Application of Tma12 protein in control of stinkbug pests and thrip pests

Abstract

The invention relates to an application of an insecticidal protein, in particular to an application of a Tma12 protein in controlling stinkbug pests and thrip pests. The Tma12 protein comprises an amino acid sequence as shown in SEQ ID NO: 1, has excellent insect-resistant characteristic, particularly can be used for protecting the whole growth period and whole plant of plants so as to prevent and control the invasion of pests such as bugs, miridae, marginaceae and Frankliniella occidentalis, and is pollution-free, residue-free, stable and thorough in effect, simple, convenient and economical.

Description

Technical Field

[0001] This invention relates to the use of an insecticidal protein, and more particularly to the application of a Tma12 protein in controlling stink bugs and thrips. Background Technology

[0002] Stink bugs and thrips are serious agricultural pests that cause severe losses to agricultural production. Current control methods primarily rely on physical and chemical control. Physical control includes clearing weeds from fields and roadsides, and deep tilling in autumn to kill some eggs, but the results are not ideal. Chemical control mainly involves spraying insecticides, but problems such as phytotoxicity and pesticide residues necessitate the search for better and more environmentally friendly pest control methods. Therefore, developing and utilizing genetic engineering methods to control stink bugs and thrips has significant economic, environmental, and social value. Summary of the Invention

[0003] To address the aforementioned problems in the existing technology, this invention provides an application of Tma12 protein in controlling stink bugs and thrips pests, resulting in excellent insect-resistant properties. In particular, it can protect plants throughout their entire growth period and the entire plant to prevent damage from pests such as stink bugs, mirid bugs, vespiders, and western flower thrips. Moreover, it is pollution-free, residue-free, and has stable and thorough effects, and is simple, convenient, and economical.

[0004] The present invention provides a method for controlling stink bugs and / or thrips pests, comprising contacting the pests with at least the Tma12 protein.

[0005] In one specific embodiment, the Tma12 protein comprises the amino acid sequence shown in SEQ ID NO:1;

[0006] In one embodiment, the Tma12 protein is encoded by the nucleotide sequence shown in SEQ ID NO:2 or its complementary sequence.

[0007] In one embodiment, the Tma12 protein is present in at least the host cells that produce the Tma12 protein, and the pest comes into contact with the Tma12 protein at least by ingesting the host cells;

[0008] In one specific embodiment, the Tma12 protein is present in at least the bacteria or transgenic plants that produce the Tma12 protein, and the pest comes into contact with the Tma12 protein by ingesting the tissues of the bacteria or the transgenic plants. Upon contact, the growth of the pest is inhibited and / or it leads to death, thereby achieving control over its damage to the plant.

[0009] In one specific embodiment, the genetically modified plant is preferably soybean.

[0010] In one specific embodiment, the pest is preferably a stink bug, mirid bug, vespider, or western flower thrips pest.

[0011] In one embodiment, the transgenic plant further includes at least one second nucleotide different from the nucleotide encoding the Tma12 protein.

[0012] In another specific embodiment, the second nucleotide encodes other Cry-type insecticidal proteins, Vip-type insecticidal proteins, protease inhibitors, lectins, α-amylases, or peroxidases; or the second nucleotide is dsRNA that inhibits important genes in the target insect pest.

[0013] The present invention also provides the use of the Tma12 protein in controlling stink bugs and / or thrips pests.

[0014] In one specific embodiment, the Tma12 protein comprises the amino acid sequence shown in SEQ ID NO:1;

[0015] In one embodiment, the Tma12 protein is encoded by the nucleotide sequence shown in SEQ ID NO:2 or its complementary sequence.

[0016] The present invention also provides a method for producing a plant for controlling stink bugs and / or thrips pests, comprising introducing a nucleotide sequence encoding the Tma12 protein into the genome of said plant.

[0017] In one specific embodiment, the Tma12 protein comprises the amino acid sequence shown in SEQ ID NO:1;

[0018] In one embodiment, the Tma12 protein is encoded by the nucleotide sequence shown in SEQ ID NO:2 or its complementary sequence.

[0019] The present invention also provides a method for producing plant seeds for controlling stink bugs and / or thrips pests, comprising crossing a first plant obtained by the method with a second plant to produce seeds containing a nucleotide sequence encoding the Tma12 protein.

[0020] The present invention also provides a method for cultivating plants for controlling stink bugs and / or thrips pests, comprising: planting at least one plant seed, wherein the genome of the plant seed includes a nucleotide sequence encoding the Tma12 protein;

[0021] To allow the plant seeds to grow into plants;

[0022] The plants are grown under conditions of artificial inoculation with the pest and / or natural pest damage, and the resulting plants are harvested that exhibit reduced plant damage and / or increased plant yield compared to other plants that do not have a nucleotide sequence encoding the Tma12 protein.

[0023] In one specific embodiment, the Tma12 protein comprises the amino acid sequence shown in SEQ ID NO:1;

[0024] In one embodiment, the Tma12 protein is encoded by the nucleotide sequence shown in SEQ ID NO:2 or its complementary sequence.

[0025] Some of the terms used in this specification are defined as follows.

[0026] In this invention, "plant" should be understood as any differentiated multicellular organism capable of photosynthesis, especially monocotyledonous or dicotyledonous plants.

[0027] In this invention, the term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant callus, plant blocks, as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, spikes, roots, root tips, anthers, etc.

[0028] In this invention, "plant cell" should be understood as any cell derived from or found in a plant that is capable of forming, for example, undifferentiated tissues such as callus, differentiated tissues such as embryos, components of a plant, or a seed.

[0029] In this invention, "host organism" should be understood as any single-celled or multi-celled organism into which mutant protein-encoding nucleic acids can be introduced, including, for example, bacteria such as Escherichia coli, fungi such as yeast (e.g., Saccharomyces cerevisiae), molds (e.g., Aspergillus), plant cells, and plants.

[0030] The terms "protein," "polypeptide," and "peptide" are used interchangeably in this invention to refer to polymers of amino acid residues, including polymers in which one or more amino acid residues are chemical analogs of natural amino acid residues. The proteins and polypeptides of this invention can be generated through recombinant synthesis or through chemical synthesis.

[0031] The term "transgenic" plant refers to a plant containing heteropolynucleotides. Preferably, the heteropolynucleotides are stably integrated into the genome, allowing the polynucleotides to be passed on to successive generations. Heteropolynucleotides may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" herein refers to any cell, cell line, callus, tissue, plant part, or plant whose genotype has been altered due to the presence of heteronucleotides, including those originally altered transgenic organisms or cells, and those produced from hybridization or asexual reproduction of the initial transgenic organism or cell. As used herein, the term "transgenic" is not intended to include changes to the genome (chromosomal or extrachromosomal) by conventional plant breeding methods (e.g., hybridization) or by naturally occurring events (e.g., self-fertilization, random hybridization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation). The transgenic plant may be at any stage of growth.

[0032] In this invention, "contact" refers to insects and / or pests touching, staying on, and / or feeding on plants, plant organs, plant tissues, or plant cells. The plants, plant organs, plant tissues, or plant cells may express insecticidal proteins within themselves, or they may have insecticidal proteins on their surface and / or have microorganisms that produce insecticidal proteins.

[0033] The step preceding the contact step is to grow a plant containing a polynucleotide encoding the Tma12 protein.

[0034] In this invention, "control" and / or "prevention" refers to pests coming into contact with the Tma12 protein at least, resulting in inhibited growth and / or death of the pests after contact. Further, the pests ingest plant tissues and come into contact with the Tma12 protein at least, resulting in the inhibition of all or part of the pests' growth and / or death after contact. Inhibition refers to sublethality, meaning it does not cause death but induces some effect on growth, development, behavior, physiology, biochemistry, and tissue aspects, such as slowed and / or stopped growth. Simultaneously, the plant should be morphologically normal and culturable under conventional methods for product consumption and / or generation. Furthermore, plants and / or seeds containing the polynucleotide sequence encoding the Tma12 protein that control pests, under conditions of artificial inoculation with pests and / or natural pest damage, exhibit reduced plant damage compared to non-transgenic wild-type plants, specifically manifested in, but not limited to, improved stem resistance, and / or increased grain weight, and / or increased yield. The "control" and / or "prevention" effect of the Tma12 protein on pests can exist independently and is not weakened or eliminated by the presence of other substances that can "control" and / or "prevent" pests. Specifically, if any tissue of a transgenic plant (containing a polynucleotide sequence encoding the Tma12 protein) simultaneously and / or asynchronously contains and / or produces the Tma12 protein and / or another substance that can control pests, then the presence of the other substance neither affects the "control" and / or "prevention" effect of the Tma12 protein on pests, nor causes the "control" and / or "prevention" effect to be wholly and / or partially achieved by the other substance, and is unrelated to the Tma12 protein. Typically, in the field, the process of pests feeding on plant tissues is brief and difficult to observe with the naked eye. Therefore, under conditions of artificial inoculation of pests and / or natural pest damage, such as the presence of dead pests in any tissue of a transgenic plant (containing a polynucleotide sequence encoding the Tma12 protein), and / or pests with inhibited growth remaining on it, and / or reduced plant damage compared to non-transgenic wild-type plants, the method and / or use of the present invention is achieved, namely, the method and / or use of controlling pests by having the pests at least come into contact with the Tma12 protein.

[0035] In this invention, the expression of the Tma12 protein in a transgenic plant can be accompanied by the expression of one or more Cry-type insecticidal proteins and / or Vip-type insecticidal proteins. The co-expression of more than one insecticidal protein in the same transgenic plant can be achieved through genetic engineering to include and express the desired genes in the plant. Alternatively, one plant (the first parent) can be genetically engineered to express the Tma12 protein, and a second plant (the second parent) can be genetically engineered to express Cry-type and / or Vip-type insecticidal proteins. Offspring plants expressing all genes introduced from both the first and second parents are obtained through hybridization.

[0036] The Hemiptera described in this invention belong to the class Insecta, subclass Pterygota, and class Hemimetamorphosis, including bugs and homoptera (cicadas, aphids, scale insects, etc.). According to M. Carver (1991), they are divided into three suborders: Sternorrhyncha, Auchenorrhyncha, and Heteroptera. Sternorrhyncha and Auchenorrhyncha are sometimes combined into Homoptera, or they may be separate orders. They have a postcephalic mouthpart, with piercing-sucking mouthparts located at the front of the head. The beak formed by the labium is usually four-segmented, extending from the front of the head, away from the coxae of the forelegs. They are distributed throughout all major zoogeographical regions of the world, with the richest diversity in tropical and subtropical regions. A few are carnivorous. Most are herbivorous, harming crops, fruit trees, forest trees, or weeds by sucking the sap from their stems, leaves, or fruits, causing a certain degree of damage to agriculture.

[0037] The stink bug described in this invention belongs to the class Insecta, order Hemiptera. Its main characteristics are: the basal half of the forewing is leathery and thickened, while the distal half is membranous. Stink bugs have piercing-sucking mouthparts, forming long, beak-like structures suitable for sucking plant sap or animal body fluids. The prothorax is well-developed, with the pronotum typically exhibiting various hexagonal shapes. A scent gland pore is often located on the metathorax near the coxae of the midlegs. This pore contains a scent gland that secretes a foul-smelling liquid that overflows from the pore and evaporates into the air, creating a strong, unpleasant odor, hence the nickname "Stinky Sister." Stink bugs undergo incomplete metamorphosis and are mostly pests, damaging crops such as soybeans, sesame, and peanuts. They belong to families such as Pentatomidae, Lysimachiidae, Elapidae, Tractidae, and Miridae.

[0038] The thrips described in this invention refer to the general term for insects belonging to the order Thysanoptera. Adult thrips have yellow bodies with marginal bristles along the posterior margin of the prothorax, and long, transparent wings with numerous fine hairs along the periphery. Eggs are oblong, initially white and slightly transparent, later turning orange-red. Nymphs are pale yellow, turning pinkish-red when mature, and include species such as the western flower thrips, bean thrips, and tobacco thrips.

[0039] The genome of a plant, plant tissue, or plant cell as described in this invention refers to any genetic material within a plant, plant tissue, or plant cell, including the nucleus and plastid genome and the mitochondrial genome.

[0040] The terms "polynucleotide," "nucleic acid," "nucleic acid molecule," or "nucleic acid sequence" used in this invention are used interchangeably and refer to oligonucleotides, nucleotides, or polynucleotides and fragments or portions thereof, which may be single-stranded or double-stranded, and indicate sense or antisense strands. Nucleic acids include DNA, RNA, or hybrids thereof, and may have natural or synthetic origins. For example, nucleic acids may include mRNA or cDNA. Nucleic acids may include nucleic acids that have been amplified (e.g., using polymerase chain reaction). The nucleotide name "R" indicates a purine, such as guanine or adenine; "Y" indicates a pyrimidine, such as cytosine or thymine (or uracil if it is RNA); "M" indicates adenine or cytosine; "K" indicates guanine or thymine; and "W" indicates adenine or thymine.

[0041] In this invention, "isolation," when referring to nucleic acids, means nucleic acids that are separated from the substantial portion of the genome in which they are naturally present and / or substantially separated from other cellular components naturally accompanying the nucleic acid. For example, any nucleic acid that has been synthesized (e.g., by sequential base condensation) is considered isolated. Similarly, recombinantly expressed nucleic acids, cloned nucleic acids, nucleic acids produced by primer extension reactions (e.g., PCR), or other nucleic acids excised from the genome are also considered isolated.

[0042] Those skilled in the art will readily understand that, due to the degeneracy of the genetic code, a variety of different nucleic acid sequences can encode the amino acid sequences disclosed herein. Generating other nucleic acid sequences encoding the same protein is within the capabilities of those skilled in the art; therefore, this invention covers nucleic acid sequences encoding the same amino acid sequence due to the degeneracy of the genetic code. For example, to achieve high expression of a heterologous gene in a target host organism such as a plant, the gene can be optimized using codons preferred by the host organism to improve its expression.

[0043] The genes and proteins described in this invention include not only specific example sequences, but also portions and / or fragments (including deletions at the ends compared to the full-length protein), variants, mutants, substitutes (proteins with substituted amino acids), chimeras, and fusion proteins that preserve the insecticidal activity characteristics of the specific example proteins. The term "variant" or "mutation" refers to a nucleotide sequence encoding the same protein or an equivalent protein with insecticidal activity. The term "equivalent protein" refers to a protein having the same or substantially the same biological activity against hemiptera pests as the protein of the claims.

[0044] The gene encoding the Tma12 protein described in this invention can be introduced into plants using methods commonly used in the industry, and can be transgenic through appropriate plant transformation expression vectors.

[0045] Using any appropriate promoter, including vectors, is a common practice in the plant transgenic industry. For example, commonly used promoters in plant transgenics include, but are not limited to, the SP6 promoter, T7 promoter, T3 promoter, PM promoter, maize ubiquitin promoter, cauliflower mosaic virus (CaMV) 35S promoter, alpha-linolenic acid synthase (NOS) promoter, Scrophularia mosaic virus 35S promoter, sugarcane stalk virus promoter, bamboo mottle virus promoter, light-induced ribulose-1,5-ketocarboxylase (ssRUBISCO small subunit) promoter, rice cytoplasmic triose phosphate isomerase (TPI) promoter, Arabidopsis thaliana adenine transphosphoribosylase (APRT) promoter, octopine synthase promoter, and BCB (copper-binding protein) promoter.

[0046] Plant transgenic vectors include polyadenylated signal sequences that can induce 3'-terminal polyadenylation. Examples include, but are not limited to, the NOS 3'-terminal derivative of the Agrobacterium tumefaciens alpha-lipoic acid synthase gene, the 3'-terminal derivative of the octopine synthase gene of Agrobacterium tumefaciens, the 3'-terminus of the tomato or potato protease resistance I or II gene, the CaMVPoly A signal sequence, the 3'-terminus of the rice α-amylase gene, and the 3'-terminus of the betaine gene.

[0047] Vectors also include coding genes that can be selectively labeled as reporter molecules. Examples of selective labeling include, but are not limited to, antibiotic (e.g., neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, bleomycin, chloramphenicol, etc.) or herbicide resistant (glyphosate, glufosinate, glufosinate, etc.) genes.

[0048] The vector transformation methods include Agrobacterium-mediated transformation, electroporation, microparticle bombardment, and polyethylene glycol-medium absorption to introduce recombinant plasmids into plants. In this invention, plant transformation recipients include plant cells (including suspension culture cells), protoplasts, callus tissue, hypocotyls, seeds, cotyledons, buds, and mature plants.

[0049] The scope of transgenic plants includes not only contemporary plants from which genes have been introduced, but also their clones and offspring (T1, T2, or subsequent generations). The scope of this invention also includes all mutants and variants of the aforementioned transgenic plants that exhibit characteristics of the primary transgenic plant after hybridization and fusion. The scope of this invention also includes parts of a plant, such as seeds, flowers, stems, fruits, leaves, roots, tubers, or rhizomes, derived from a plant that has been genetically modified in advance using the methods mentioned in this invention, or its offspring, and which must consist at least of a portion of genetically modified cells.

[0050] In this invention, "insecticide" or "insect-resistant" refers to insects that are toxic to crop pests, thereby achieving "control" and / or "prevention" of crop pests. Preferably, "insecticide" or "insect-resistant" means killing crop pests. More specifically, the target insects are hemiptera pests such as the spotted leaf bug and the green mirid bug.

[0051] In this invention, the Tma12 protein is toxic to pests. The plants used in this invention, particularly soybeans, contain exogenous DNA in their genome, which includes a nucleotide sequence encoding the Tma12 protein. Pests come into contact with this protein by ingesting plant tissues, and upon contact, their growth is inhibited and / or they die. Inhibition refers to lethality or sublethality. Simultaneously, the plants should be morphologically normal and can be cultured using conventional methods for product consumption and / or generation. Furthermore, this plant essentially eliminates the need for chemical or biological pesticides (specifically, pesticides targeting pests targeted by the Tma12 protein).

[0052] The expression level of insecticidal proteins in plant materials can be detected by a variety of methods described in the art, such as quantifying the mRNA encoding insecticidal proteins produced in tissues by applying specific primers, or directly and specifically detecting the amount of insecticidal proteins produced.

[0053] Different experiments can be used to determine the insecticidal effect of insecticidal proteins in plants. The target insects in this invention are mainly hemiptera pests such as stink bugs, mirid bugs, and vespiders, or thrips such as western flower thrips and bean thrips.

[0054] In this invention, the Tma12 protein may have the amino acid sequence shown in SEQ ID NO:1 of the sequence listing. In addition to the coding region of the Tma12 protein, it may also contain other elements, such as proteins encoding selective markers.

[0055] Furthermore, the expression cassette containing the nucleotide sequence encoding the Tma12 protein of the present invention can also be expressed in plants along with at least one protein encoding a herbicide resistance gene, including but not limited to glufosinate resistance genes (such as bar genes, pat genes), benzyladenine resistance genes (such as pmph genes), glyphosate resistance genes (such as EPSPS genes), bromoxynil resistance genes, sulfonylurea resistance genes, herbicide resistance genes, ammonia nitrile resistance genes, or glutamine synthetase inhibitor resistance genes (such as PPT), thereby obtaining transgenic plants that have both high insecticidal activity and herbicide resistance.

[0056] In this invention, exogenous DNA is introduced into plants, such as introducing the gene encoding the Tma12 protein, expression cassette, or recombinant vector into plant cells. Conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, microemission bombardment, direct DNA uptake into protoplasts, electroporation, or whisker-silicon-mediated DNA introduction.

[0057] The terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting. Unless otherwise expressly stated herein, the terms “a,” “an,” and “the” as used in the foregoing include their plural forms. The terms “comprises” and / or “comprising,” or “includes” and / or “including” as used herein specifically refer to the presence of the features, factors, and / or ingredients described herein, without excluding the presence and addition of one or more other features, factors, and ingredients. The term “and / or” as used above includes all or one of the items in the list of combinations.

[0058] This invention has been described in detail through a series of embodiments, but the invention is not limited to the disclosed embodiments. Any variations, substitutions, or replacements that fall within the scope of this invention, not described herein, may be modified according to public needs. Attached Figure Description

[0059] Figure 1 Image of soybean transformation vector pQY007185.

[0060] Figure 2 A comparison of the consumption of western flower thrips on transgenic Tma12 soybean (pQY7185). CK represents the non-transgenic soybean recipient, and event-1-event-3 correspond to the three transgenic event numbers in Table 1.

[0061] Sequence Description

[0062] Serial Number name SEQ ID NO:1 Tma12 protein amino acid sequence SEQ ID NO:2 Tma12 nucleotide sequence Detailed Implementation

[0063] The following embodiments are provided to provide those skilled in the art with a complete disclosure and description of how to prepare and use the invention, and these embodiments are not intended to limit the scope of the invention as viewed by the inventors, nor are they intended to represent or imply that the experiments described below are all or only the experiments performed. Those skilled in the art will understand that many variations and / or modifications can be made to the invention shown in specific aspects without departing from the spirit or scope broadly described herein. Therefore, aspects herein are to be considered illustrative rather than restrictive in all respects.

[0064] Example 1: Construction of a recombinant expression vector containing Tma12 protein

[0065] The synthesized Tma12 nucleotide sequence (SEQ ID NO:2) was ligated into the protein expression vector pET28a (Novagen, USA, CAT: 69864-3) according to the instructions for the Novagen pET28a vector, resulting in the recombinant expression vector pQY-Tma12.

[0066] Example 2: Obtaining Tma12 protein by transforming Escherichia coli with recombinant expression vector.

[0067] The recombinant expression vector pQY-Tma12 was transformed into Escherichia coli BL21(DE3) competent cells (Transgen, China, CAT: CD501) using the heat shock method. Positive clones were picked, cultured, and the cells were collected. The supernatant was discarded, and the cells were resuspended in PBS and sonicated. The cells were purified using a nickel column, and the expressed protein was detected by SDS-PAGE to estimate the protein concentration. The cells were then stored at -20°C for later use.

[0068] Example 3: Identification of the insect-resistant effect of feeding Tma12 protein on spotted leaf bugs and green mirid bugs.

[0069] The Tma12 protein obtained in Example 2 was used to test its insecticidal effect against the spotted cricket bug (family Ceratopodidae) and the green mirid bug (family Mirididae). The Tma12 protein solution was diluted to different concentration gradients, and equal volumes of each dilution were mixed into the feed. An equal volume of buffer solution was mixed with the feed as the control (CK). Each treatment had five replicates, with 15 nymphs per replicate. Mortality rates were assessed on days 3 and 6, with the feed containing the protein dilution replaced on day 3. Results showed that on day 3, when fed 400 μg / g Tma12 protein, the mortality rate of the green mirid bug reached 100%, and that of the spotted cricket bug reached 71%. On day 6, the mortality rate of the spotted cricket bug also reached 100%. On day 6, when fed 200 μg / g Tma12 protein, the mortality rates of both the green mirid bug and the spotted cricket bug reached 100%. The above results indicate that the Tma12 protein has excellent insecticidal activity against the spotted leaf bug and the green mirid bug.

[0070] Example 4: Identification of the insecticidal effect of feeding Tma12 protein on western flower thrips

[0071] The Tma12 protein obtained in Example 2 was used to test its insecticidal effect against western flower thrips. The Tma12 protein solution was diluted to different concentration gradients, and equal volumes of each dilution were mixed into the feed. An equal volume of buffer solution was mixed with the feed as the control (CK). Each treatment group had six replicates, with ten nymphs per replicate. Mortality rates were assessed on days 3 and 6, with the feed containing the protein dilution replaced on day 3. The results showed that the mortality rate of western flower thrips reached 41.7% on day 3 and 98.3% on day 6 after feeding with 750 μg / ml Tma12 protein, indicating that the Tma12 protein has excellent insecticidal activity against western flower thrips.

[0072] Example 5: Detection of the insecticidal activity spectrum of Tma12 protein

[0073] In addition to *Echinochloa crus-galli*, *Echinochloa chinensis*, and *Thrips serrata*, this invention also tested the insecticidal activity of Tma12 protein against other pests, such as the hemiptera pests *Echinochloa crus-galli*, *Echinochloa chinensis*, *Planthopper*, and *Brassica napus*, and the lepidopteran pests *Fall Armyworm* and *Armorworm*, using an in vitro feeding method. It was found that compared to the buffer control, Tma12 protein could inhibit the growth and development of *Echinochloa crus-galli* and *Echinochloa chinensis*, but had no insecticidal activity against the hemiptera pests *Planthopper* and *Brassica napus*, and the lepidopteran pests *Fall Armyworm* and *Armorworm*.

[0074] Example 6: Test of insect resistance in genetically modified soybeans

[0075] 1. Carrier construction and transformation

[0076] Construct the soybean Tma12 protein expression vector pQY007185, as follows: Figure 1 As shown, using pCambia1300 as the vector backbone, its hygromycin expression cassette and lacZ gene were removed, and two Bsa1 restriction sites were introduced. After Bsa1 digestion, a Pat selection marker expression cassette was introduced. Based on this vector, the Tma12 gene expression cassette was introduced through seamless cloning of three fragments with the backbone vector. The above expression vector was transformed into soybean recipients using Agrobacterium-mediated cotyledonary node transformation to obtain transgenic soybean plants.

[0077] 2. Insect resistance test of T0 generation genetically modified soybeans

[0078] Cut cotton wool and filter paper and place them at the bottom of the test box, soaking them until the surface is dry. Select healthy, disease-free leaves, taking one compound leaf with petiole from each plant, wrapping it in moistened cotton wool, sealing it tightly with film, and placing it in the box. Select healthy second-instar nymphs of western flower thrips with uniform growth, 30 per box. Investigate the survival and development of the test insects after 7 days. Use non-transgenic soybean recipient (CK) as the control group, and use univariate analysis to compare the differences in survival rates between different treatments. The survival and development of western flower thrips on transgenic soybean pQY7185 are shown in Table 1. The feeding of western flower thrips on transgenic soybean pQY7185 and non-transgenic soybean recipient (CK) are compared as follows. Figure 2 As shown.

[0079] Table 1. Survival and development of western flower thrips on transgenic soybean pQY7185

[0080]

[0081] The results showed that the survival rate and development of western flower thrips that fed on Tma12 soybean (pQY7185) were significantly lower than those of the control group (as shown in Table 1). The amount of western flower thrips consumed from Tma12 soybean was significantly lower than that from non-transgenic soybean. Figure 2 This indicates that the introduction of Tma12 endows soybean plants with excellent resistance to western flower thrips.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for controlling stink bugs and / or thrips, characterized in that, This includes contacting the pest with at least the Tma12 protein; preferably, the Tma12 protein comprises the amino acid sequence shown in SEQ ID NO:1; more preferably, the Tma12 protein is encoded by the nucleotide sequence shown in SEQ ID NO:2 or its complementary sequence.

2. The method according to claim 1, characterized in that, The Tma12 protein is present in at least the host cells that produce the Tma12 protein, and the pest comes into contact with the Tma12 protein by ingesting the host cells.

3. The method according to claim 1, characterized in that, The Tma12 protein is present in at least the bacteria or transgenic plants that produce the Tma12 protein. The pest comes into contact with the Tma12 protein by ingesting the tissues of the bacteria or the transgenic plants. Upon contact, the growth of the pest is inhibited and / or it leads to death, thereby controlling its damage to the plant.

4. The method according to claim 3, characterized in that, The genetically modified plant is preferably soybean; or the pest is preferably a stink bug, mirid bug, vespider, or western flower thrips pest.

5. The method according to claim 3 or 4, characterized in that, The transgenic plant also includes at least one second nucleotide that is different from the nucleotide encoding the Tma12 protein.

6. The method according to claim 5, characterized in that, The second nucleotide encodes other Cry-type insecticidal proteins, Vip-type insecticidal proteins, protease inhibitors, lectins, α-amylases, or peroxidases; or the second nucleotide is a dsRNA that inhibits important genes in the target insect pest.

7. Use of a Tma12 protein in controlling stink bugs and / or thrips pests; preferably, the Tma12 protein comprises the amino acid sequence shown in SEQ ID NO:1; more preferably, the Tma12 protein is encoded by the nucleotide sequence shown in SEQ ID NO:2 or its complementary sequence.

8. A method for producing plants for controlling stink bugs and / or thrips pests, characterized in that, This includes introducing a nucleotide sequence encoding a Tma12 protein into the genome of the plant; preferably, the Tma12 protein comprises an amino acid sequence as shown in SEQ ID NO:1; more preferably, the nucleotide sequence encoding the Tma12 protein is shown in SEQ ID NO:

2.

9. A method for producing plant seeds for controlling stink bugs and / or thrips pests, characterized in that, This includes hybridizing a first plant obtained by the method of claim 8 with a second plant to produce seeds containing a nucleotide sequence encoding the Tma12 protein.

10. A method for cultivating plants for controlling stink bugs and / or thrips pests, characterized in that, include: Plant at least one plant seed, the genome of which includes a nucleotide sequence encoding the Tma12 protein; To allow the plant seeds to grow into plants; The plants are grown under conditions of artificial inoculation with the pest and / or natural occurrence of the pest, and the harvested plants exhibit reduced plant damage and / or increased plant yield compared to other plants that do not have the nucleotide sequence encoding the Tma12 protein. Preferably, the Tma12 protein comprises the amino acid sequence shown in SEQ ID NO:1; more preferably, the nucleotide sequence encoding the Tma12 protein is shown in SEQ ID NO:2.

Images