Application of insect inhibitory protein and method for improving plant resistance

Abstract

The invention relates to application of Col05, Col06 or Col29 protein, the Col05, Col06 or Col29 protein is used for inhibiting or killing insects, the Col05, Col06 or Col29 protein respectively has an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, dicotyledonous plant tobacco is selected as a first host, monocotyledonous plant corn is selected as a second host, the Col05, Col06 or Col29 protein is subjected to plant codon optimization, and the Col05, Col06 or Col29 protein is obtained. And respectively obtaining nucleotide sequences as shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. Compared with the prior art, the insect inhibition protein Col05, Col06 or Col29 and the application of the coding gene have the advantages that a more effective insect inhibition method, particularly a prevention and control method for diplodia juncoides, is provided, the use of insecticides can be effectively reduced, and the ecological risk can be remarkably reduced. The invention provides a brand-new inhibition method for the diplodia juncosa, the existing genes for resisting the diplodia juncosa are mainly RNAi, and the method provided by the invention is completely different from a method for resisting insects by using proteins in the aspect of insect resistance mechanism.

Description

Technical Field

[0001] This invention relates to the use of insect inhibitory proteins and methods for improving plant resistance. In particular, this invention relates to the use of Col05, Col06, or Col29 proteins for inhibiting Coleoptera pests, especially the two-spotted leaf beetle, and methods for improving the resistance of plants, particularly tobacco, to Coleoptera pests, especially the two-spotted leaf beetle. Background Technology

[0002] Corn, a pillar crop of Chinese agriculture, has long ranked among the top grain crops in terms of planting area and yield. It is not only a crucial cornerstone for ensuring national food security but also a core resource supporting the development of animal husbandry and processing industries. In recent years, with the continuous expansion of the pilot area for genetically modified corn, farmers have gradually recognized the significant advantages of genetically modified insect-resistant corn in planting management and yield improvement. This shift has led scientists to note that, in addition to the first generation of genetically modified insect-resistant corn effectively controlling lepidopteran pests, the two-spotted leaf beetle (a type of coleopteran) is showing a year-on-year increasing trend, becoming a new challenge for industrial development. Currently, the Agricultural Technology Extension Center of the Ministry of Agriculture and Rural Affairs has observed that the two-spotted leaf beetle is severely damaging corn and soybeans in major corn-growing areas in northern China, from Heilongjiang in the east to Xinjiang in the west, and from Inner Mongolia in the north to Henan in the south, causing serious impacts on corn and soybean production.

[0003] The two-spotted leaf beetle, a representative of Coleoptera pests, exhibits a significantly different life cycle compared to Lepidoptera pests: it has only one generation per year, overwintering as diapause eggs in the soil. These diapause eggs can withstand extreme environments to maintain population size for the following year. In spring, the larvae emerge from the soil and bore into the roots of corn, causing damage to the seedling roots and poor development, severely impacting early corn growth. In summer, the adults feed on corn leaves and silks, resulting in serious problems such as missing kernels and pointed kernels, directly threatening corn yield. To effectively combat the damage caused by the two-spotted leaf beetle, agricultural technology departments in various regions have guided farmers to conduct deep plowing and tilling in winter. However, due to the extremely strong environmental tolerance of the diapause eggs, this method has limited control effectiveness. Summer campaigns involving concentrated aerial spraying in townships have also been implemented, but chemical control is not only delayed but also poses serious environmental and ecological problems.

[0004] Currently, the genes resisting the two-spotted leaf beetle are mainly RNAi-based, a method whose resistance mechanism is entirely different from that using protein-based insect resistance methods. No effective proteins against Coleoptera pests, especially those against the two-spotted leaf beetle, have yet been discovered. Summary of the Invention

[0005] To address the problems existing in the prior art, in a first aspect of the present invention, the use of an insect inhibitor protein is provided, wherein the protein is a Col05, Col06, or Col29 protein, for inhibiting or killing insects that infest plants.

[0006] The Col05 protein has SEQ ID The amino acid sequence shown in NO:1, namely: MKELQEITNAWGAWKAKQLGTSLHFTASTKYGGNSHLSPYTKYEVQSDLEAGGFHFVNKRELPDGVGFDLSHTVHNSTGEKQTMDVTFTEKKTSELTISVTEAVKIGNKVSGGVNVPFFADGKVEITAELSVSSTQS WKNSKDNGLSIKVPTVVPAHSSVLVKSTWVINRLQADWVADVVMRGYVAVRFNDYVYYDNYKENGWHALWFIPIKSVFDEIIQNNIIDTTGYIRQYNSVIAQAKGTLETATTAFGKTSFSPIDSASVIGNVRYNKEEPVDYVLIGADNEEGV.

[0007] The Col06 protein has the amino acid sequence shown in SEQ ID NO:3, namely: MNSLQQITDAWGKWYSQQHGTTCRFTASTDYSSQSFLDDYHQYQVSTTAQNIVYDDNSLPTNGSEIAFKTIYNNNTQAANQQSLIETATSTQSFEWSITEAVSIGVEISATEGVPAVASSTQKVTVNLSLSSTQKSTVTNTQSWSVNTILTIPPQSTIKADIVIGTQSYNINFTLSVMLNGYVAIWNNDKVNGHWLWFIPITQVFSDCIANNIIDTSGYDFVGGGISTTASGVFTGSQGISVGVNTTQYPLNSNTDAAKEPGFIDTPAVSKIVAMAGKE.

[0008] The Col29 protein has SEQ ID The amino acid sequence shown in NO:5, namely: MESNNQQNKMVEVNGTTSNSFILSPITSNIITDVDQQMNKISDYYYNNNLKLKDIGDYYHIIRLENKNTTMSFDLNADDIKNLHYNDLQPQYIGENEFKNTTDQEQTFTTASYSQAVTNSVSSTVIQGFKATSTTSLLKIPIL LPGGINLNAEFNSASNTTTTNTTTETLTAPPQNIKVPAGRTYKVEVNLLKKKFTGDIDFHGKGTNVKSNLKVRATYYGPGFPRPTKYPTYTYSTADMWRGLTTEQKKQITGVNFNNNKDLTIDGTTKVEGIYGSNLEVVVYDITNKNTPKIVETRTFK.

[0009] The insect is a two-spotted leaf beetle; the plant is tobacco.

[0010] In a preferred embodiment, the coding gene of the Col05 protein has the nucleotide sequence shown in SEQ ID NO: 2, that is: atgaaagaactacaagagataacaaatgcttggggtgcttggaaagcaaaacaactgggtacgagcctgcatttcaccgccagcacgaaatacggcggtaactcccacctgtcaccgtacaccaaatacgaagttcagtctgatcttgaggcgggtggcttccacttcgtgaataagcgcgagctcccggatggcgtgggtttcgacttgtcgcacaccgtgcacaattctacgggtgaaaaacaaaccatggatgtgacctttactgagaagaagaccagcgaattgaccattagcgtgaccgaggcggttaagatcggcaacaaggtgagcggtggtgttaatgttccgtttttcgcggacggcaaggtcgagatcaccgctgaattgtccgtgagctccacgcagagctggaagaactccaaagataacggtctgtcgattaaggtgccgaccgtggttccagcacatagcagcgttctggttaaaagcacctgggtcatcaaccgcctgcaagcggactgggttgccgacgttgttatgcgtggttatgtggccgttcgttttaacgattacgtgtattacgacaactataaagagaacggctggcatgcgctgtggttcatcccgattaagtccgtctttgacgaaatcatccagaataacattatcgataccaccggttacattcgtcagtataactctgtcatcgcgcaggctaaaggtacactggaaaccgcgaccactgcgtttggcaaaacctctttcagcccgattgacagcgcaagtgtgatcgggaacgtgcgttataataaggaggaaccggttgattacgttctgattggcgcggacaatgaagagggcgta。

[0011] The coding gene of the Col06 protein has the nucleotide sequence shown in SEQ ID NO:4, i.e.: atgaattcactacaacagataacagatgcttggggtaagtggtatagccaacaacacggcacaacgtgccgttttaccgcgagcaccgactactcgagccagtccttcctggatgactaccaccagtaccaggtttctaccactgcgcaaaacatcgtctatgacgacaacagcttaccgacgaacggcagcgagatcgcgtttaaaaccatttataataacaacacccaggcggcgaatcagcagtccttgatcgaaaccgcgacctccacccagtcgttcgagtggtcaatcaccgaagcagtttctattggcgttgaaatttctgcaaccgaaggtgttccggctgtggccagcagcacgcaaaaggtcaccgtgaacctgtcccttagtagcacgcaaaaaagcaccgttaccaatactcaatcttggagcgtcaacacgatcttgacgattccgccacagtccacgatcaaagcagatattgtgatcggcacccagagttataacatcaattttaccctgagcgtgatgctgaatggctacgttgctatctggaataatgataaggtgaacggccattggctgtggttcatcccgattacccaagttttcagcgattgtattgccaacaacattatcgacaccagcggttacgactttgttggtggtggcattagcacgactgcgtccggtgtgttcaccggtagccaaggtattagcgttggcgtgaacacaacccagtacccgctgaacagcaatactgacgccgcgaaagagccgggttttatcgataccccggcagtatcgaaaatcgtggctatggcgggtaaggag

[0012] The coding gene of the Col29 protein has the nucleotide sequence shown in SEQ ID NO:6, i.e.: atggaaagcaacaaccagcagaacaagatggtcgaggtgaacggcaccaccagcaacagcttcatcctgtctccgatcacctccaacatcatcaccgacgtggaccagcagatgaacaagatcagcgactactactataacaacaacctgaagctgaaggacatcggcgattactaccacatcatcaggctcgagaacaagaacaccaccatgagcttcgacctgaacgccgacgacatcaagaacctgcactacaacgacctgcagccgcagtacattggcgagaacgagttcaagaacacgaccgaccaagagcagaccttcaccaccgctagctacagccaggctgtgaccaactctgtgagcagcactgtgatccagggcttcaaggctacctctaccaccagcctgctgaagatcccgatcttgcttccaggcggcatcaacctcaacgccgagttcaacagcgccagcaacaccacgaccaccaacaccactaccgagactctgaccgctccgccgcagaacatcaaggttccagctggcaggacctacaaggtcgaggtcaacctgctcaagaagaagttcaccggcgacatcgacttccacggcaagggcaccaacgtgaagtccaacctcaaggtgagggccacctactacggcccaggctttccaaggccgactaagtacccgacctacacctacagcaccgccgatatgtggcgcggtctgaccactgagcagaagaaacagatcaccggcgtgaacttcaacaacaacaaggacctgaccatcgacggcacgaccaaggttgagggcatctacggcagcaatctcgaggtggtggtgtacgacatcaccaacaagaataccccgaagatcgtcgagactaggaccttcaag。

[0013] In a preferred embodiment, dicotyledonous tobacco is selected as the first host and monocotyledonous maize as the second host. Plant codon optimization is performed on the Col05, Col06 or Col29 proteins to obtain nucleotide sequences as shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

[0014] In a second aspect of the invention, a method for improving plant resistance to insects is provided, the method comprising the step of expressing a gene encoding a Col05, Col06, or Col29 protein in the plant, wherein the Col05 protein has the amino acid sequence shown in SEQ ID NO:1, the Col06 protein has the amino acid sequence shown in SEQ ID NO:3, and the Col29 protein has the amino acid sequence shown in SEQ ID NO:5. The plant is tobacco.

[0015] In a preferred embodiment, the gene encoding the Col05 protein has the nucleotide sequence shown in SEQ ID NO:2, the gene encoding the Col06 protein has the nucleotide sequence shown in SEQ ID NO:4, and the gene encoding the Col29 protein has the nucleotide sequence shown in SEQ ID NO:6.

[0016] The dicotyledonous plant tobacco was selected as the first host and the monocotyledonous plant maize as the second host. Plant codon optimization was performed on the Col05, Col06 or Col29 proteins to obtain the nucleotide sequences shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 respectively.

[0017] In a preferred embodiment, the method includes the following steps: constructing a transient expression vector using the Col05, Col06, or Col29 protein-coding gene, introducing it into Agrobacterium, and obtaining Agrobacterium expressing the gene; applying Agrobacterium to a plant to cause the plant to express the Col05, Col06, or Col29 protein-coding gene.

[0018] In a preferred embodiment, applying Agrobacterium to the plant means applying Agrobacterium to the plant leaves.

[0019] In a preferred embodiment, the transient expression vector includes two expression cassettes: the first expression cassette contains the gene encoding the Col05, Col06, or Col29 protein; and the second expression cassette is used to screen marker expression units to identify whether the Col05, Col06, or Col29 protein is expressed in plant cells.

[0020] In a preferred embodiment, the insect is a Coleoptera pest, more preferably a two-spotted leaf beetle.

[0021] In a third aspect of the invention, a plant obtained according to the method described above is provided, said plant being tobacco.

[0022] The insect inhibitory proteins Col05, Col06, or Col29 and their encoding genes described above, provided by this invention, offer a more effective insect control method compared to existing technologies, particularly for the control of the two-spotted leaf beetle. This method effectively reduces pesticide use and significantly lowers ecological risks. This invention provides a novel method for inhibiting the two-spotted leaf beetle, as existing resistance genes for this beetle are primarily RNAi-based. The method of this invention differs entirely in its insect-resistance mechanism from methods using protein-based insect resistance.

[0023] Unless otherwise described, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of interpreting this specification, the following terminology will be applied, and where appropriate, terms used in the singular will also include the plural form, and vice versa. All patents, applications, published applications, and other publications are incorporated herein by reference in their entirety. If any description of terminology set forth herein conflicts with any document incorporated herein by reference, the terminology set forth herein shall prevail. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The pGRB-V1.1 vector spectrum; Figure 2 The spectrum of the Col-B05 vector; Figure 3 The spectrum of the Col-B06 vector; Figure 4 The spectrum of the Col-B29 vector; Figure 5 This is a map of the Red-VC vector. Detailed Implementation

[0026] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0027] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Example 1: Obtaining insect inhibitory proteins and their encoding genes

[0028] The amino acid sequences of the Col05, Col06 and Col29 proteins of the present invention were obtained as shown in SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, respectively.

[0029] The dicotyledonous plant tobacco was selected as the first host and the monocotyledonous plant maize as the second host. Plant codon optimization was performed on the Col05, Col06 and Col29 proteins to obtain the nucleotide sequences shown in SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6, respectively. Example 2: Constructing a Carrier

[0030] A tobacco transient transduction expression vector containing two expression cassettes was constructed based on the plant codon-optimized nucleotide sequence of Example 1.

[0031] The first expression cassette contains a promoter from Arabidopsis thaliana Ubi10 (which has constitutive expression characteristics and can be expressed at high intensity in tobacco leaves), with a codon-optimized nucleotide sequence of Col05, Col06, or Col29 protein inserted in the middle (to ensure efficient ribosome recognition and improve protein transcription and translation efficiency), and a Nos terminator from Agrobacterium tumefaciens Ti plasmid tandem at the 3' end (which can precisely terminate transcription and reduce the impact of readthrough on downstream sequences). All elements are operatively linked.

[0032]

[0033] The Nos terminator of the Agrobacterium tumefaciens Ti plasmid has the nucleotide sequence shown in SEQ ID NO:8, namely, gatcgttcaaacatttggcaataaagtttcttaagattgaatcctgttgccggtcttgcgatgattatcatataatttctgttgaattacgttaagcatgtaataattaacatgtaatgcatgacgttatttatgagatgggtttttatgattagagtcccgcaattatacatttaatacgcgatagaaaacaaaatatagcgcgcaaactaggataaattatcgcgcgcgcggtgtcatctatgttactagatc.

[0034] The second expression cassette is a selection marker expression unit used to identify whether the target protein is expressed in tobacco cells. The promoter selected is the 35S promoter of tobacco mosaic virus (TMV) (which has extremely strong initiation activity and can efficiently drive the expression of the selection gene). The target gene is introduced with the phosphinic acid acetyltransferase gene (i.e., the PAT gene, which encodes a protein that can detoxify the herbicide glufosinate, confer glufosinate resistance to transformed cells, and facilitate the exclusion of untransformed cells through herbicide screening). The 3' end is matched with the 35S terminator of tobacco mosaic virus (consistent with the promoter source, which can further enhance the transcription termination efficiency).

[0035] Among them, the tobacco mosaic virus 35S promoter has the nucleotide sequence shown in SEQ ID NO:9, that is.

[0036] The 35S terminator of tobacco mosaic virus has a nucleotide sequence as shown in SEQ ID NO:10, namely ctgaaatcaccagtctctctctacaaatctatctctctctataataatgtgtgagtagttcccagataagggaattagggttcttatagggtttcgctcatgtgttgagcatataagaaacccttagtatgtatttgtatttgtaaaatacttctatcaataaaatttctaattcctaaaaccaaaatccagtgg.

[0037] The phosphinothricin acetyltransferase gene (i.e., the PAT gene) has an amino acid sequence as shown in SEQ ID NO:11, i.e., MSPERRPVEIRPATAADMAAVCDIVNHYIETSTVNFRTEPQTPQEWIDDLERLQDRYPWLVAEVEGVVAGIAYAGPWKARNAYDWTVESTVYVSHRHQRLGLGSTLYTHLLKSMEAQGFKSVVAVIGLPNDPSVRLHEALGYTARGTLRAAGYKHGGWHDVGFWQRDFELPAPPRPVRPVTQI; it has a nucleotide sequence as shown in SEQ ID NO:12, i.e., atgtctcctgaaagacgccctgtggaaattagacctgcaaccgctgccgatatggctgccgtttgcgacattgtcaatcactatattgaaacttctacagtgaatttcagaactgagcctcaaactcctcaagagtggattgatgaccttgagagattgcaggataggtatccttggttggttgctgaagtggagggtgttgtcgccggtatagcttacgctggtccttggaaggctaggaacgcttacgattggactgttgaatctacagtgtacgtttcacataggcatcaaaggcttggcttgggttctaccctttatactcatctcttgaaatctatggaggcacaaggatttaagagtgttgtggctgttatcggccttccaaatgatccttcagttaggttgcatgaagctcttggatacacagcaaggggaactctaagggccgctggatataaacacggtggatggcatgatgttggtttttggcagagggattttgagttgccagcacctcctagacctgttagaccagtcactcaaatttga。

[0038] The backbone vector pGRB-V1.1 was digested with SpeI and AscI restriction endonucleases, and after recovery and purification, a backbone fragment of approximately 8.7 kb in size was obtained. This backbone fragment was then combined with the target genes Col05, Col06, or Col29, and DsRed, which were obtained by PCR amplification and contained homologous arms, using In-Fusion homologous recombination technology (reagent: In-Fusion® Snap Assembly Master Mix, purchased from Takara Clontech, catalog number: 638949). The recombination products were heat-shocked at 42°C and transformed into DH5α competent cells (purchased from Shanghai Weidi Biotechnology Co., Ltd., catalog number: DL1001), followed by plate culture. Positive transformants were screened by PCR, and positive clones were sequenced for verification. Finally, the vectors Col-B05, Col-B06, or Col-B29 (tobacco transient expression vector), and Red-VC (experimental negative control vector) were successfully constructed.

[0039] The red fluorescent protein gene (DsRed) has the amino acid sequence shown in SEQ ID NO: 13, i.e., MASSENVITEFMRFKVRMEGTVNGHEFEIEGEGEGRPYEGHNTVKLKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGVATVTQDSSLQDGCFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGETHKALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDAKLDITSHNEDYTIVEQYERTEGRHHLFL; and has the nucleotide sequence shown in SEQ ID NO: 14, i.e., atggcctcctccgagaacgtcatcaccgagttcatgcgcttcaaggtgcgcatggagggcaccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggccacaacaccgtgaagctgaaggtgacgaagggcggccccctgcccttcgcctgggacatcctgtccccccagttccagtacggctccaaggtgtacgtgaagcaccccgccgacatccccgactacaagaagctgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggcgaccgtgacccaggactcctccctccaggacggctgcttcatctacaaggtgaagttcatcggcgtgaacttcccctccgacggccccgtgatgcagaagaagaccatgggctgggaggcctccaccgagcgcctgtacccccgcgacggcgtgctgaagggcgaaacccacaaggccctgaagctgaaggacggcggccactacctggtggagttcaagtctatctacatggccaagaagcccgtgcagctgcccggctactactacgtggacgccaagctggacatcacctcccacaacgaggactacaccatcgtggagcagtacgagcgcaccgagggccgccaccacctgttcctgtag。 Example 3: The vector constructed in Example 2 was introduced into Agrobacterium.

[0040] Take GV3101 Agrobacterium competent cells (Shanghai Weidi Biotechnology Co., Ltd., catalog number: AC1001) stored at -80℃, place them at room temperature for a short time until they partially thaw and are in an ice-water mixture, then quickly transfer them to an ice bath. Add the constructed Col-B05, Col-B06, and Col-B29 plasmids and Red-VC plasmids to the corresponding Agrobacterium competent cells, and gently tap the bottom of the tube to thoroughly mix the contents. Then perform the following treatments in sequence: stand on ice for 5 minutes, incubate in liquid nitrogen for 5 minutes, heat shock in a 37℃ water bath for 5 minutes, and cool on ice for 5 minutes. After treatment, add 700 μl of antibiotic-free LB or YEB liquid medium to each tube and incubate at 28℃ with shaking for 2-3 hours. Spread the bacterial culture onto YEP solid medium, invert the plate and incubate at 28℃ for 2-3 days. Positive transformants were screened using PCR technology, and positive clones were sequenced for verification. Col-B05, Col-B06, and Col-B29 and Red-VC positive Agrobacterium strains were obtained and stored in an ultra-low temperature freezer at -80°C for later use. Example 4: Transforming tobacco using the Agrobacterium strain obtained in Example 3.

[0041] The Col-B05, Col-B06, and Col-B29 strains and the Red-VC positive Agrobacterium strain obtained in Example 3 and cryopreserved at -80℃ were thawed in an ice-water bath and inoculated into YEP liquid medium containing the corresponding antibiotics. The cultures were then incubated at 28℃ and 220 rpm in the dark for 16-18 hours with shaking. The cultures were centrifuged to obtain bacterial precipitates, which were resuspended in sterile infection buffer (10 mM MES, 10 mM MgCl2, 200 μM acetylsyleugenone). The OD600 of the bacterial suspension was adjusted to 1.0 ± 0.05 using spectrophotometry and allowed to stand in the dark at room temperature for at least 30 minutes.

[0042] Select 4-6 week old Nicotiana benthamiana plants with intact leaves and free from pests and diseases. Water the plants one day in advance and mark the leaves to be injected with a bright marker. Using a sterile syringe (without the needle), draw up the Agrobacterium tumefaciens solution that has been allowed to stand and gently inject it into the underside of the tobacco leaves, ensuring complete penetration of the solution into the entire leaf; infect 3-4 leaves per tobacco plant. After infection, place the tobacco plants in the dark at 25°C for 12 hours, then begin greenhouse cultivation with 16 hours of light / 8 hours of darkness for 2-3 days. Use the leaves to be tested for bioassay. Example 5: Testing of Two-Spotted Leaf Beetle Larvae

[0043] Tobacco leaves after transient expression in Example 4 were selected, and leaf discs were vertically punched from the non-midrib area of ​​the leaves using a sterilized 1cm×1cm punch, ensuring that the leaf discs were intact, undamaged, and free from pests and diseases. The prepared leaf discs were then laid flat in 6cm diameter petri dishes lined with sterilized moisturizing filter paper, with one leaf disc placed in each dish.

[0044] Newly hatched larvae of the two-spotted leaf beetle with uniform physiological condition and good vitality were selected, with 15 larvae precisely chosen per dish. Using the tip of a sterile brush, the larvae were gently lifted and transferred to a corner of the culture dish, avoiding squeezing or damaging the larvae during the process. After all culture dishes had been inoculated with larvae, the vitality of the larvae was checked in each dish. If any individuals were found not moving independently or curled up, they were immediately replaced with spare, healthy larvae to ensure that the initial condition of the larvae in each dish was consistent.

[0045] The Red-VC vector expressing dsRed was set up as a negative control in the experiment, and four biological replicates were set up for each test vector and control vector.

[0046] After inoculating the larvae, the culture dishes were placed in a constant temperature and humidity incubator, and the culture conditions were strictly controlled as follows: 25℃, 16h light / 8h dark light cycle, and 70% relative humidity, and cultured for 3 days.

[0047] After the cultivation was completed, the survival status of the two-spotted leaf beetle larvae and the feeding area of ​​tobacco leaf discs were investigated simultaneously: survival was determined by gently touching the larvae with the tip of a sterile brush; if the larvae showed spontaneous activity, they were considered alive, and if they showed no stress response, they were considered dead. The remaining area of ​​the leaf discs was measured using grid paper, the actual feeding area was calculated, and the relevant data were recorded and statistically analyzed. See the table below for details.

[0048]

[0049] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. The use of an insect inhibitory protein, characterized in that: The protein is Col05, Col06, or Col29, used to inhibit or kill insects that damage plants; The Col05 protein has the amino acid sequence shown in SEQ ID NO:1, the Col06 protein has the amino acid sequence shown in SEQ ID NO:3, and the Col29 protein has the amino acid sequence shown in SEQ ID NO:

5. The insect in question is the two-spotted firefly beetle; The plant in question is tobacco.

2. The use according to claim 1, characterized in that: The gene encoding the Col05 protein has the nucleotide sequence shown in SEQ ID NO:2, the gene encoding the Col06 protein has the nucleotide sequence shown in SEQ ID NO:4, and the gene encoding the Col29 protein has the nucleotide sequence shown in SEQ ID NO:

6.

3. The use according to claim 2, characterized in that: The dicotyledonous plant tobacco was selected as the first host and the monocotyledonous plant maize as the second host. Plant codon optimization was performed on the Col05, Col06 or Col29 proteins to obtain nucleotide sequences as shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 respectively.

4. A method for improving plant resistance to insects using insect-inhibiting proteins, characterized in that: The protein is Col05, Col06, or Col29 protein, and the method includes the step of expressing a gene encoding Col05, Col06, or Col29 protein in a plant, wherein the Col05 protein has the amino acid sequence shown in SEQ ID NO:1, the Col06 protein has the amino acid sequence shown in SEQ ID NO:3, and the Col29 protein has the amino acid sequence shown in SEQ ID NO:5; The plant in question is tobacco.

5. The method according to claim 4, characterized in that: The gene encoding the Col05 protein has the nucleotide sequence shown in SEQ ID NO:2, the gene encoding the Col06 protein has the nucleotide sequence shown in SEQ ID NO:4, and the gene encoding the Col29 protein has the nucleotide sequence shown in SEQ ID NO:

6. The dicotyledonous plant tobacco was selected as the first host and the monocotyledonous plant maize as the second host. Plant codon optimization was performed on the Col05, Col06 or Col29 proteins to obtain nucleotide sequences as shown in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6 respectively.

6. The method according to claim 5, characterized in that: The method includes the following steps: constructing a transient expression vector using the Col05, Col06, or Col29 protein-coding genes, introducing Agrobacterium tumefaciens into it to obtain Agrobacterium tumefaciens expressing the genes; applying Agrobacterium tumefaciens to plants to induce the plants to express the Col05, Col06, or Col29 protein-coding genes.

7. The method according to claim 6, characterized in that: The application of Agrobacterium to plants refers to the application of Agrobacterium to plant leaves.

8. The method according to claim 6 or 7, characterized in that: The transient expression vector includes two expression cassettes. The first expression cassette contains the gene encoding the Col05, Col06, or Col29 protein. The second expression cassette is used to screen marker expression units and identify whether the Col05, Col06, or Col29 protein is expressed in plant cells.

9. The method according to any one of claims 4 to 8, characterized in that: The insect is a Coleoptera pest, preferably the two-spotted firefly beetle.

10. A plant obtained according to any one of claims 4 to 9, wherein the plant is tobacco.

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