conventional usage by those skilled in the art.

[0020] In the present invention, the GRIC23 protein is delivered to the two-spotted firefly leaf beetle, which means that the GRIC23 protein can be contacted with the two-spotted firefly leaf beetle in many well-known ways, including but not limited to: (1) providing the GRIC23 protein in a transgenic plant, wherein the two-spotted firefly leaf beetle eats (ingests) one or more parts of the transgenic plant, thereby ingesting the GRIC23 protein expressed in the transgenic plant; (2) providing the GRIC23 protein in a formulated protein composition that can be applied to or incorporated into, for example, an insect growth medium; (3) providing the GRIC23 protein in a protein composition that can be applied to a surface, such as spraying onto the surface of one or more parts of a plant, and the two-spotted firefly leaf beetle eats (ingests) one or more of the sprayed plant parts; (4) bait matrix; or (5) any other protein delivery system known in the art. Therefore, in the present invention, any method of delivery to insects can be used to deliver the GRIC23 protein of the present invention.

[0021] The GRIC23 protein of the present invention is delivered to the two-spotted firefly leaf beetle, and the composition containing the GRIC23 protein can be sprayed onto the surface of one or more parts of the plant. The composition containing the GRIC23 protein is delivered to the plant surface by using any method known to those skilled in the art for applying compounds, compositions, formulations, etc. to the plant surface. Some non-limiting examples of delivery to or contacting plants or parts thereof include spraying, dusting, sprinkling, spreading, misting, atomizing, broadcasting, soaking, soil perfusion, soil incorporation, soaking (e.g., root, soil treatment), dipping, irrigation, coating, leaf or stem penetration, side application or seed treatment, etc. and combinations thereof. Methods for contacting plants or parts thereof with compounds, compositions or formulations are well known in the art.

[0022] The GRIC23 protein of the present invention is delivered to the two-spotted firefly leaf beetle, and the two-spotted firefly leaf beetle can also ingest one or more parts of the transgenic plant expressing the GRIC23 protein.

[0023] The term "control" and / or "prevention and treatment" of the present invention refers to at least delivering the GRIC23 protein to the two-spotted firefly leaf beetle, and after delivery, the growth of the two-spotted firefly leaf beetle is inhibited and / or death is caused. Further, the two-spotted firefly leaf beetle at least contacts the GRIC23 protein by feeding on bacteria or plant tissues, and after contact, the growth of all or part of the two-spotted firefly leaf beetle is inhibited and / or death is caused. Inhibition refers to sublethal, that is, it is not lethal but can cause certain effects in growth and development, behavior, physiology, biochemistry and organization, such as slow growth and / or cessation. At the same time, the plant should be normal in morphology and can be cultured under conventional methods for consumption and / or production of products. In addition, the plant and / or plant seeds for controlling the two-spotted firefly leaf beetle containing the polynucleotide sequence encoding the GRIC23 protein have reduced plant damage compared with non-transgenic wild-type plants under the conditions of artificial inoculation of the two-spotted firefly leaf beetle and / or natural occurrence of the two-spotted firefly leaf beetle, and the specific performance includes but is not limited to improved stem resistance, increased grain weight, and / or increased yield. The "control" and / or "prevention" effect of the GRIC23 protein on the two-spotted firefly leaf beetle can exist independently, and will not be weakened and / or disappear due to the presence of other substances that can "control" and / or "prevent" the two-spotted firefly leaf beetle. Specifically, any part of the transgenic plant (containing a polynucleotide sequence encoding the GRIC23 protein) simultaneously and / or asynchronously exists and / or produces the GRIC23 protein and / or another substance that can control the two-spotted firefly leaf beetle, then the presence of the other substance will neither affect the "control" and / or "prevention" effect of the GRIC23 protein on the two-spotted firefly leaf beetle, nor cause the "control" and / or "prevention" effect to be completely achieved by the other substance, and has nothing to do with the GRIC23 protein. Normally, in the field, the process of two-spotted firefly leaf beetles feeding on plants or parts thereof is short and difficult to observe with the naked eye. Therefore, under conditions of artificial inoculation of two-spotted firefly leaf beetles and / or natural damage by two-spotted firefly leaf beetles, if there are dead two-spotted firefly leaf beetles on any part of the transgenic plant (containing a polynucleotide sequence encoding the GRIC23 protein), and / or two-spotted firefly leaf beetles whose growth is inhibited thereon, and / or the plant has reduced damage compared to non-transgenic wild-type plants, then the method and / or use of the present invention is achieved, that is, the method and / or use of controlling two-spotted firefly leaf beetles is achieved by delivering at least the GRIC23 protein to the two-spotted firefly leaf beetles.

[0024] The Monolepta hieroglyphica (Motschulsky) described in the present invention is a holometabolous insect of the family Chrysomelidae of the order Coleoptera. The adult body is 3.6-4.8 mm long and 2-2.5 mm wide, with 11 filamentous antennae, the ends of which are black and are 2 / 3 of the body length; the compound eyes are large and oval; the pronotum is wider than it is long, with a raised surface and densely covered with many fine punctures; the scutellum is black and triangular; the elytra are covered with fine linear punctures, and each elytra base half has a nearly circular light spot surrounded by black, and the posterior and lateral sides of the light spot are mostly not completely closed, and the rear ends of the two wings are fused into a circle.

[0025] The two-spotted leaf beetle is widely distributed in my country, mainly in Northeast China, North China, Jiangsu, Zhejiang, Hubei, Jiangxi, Fujian, Guangdong, Guangxi, Ningxia, Gansu, Shaanxi, Sichuan, Yunnan, Guizhou, Taiwan and other provinces and regions. It is an omnivorous pest, mainly harming beans, potatoes, alfalfa, corn, chrysanthemum, carrots, cruciferous vegetables, sunflowers, apricots, apples and other crops. The larvae mainly feed on the roots of crops in the field, affecting the root support of the crops, thus affecting the growth of crops; from mid-July every year, its adults can be found in corn and soybean fields to harm leaves; from the end of July to the beginning of August, a large number of adults mainly harm corn silks, bite off the corn silks, seriously affecting pollination, resulting in pointed and spindle-shaped ears, resulting in reduced corn production; then, the two-spotted leaf beetle moves to soybean fields to feed on soybean leaves, and can also move to surrounding vegetable fields to harm vegetables. From 2009 to 2016, the area of ​​corn damaged by the two-spotted leaf beetle increased from 16 million mu to nearly 40 million mu, and the damaged areas also spread from the northwest to major corn producing areas such as the northeast and north China.

[0026] Bacteria described in the present invention include Agrobacterium, Rhizobium, Bacillus, Brevibacillus, Escherichia, Pseudomonas, Klebsiella, Pantoec and Erwinia. The Bacillus species can be Bacillus cereus or Bacillus thuringiensis, and the Brevibacillus can be Brevibacillus laterosperous. The Escherichia can be Escherichia coli.

[0027] The GRIC23 protein of the present invention can be used in combination with insecticidal chemicals, including but not limited to organic chlorides, carbamates, pyrethroids, neonicotinoids and ryanodine.

[0028] The GRIC23 protein of the present invention can be expressed in a host organism by genetic transformation techniques. Genetic transformation techniques known in the art include, but are not limited to, Agrobacterium-mediated method, gene gun method, micro-emission bombardment, direct DNA uptake into protoplasts, electroporation or silicon whisker-mediated DNA introduction. For example, the nucleotide sequence encoding the GRIC23 protein described in the present invention is cloned and expressed in Escherichia coli to produce a recombinant protein for bioassay.

[0029] The GRIC23 protein of the present invention can be transformed to express the GRIC23 protein by recombinant E. coli or any other recombinant bacterial cells under conditions suitable for expressing the GRIC23 protein, thereby preparing an insect inhibitory composition. The insect inhibitory composition can be prepared by drying, lyophilizing, homogenizing, extracting, filtering, centrifuging, settling or concentrating the culture of recombinant cells expressing / producing the GRIC23 protein. Such a process can produce E. coli or other insect pathogenic bacterial cell extracts, cell suspensions, cell homogenates, cell lysates, cell supernatants, cell filtrates or cell pellets. The insect inhibitory composition containing the GRIC23 protein can include bacterial cells, bacterial spores and parasporal inclusion bodies, and can be formulated for a variety of uses, including as an agricultural insect inhibitory spray product or as an insect inhibitory formulation in a dietary bioassay.

[0030] To reduce the possibility of resistance development, the insect inhibitory composition comprising the GRIC23 protein of the present invention may also comprise at least one additional protein. Additional proteins for use in insect inhibitory compositions include any insect inhibitory protein or insect inhibitory dsRNA molecule known to those of ordinary skill in the art.

[0031] For example, in the present invention, the expression of the GRIC23 protein in a transgenic plant can be accompanied by the expression of one or more Cry insecticidal proteins and / or Vip insecticidal proteins. The co-expression of more than one insecticidal protein in the same transgenic plant can be achieved by genetic engineering to make the plant contain and express the required genes. In addition, one plant (the first parent) can express the GRIC23 protein through genetic engineering, and another plant (the second parent) can express Cry insecticidal proteins and / or Vip insecticidal proteins through genetic engineering. By hybridizing the first parent and the second parent, an offspring plant expressing all the genes introduced into the first parent and the second parent is obtained.

[0032] In the present invention, the term "plant part" or "plant tissue" includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clusters, and intact plant cells in plants or parts of the following plants, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, kernels, ears (male or female spikes), cobs, shells, stems, roots, root tips, anthers, filaments, etc.

[0033] The term "transgenic" in the present invention refers to a DNA molecule that is incorporated into the genome of an organism due to human intervention (e.g., genetic transformation technology or gene editing technology). The term "transgenic plant" includes plants into which exogenous nucleic acid molecules have been introduced. Typically, the exogenous nucleic acid sequence is stably integrated into the plant genome so that the nucleic acid sequence is passed on to successive generations. The exogenous nucleic acid sequence can be integrated into the genome alone or as part of a recombinant expression cassette. The "transgenic" of the present invention includes: any cell, cell line, callus, tissue, plant part or plant whose genotype has been changed due to the presence of an exogenous nucleic acid sequence.

[0034] The plants of the present invention include dicotyledonous plants and monocotyledonous plants, such as soybean, wheat, barley, corn, tobacco, rice, rapeseed, cotton or sunflower.

[0035] In the present invention, the term "recombination" refers to a non-natural DNA, protein, cell, seed or organism that is caused by genetic engineering and is not usually present in nature. "Recombinant protein" refers to a protein that is contained in nature and is produced by human intervention, such as a protein consisting of at least two DNA molecules that are heterologous to each other. Wherein, "genetic engineering" refers to the creation of non-natural DNA, protein or organism that is not usually present in nature and requires human intervention. Genetic engineering can be used to use biotechnology, such as one or more techniques in molecular biology, protein biochemistry, bacterial transformation, and plant transformation to conceive and create engineered DNA, protein or organism.

[0036] The substitution, deletion or addition of the amino acid sequence in the present invention is a routine technique in the art, and preferably the amino acid changes are: small property changes, i.e. conservative amino acid substitutions that do not significantly affect the folding and / or activity of the protein; small deletions, usually deletions of about 1-30 amino acids; small amino or carboxyl terminal extensions, such as an amino terminal extension of a methionine residue; a small connecting peptide, such as about 20-25 residues long.

[0037] Examples of conservative substitutions are substitutions within the following amino acid groups: 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 molecule amino acids (such as glycine, alanine, serine, threonine and methionine). Those amino acid substitutions that generally do not change a specific activity are well known in the art, and 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, as well as their reverse interchanges.

[0038] It is obvious to those skilled in the art that such substitutions can occur outside of regions that are important for the function of the molecule and still produce active protein. For the GRIC23 protein of the present invention, the amino acid residues that are essential for its activity and therefore selected not to be substituted can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis. The latter technique is to introduce mutations at each positively charged residue in the molecule and test the insect-resistant activity of the resulting mutant molecule to determine the amino acid residues that are important for the activity of the molecule. The substrate-enzyme interaction site can also be determined by analysis of its three-dimensional structure, which can be determined by techniques such as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling.

[0039] Therefore, amino acid sequences having certain homology with the amino acid sequence shown in the sequence 1 of the present invention are also included in the present invention. These sequences have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the sequence of the present invention.

[0040] Identity, generally, "sequence identity" refers to the degree to which two optimally aligned DNA or amino acid sequences are invariant over the entire range of the alignment window of components (e.g., nucleotides or amino acids). The "identity score" for the aligned segments of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence segment (i.e., the entire reference sequence or a smaller defined portion of the reference sequence). "Identity%" is the identity score multiplied by 100. For example, the BLAST algorithm available from the National Center for Biotechnology Information (National Center for Biotechnology Information; www.ncbi.nlm.nih.gov / ) is determined using default parameters.

[0041] The term "optimized nucleic acid sequence" as used herein means that, where appropriate, the nucleic acid can be optimized to increase expression in the host organism. Thus, where the host organism is a bacterium or a plant, the synthetic nucleic acid can be synthesized using bacterial or plant-preferred codons to improve expression.

[0042] The selection of regulatory sequences in the vector of the present invention is well known to those skilled in the art. The regulatory sequences include, but are not limited to, promoters (e.g., CaMV 35S promoter, maize Ubi promoter, promoter of rice GOS2 gene, etc.), transit peptides (e.g., transit peptides targeting chloroplasts, etc.), terminators (e.g., polyadenylation signal sequence of NOS gene, polyadenylation signal sequence of pinⅡ gene, polyadenylation signal sequence of E9 gene, etc.), enhancers (e.g., CaMV enhancer, FMV enhancer, MMV enhancer, etc.), leaders (e.g., TMV leader, MDMV leader, potyvirus leader, etc.), introns (e.g., maize hsp70 intron, maize ubiquitin intron, CAT-1 intron, etc.), and other regulatory sequences effectively linked to the insecticidal gene.

[0043] In addition, the expression cassette containing the nucleotide sequence encoding the GRIC23 protein of the present invention can also be expressed in plants together with at least one protein encoding a herbicide-tolerant gene, wherein the herbicide-tolerant gene includes, but is not limited to, a glufosinate-tolerant gene (such as a bar gene, a pat gene), a benzylpyridinium-tolerant gene (such as a pmph gene), a glyphosate-tolerant gene (such as an EPSPS gene), a bromoxynil-tolerant gene, a sulfonylurea-tolerant gene, a gene tolerant to the herbicide dalapon, a gene tolerant to cyanamide, or a gene tolerant to glutamine synthetase inhibitors (such as PPT), thereby obtaining a transgenic plant having both high insecticidal activity and herbicide tolerance.

[0044] The articles "a" and "an" used in the present invention refer to one or more than one (ie, at least one). For example, "an element" means one or more elements.

[0045] The term "comprise" or variations thereof, such as "comprising", "including" or "including", "comprising", means including stated elements, integers or steps, or groups of elements, integers or steps, but not excluding any other elements, integers or steps, or groups of elements, integers or steps.

[0046] The present invention provides an insecticidal use of a protein, which has the following beneficial effects:

[0047] 1. Insecticidal activity: The GRIC23 protein of the present invention exhibits insecticidal activity against the coleopteran insect Diabrotica bipunctata.

[0048] 2. Increase the insect resistance mechanism: The GRIC23 protein described in the present invention supplements the insect resistance mechanism of the existing insecticidal protein against the two-spotted firefly leaf beetle, and can effectively delay the development of insect resistance.

[0049] The technical solution of the present invention is further described in detail below through embodiments. DETAILED DESCRIPTION

[0050] The following specific examples further illustrate the technical scheme of the insecticidal use of the protein of the present invention. The methods and operations described in the following examples are exemplary and should not be construed as limiting.

[0051] First Example: Obtaining the GRIC23 protein sequence of the present invention

[0052] The amino acid sequence of the GRIC23 protein is shown in SEQ ID NO: 1 in the sequence listing; the nucleotide sequence thereof is optimized according to the preferred codons of Escherichia coli, as shown in SEQ ID NO: 2 in the sequence listing.

[0053] Second Example: Expression of GRIC23 protein in Escherichia coli

[0054] Synthesize SEQ ID NO:2 in the first embodiment, and connect the aforementioned nucleic acid sequence into the protein expression vector pET28a (Novagen, USA, CAT:69864-3) according to the instructions to obtain a recombinant expression vector. The recombinant expression vector containing the nucleotide sequence of the protein GRIC23 was transformed into Escherichia coli BL21 (DE3) competent cells (Transgen, China, CAT:CD501) using a heat shock method known in the art to express the GRIC23 protein. The expressed protein was extracted using a protein extraction method known in the art and finally dissolved in a CBS solution (Sigma-Aldrich, China, CAT:3041). The BSA quantitative method was used to estimate the concentration of the dissolved protein solution.

[0055] Example 3: Determination of insecticidal activity of GRIC23 protein

[0056] The protein solution after the concentration determined in the second embodiment is used for testing. Artificial feed (feed formula reference Development of an artificial diet for the western corn rootworm, Entomologia Experimentalis et Applicata 105: 1-11, 2002.) is added to the culture dish, and about 100 μl of protein solution is added to each gram of artificial feed, so that the final concentration is 50 μg / g-200 μg / g, and the protein solution expressing GFP fluorescent protein is used as a negative control. For the protein solution of GRIC23 protein and GFP fluorescent protein, the two-spotted firefly leaf beetle is tested, and 15 two-spotted firefly leaf beetle larvae are placed in each culture dish. On the 4th, 6th and 8th days respectively, the mortality rate was investigated, and the mortality rate = the number of dead insects / total number of insects × 100%, and the mortality rate data are shown in Table 1.

[0057] Table 1. Insect resistance results of feeding GRIC23 protein to the two-spotted leaf beetle

[0058]

[0059] The bioassay results in Table 1 show that the GRIC23 protein has significantly better insecticidal activity than the GFP fluorescent protein for the Coleoptera: Diplocaulus bipunctatus, and the lethality rate on the 8th day can reach 90%.

[0060] In summary, the present invention discloses for the first time that the GRIC23 protein is active against the two-spotted firefly leaf beetle, can kill the two-spotted firefly leaf beetle or have adverse effects on its growth and development, thereby controlling it in the field, and at the same time supplements a new insect resistance mechanism for delaying the development of insect resistance.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for controlling the two-spotted leaf beetle, characterized in that: The method comprises delivering at least GRIC23 protein to the two-spotted firefly leaf beetle, wherein the GRIC23 protein has an amino acid sequence shown in SEQ ID NO:

1.

2. The method for controlling the two-spotted leaf beetle according to claim 1, characterized in that: The delivery method is: the two-spotted firefly leaf beetle contacts the GRIC23 protein by ingesting a formulated protein composition containing at least the GRIC23 protein, and after contact, the growth of the two-spotted firefly leaf beetle is inhibited and / or the beetle dies.

3. The method for controlling the two-spotted leaf beetle according to claim 1, characterized in that: The delivery method is: the GRIC23 protein exists in at least the bacteria that produce the GRIC23 protein on the surface of the plant part, the two-spotted firefly leaf beetle contacts the GRIC23 protein by feeding on the plant part to which the bacteria are attached, and after contact, the growth of the two-spotted firefly leaf beetle is inhibited and / or it dies.

4. The method for controlling the two-spotted leaf beetle according to claim 1, characterized in that: The delivery method is: the two-spotted firefly leaf beetle contacts the GRIC23 protein by ingesting at least the transgenic plant part that produces the GRIC23 protein, and after the contact, the growth of the two-spotted firefly leaf beetle is inhibited and / or the beetle dies.

5. The method for controlling the two-spotted leaf beetle according to claim 4, characterized in that: The transgenic plant also produces at least one protein selected from the following group: Cry insecticidal proteins, Vip insecticidal proteins, protease inhibitors, lectins, α-amylases, peroxidases and herbicide tolerance proteins.

6. The method for controlling the two-spotted leaf beetle according to claim 4, characterized in that: The transgenic plants also produced dsRNA that inhibited important genes in the two-spotted leaf beetle.

7. The method for controlling the two-spotted leaf beetle according to any one of claims 1 to 6, characterized in that: The nucleic acid molecule encoding the GRIC23 protein has a nucleotide sequence shown in SEQ ID NO:

2.

8. Use of a GRIC23 protein for controlling the two-spotted leaf beetle, wherein the GRIC23 protein has the amino acid sequence shown in SEQ ID NO:

1.

9. A method for producing a plant for controlling the two-spotted leaf beetle, characterized in that: The method comprises introducing a nucleic acid molecule encoding the GRIC23 protein into the genome of the plant, wherein the GRIC23 protein has an amino acid sequence shown in SEQ ID NO:

1.

10. A method for producing plant seeds for controlling the two-spotted leaf beetle, characterized in that: The method comprises selfing the plant obtained by the method of claim 9, or crossing the plant obtained by the method of claim 9 as a first plant with a second plant, thereby producing plant seeds containing the nucleic acid molecule encoding the GRIC23 protein.

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