Predatory mite poison protein and spider poison protein and application thereof

Abstract

The invention provides a predatory mite venom protein or a spider venom protein. The amino acid sequences of the predatory mite venom protein and the spider venom protein are selected from SEQ ID NO.1-8 or any one of SEQ ID NO.1-8 with 95% or more of identity. Tetranychus urticae tests show that after spraying treatment, the predatory mite poison protein or spider poison protein provided by the invention achieves a 100% lethal effect within 48 hours, and is a known protein pesticide with the highest tetranychus urticae lethal rate at present. The predatory mite poison protein or spider poison protein provided by the invention has high specificity to spider mites, is mild and harmless to human bodies and other organisms in the environment, can be used in farmlands, gardens and even family potted plants, and can be naturally degraded; the production process is simple and efficient, and has the potential of large-scale application.

Description

Invention Field

[0001] This application belongs to the field of biotechnology, and specifically provides predatory mite venom proteins and spider venom proteins and their applications. Background Technology

[0002] Spider mites, represented by the two-spotted spider mite (Tetranychusurticae), are a typical agricultural pest. They parasitize thousands of agricultural and garden plants, feeding on leaves and cellular contents, causing rapid damage and wilting of plant leaves. Spider mites cause devastating damage to global agriculture. Due to their small size, short lifespan, rapid reproduction rate, parthenogenesis, and ability to adapt to harsh environments by spinning webs, spider mites have become one of the most troublesome agricultural pests worldwide.

[0003] Currently, common methods for controlling spider mites primarily employ chemical pesticides: broad-spectrum insecticides or specific acaricides—such as bizenafate and etoxazole. However, broad-spectrum insecticides often kill the spider mite's natural predators, making plants more susceptible to spider mite infestations. Furthermore, spider mites are gradually developing tolerance to specific acaricides, leading to large-scale resistance. Literature indicates (https: / / doi.org / 10.1007 / s10493-016-0054-1) that the resistance of *Tetranychus spp.* to etoxazole can increase tenfold within just five generations. Besides the serious concern about mite resistance, the use of these compounds in agricultural environments still poses potential risks to humans and the natural environment. Residues or volatilized pesticides on crops can accumulate in the human body, causing harm. An effective, non-toxic, and biodegradable bio-based acaricide is the core requirement for controlling spider mite infestations.

[0004] Spider venom proteins are proteins derived from the venom of arachnids and play a crucial role in the spider's predation of insects and other arthropods. These proteins are generally small, cysteine-rich proteins that, after being injected into their prey via the spider's fangs, act on the ion channels of the arthropod's nerve and muscle cells, interfering with the prey's neural activity and thus paralyzing and killing it. Spider venom proteins are diverse, with most primarily acting on arthropods (such as insects) and exhibiting almost no toxicity to mammals. Currently, some spider venom proteins are used commercially as insecticides (https: / / www.vestaron.com / ) to kill lepidopteran insects, aphids, and other pests. However, research on the application of spider venom proteins to kill mites is still extremely rare. In June 2024, Li Chen et al. (https: / / doi.org / 10.1016 / j.pestbp.2024.105963) reported that proteins from the predatory mite *Neoseiulus barkeri* have injection toxicity against lepidopteran insects and against the carmine spider mite (*T. cinnabarinus*). However, the toxicity of the mite venom protein found in this study against the carmine spider mite only reached a 58% lethality rate within 48 hours, making it insufficient to be an effective drug in practical applications. Summary of the Invention

[0005] To address the above issues, this invention has identified several novel mite and spider venom proteins from the genomes of predatory mites specifically targeting spider mites, including *Phytoseiulus persimilis*, *Phoneutriadepilata*, *Calommatasignata*, and *Neoseiulus barkeri*. These proteins exhibit specific toxicity against *Tetranychus bicornuate*. To enhance contact toxicity, the core sequence of the selected venom protein was fused with an expression signal peptide, a SUMOylated peptide, and snowdrop lectin protein (GNA) to form a novel fusion protein. Recombinant proteins were then produced economically and efficiently using engineered *E. coli*.

[0006] On the one hand, this application provides predatory mite venom protein or spider venom protein, wherein the amino acid sequence of the predatory mite venom protein or spider venom protein is selected from SEQ ID NO.1-8 or a sequence that has more than 95% identity with any one of SEQ ID NO.1-8.

[0007] Furthermore, the amino acid sequence of the predatory mite venom protein or spider venom protein is selected from SEQ ID NO.1-8.

[0008] Furthermore, the amino acid sequence of the predatory mite venom protein is SEQ ID NO.3 or SEQ ID NO.4.

[0009] Furthermore, the amino acid sequence of the spider venom protein is SEQ ID NO.5.

[0010] On the other hand, the present invention also proposes a recombinant predatory mite venom protein or spider venom protein, wherein the recombinant predatory mite venom protein or spider venom protein comprises the predatory mite venom protein or spider venom protein shown in any one of SEQ ID NO. 1-8, or the predatory mite venom protein or spider venom protein shown in the sequence having more than 95% identity with any one of SEQ ID NO. 1-8, and snowdrop lectin protein.

[0011] Optionally, the recombinant predatory mite venom protein or spider venom egg may further include a connecting portion.

[0012] Furthermore, the recombinant predatory mite venom protein or spider venom protein comprises any one of the predatory mite venom proteins or spider venom proteins shown in SEQ ID NO.1-8, and snowdrop lectin protein;

[0013] Optionally, the recombinant predatory mite venom protein or spider venom egg further includes a linker portion. Further, the recombinant predatory mite venom protein or spider venom protein also includes sequences encoding a signal peptide and / or a SUMO peptide.

[0014] Furthermore, the amino acid sequence of the SUMO peptide is SEQ ID NO.20 or SEQ ID NO.21.

[0015] Furthermore, the amino acid sequence of the snowdrop lectin protein is SEQ ID NO.22.

[0016] Non-limiting examples of the connecting sequences are SEQ ID NO.23 used in the embodiments or other sequences rich in G and S.

[0017] Sequence identity in this application can be determined using alignment algorithms known in the art, including but not limited to blast, GenPast, etc.

[0018] On the other hand, this application provides a gene for predatory mite venom protein or spider venom protein, wherein the recombinant predatory mite venom protein or spider venom protein gene contains the aforementioned predatory mite venom protein or spider venom protein, or the coding sequence of the recombinant predatory mite venom protein or spider venom protein.

[0019] Furthermore, the predatory mite venom protein or spider venom protein gene also includes a signal peptide coding sequence and / or a SUMO peptide coding sequence.

[0020] Furthermore, the amino acid sequence of the SUMO peptide is SEQ ID NO.20 or SEQ ID NO.21.

[0021] Signal peptides can be of types known in molecular biology, such as G1M5 in the examples.

[0022] In this invention, the signal peptide and SUMO protein are used to improve the water solubility of the target protein, making it easier to express in engineered E. coli. The SUMO hydrolytic peptide, under the action of SUMO enzymes, cleaves the protein to expose the toxic protein region. The toxic protein region is linked by a linker and snowdrop lectin (GNA) protein. GNA can bind to intestinal receptors in arthropods such as insects, allowing the toxic protein to be ingested through the insect's mouth and enter the insect's hemolymph via the intestine, achieving an oral toxic effect.

[0023] Further, the nucleotide sequence of the predatory mite venom protein or spider venom protein is SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16 or SEQ ID NO.18.

[0024] Furthermore, this application provides a vector containing the aforementioned predatory mite venom protein gene.

[0025] Furthermore, this application provides a host cell containing the aforementioned vector.

[0026] The vectors and host cells used in this application are not limited to the types used in the examples. Various commercially available and specially designed vectors, as well as eukaryotic and prokaryotic host cells, can be used after appropriate codon adjustment and parameter optimization, including but not limited to Escherichia coli, Bacillus subtilis, yeast, mammalian cells, insect cells, etc.

[0027] On the other hand, this application provides a method for producing the aforementioned predatory mite venom protein or spider venom protein, including the step of culturing the aforementioned host cells.

[0028] On the other hand, this application provides the application of the above-mentioned predatory mite venom protein or spider venom protein in the preparation of pesticides that inhibit red spider mites.

[0029] Furthermore, the spider mite is a two-spotted spider mite (Tetranychusurticae).

[0030] The red spider mentioned, also known as the cotton red spider, yellow spider, etc., refers to animals of the family Tetranychidae in the superorder Erythropoda of the class Arachnida.

[0031] On the other hand, this application provides a pesticide for inhibiting spider mites, the pesticide comprising the aforementioned predatory mite venom protein or spider venom protein.

[0032] Furthermore, the spider mite is a two-spotted spider mite (Tetranychusurticae).

[0033] Furthermore, the pesticide also contains pesticide-acceptable excipients.

[0034] Furthermore, the pesticide is an aqueous solution, an oil-based solution, or a solid formulation.

[0035] Those skilled in the art can choose, as needed, methods for processing predatory acarinic proteins, such as freeze-drying, preparing microparticles or emulsions, and formulate pesticide formulations by adding various pesticide-acceptable excipients.

[0036] On the other hand, this application provides a method for suppressing spider mites, the method comprising applying the aforementioned pesticide.

[0037] This invention marks the first time that multiple predatory acaricidal or spider venom proteins have been screened from *Phytoseiulus persimilis*, *Phoneutriadepilata*, and *Calommata signata*. Testing on *Tetranychus spp.* showed that spraying the recombinant protein solution on mites achieved 100% lethality within 48 hours; this is currently the protein-based pesticide with the highest known lethality against spider mites, demonstrating excellent acaricidal activity. These proteins exhibit high specificity, are mild and harmless to humans and other organisms in the environment, and can be used in farmland, gardens, and even home potted plants. They are also biodegradable; their production process is simple and efficient, possessing the potential for large-scale application. Attached Figure Description

[0038] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0039] Figure 1 The protein structures of PpMVP1 and PpMVP2 are shown, along with their molecular alignment with the core structure of the recombinant spider venom protein rCtx-4.

[0040] Figure 2 Differential analysis of the protein sequences of PpMVP1 and PpMVP2 with the previously reported protein sequences of NpVP1 and NpVP2;

[0041] Figure 3Amino acid sequence alignment of the core region of predatory mites, herbivorous mites, and spider venom proteins;

[0042] Figure 4 This is a schematic diagram of the design of a toxic protein expression vector. The VPs region refers to cysteine-rich toxic protein sequences, including but not limited to... Figure 3 The image shows mite venom proteins MVPs and spider venom proteins SVPs.

[0043] Figure 5 The results show the expression of the venom proteins and the results after SUMO enzyme treatment. Part A characterizes the expression of rMVP2s and rMVP1s, respectively. M refers to the protein marker, S refers to the bacterial lysate supernatant, and SUMO represents the enzymatic digest after SUMO enzyme treatment. The target bands before and after enzyme digestion are marked in red, indicating the successful production of the 17-20 kDa target protein. Part B shows the successful expression of the control spider venom proteins HxTh-hv1h, rCtx-4, and Cs1A, as well as another venom protein, NbVP2F, which was previously reported. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Unless otherwise specified, all materials used in the present invention are commercially available materials.

[0045] Example 1: Sequence Acquisition and Filtering

[0046] Based on the genome and transcriptome data of *Phytoseiolus chinensis* from the NCBI database, and utilizing known spider venom protein sequences and mite venom protein CDS core sequences, sequence alignment was performed using the BLAST tool to identify mRNA sequences of venom proteins highly similar to the core regions of known venom proteins. The complete venom proteins were deduced from the mRNA sequences, resulting in the suspected venom proteins PpMVP1 (sequence 1) and PpMVP2 (sequence 3). By comparing the venom protein structures predicted by the *Phytoseiolus chinensis* venom protein AlphaFold / SWISS Model with known venom protein structures, the core regions of the two *Phytoseiolus chinensis* venom proteins were confirmed. The sequences of the full-length venom protein versions from natural *Phytoseiolus chinensis* were modified by removing structures other than the core regions to improve their expression efficiency and virulence. The truncated sequences were named PpMVp1s (sequence 2) and PpMVP2s (sequence 4). The differences between the sequences and structures of these two venom proteins and known mite venom proteins and commercially available spider venom protein sequences are shown below. Figure 1-3 As shown: Figure 1The protein structures of PpMVP1 and PpMVP2 are shown, along with their molecular alignment with the core structure of the recombinant spider venom protein rCtx-4: Part A shows the molecular structure of PpMVP1 in light blue and the core region of rCtx-4 in dark gray; Part B shows the molecular structure of PpMVP2 in light blue and the core region of rCtx-4 in dark gray. The high similarity in molecular conformation indicates evolutionary homology of the sequences.

[0047] Figure 2 The protein sequences of PpMVP1 and PpMVP2 were analyzed to differentiate them from the previously reported NpVP1 and NpVP2 protein sequences. From the perspective of the complete proteins, the PpMVP and NbVP sequences are highly similar, with PpMVP1 and NbVP1 differing by only 6 amino acids and only 3 amino acids in the core region. PpMVP2 and NbVP2, on the other hand, differ by 19 amino acids and 6 amino acids in the core region. However, the toxic proteins from the two sources have significantly different functions in killing mites.

[0048] Figure 3 The amino acid sequences of the core regions of spider venom proteins from predatory mites, herbivorous mites, and spiders were compared. As shown in the figure, the spider venom protein family exhibits high homology; both predatory and non-predatory mites possess homologous venom proteins with highly similar core sequences (e.g., the predatory NbVP2 and herbivorous GoVP2 venom proteins share more than 90% similarity, despite their vastly different biological functions). Some predatory mites use venom proteins to prey on aphids, some on spider mites, and some on plants, suggesting that the specificity and effectiveness of mite venom proteins may be determined by only a few amino acids.

[0049] Sequence 1 (complete amino acid sequence of PpMVP1):

[0050] MQSSVVHIQGSRKSRQQCLCCNSRRRSISRRSCLRRGASCDARPHDCCEYSACRCNLWGTNCRCQRAGL

[0051] LQRLG

[0052] Sequence 2 (PpMVP 1s core amino acid sequence):

[0053] RRSCLRRGASCDARPHDCCEYSACRCNLWGTNCRCQRAGLLQRLG

[0054] Sequence 3 (complete amino acid sequence of PpMVP2):

[0055] MERLVPFEILLGLLLLSAVPASLEPLPTGQSVLLQQLLEGREDSLHTFEEEAYETCKRYVALHDASRAV

[0056] ASQAFQAGSSREATSLMLADEEEPQLGPNLARDIRRPPSRIRTLSYRGQGEEGDFGGSHDVARIVASKK

[0057] RSCIRRGGSCDARPGDCCYHSSCRCNLWGTNCRCMRQGLLQRWIQGKR

[0058] Sequence 4 (PpMVP 2S core amino acid sequence):

[0059] KRSCIRRGGSCDARPGDCCYHSSCRCNLWGTNCRCMRQGLLQRWIQGKR

[0060] In addition, spider venom protein Ctx-4 was obtained from *Phoneutriadepilata*, spider venom protein Cs1a from *Calommata signata*, and the mite venom protein NbVP2F from *Neoseiulus barkeri* was extracted using the above methods. The core sequence was then extracted: sequence 5 (Ctx-4 amino acid sequence).

[0061] GKCGDINAPCQSDDCCCGYSVTCDCYWSKDCKCRESNFAAGMALRKAFCKNKI

[0062] Sequence 6 (Cs1a amino acid sequence)

[0063] GCVGLNKYCMATPCCKPFHCRGIMFLYFCAKS

[0064] Sequence 7 (complete amino acid sequence of NbVP2F)

[0065] MERLVPFEIILGLFLLSALPASLEPLPTGQSVLLQQLLEGREDSLQTFEEEAYETCRRYVALHDAGRAV

[0066] ASQAFQAGSSREATSLMQADEEEPPQGANLARDIRRPPSRARTLSYRGQGEEGDFGGSHDATRIVTSKK

[0067] RSCIRRGGSCDARPSDCCYHSACRCNLWGTNCRCMRMGLLRRWINGKR

[0068] Sequence 8 (NbVP2F core amino acid sequence)

[0069] RIVTSKKRSCIRRGGSCDARPSDCCYHSACRCNLWGTNCRCMRMGLLRRWINGKR

[0070] Example 2: Design of rMVP and Construction of Prokaryotic Expression Vector

[0071] A fusion sequence was constructed using the PpMVP 1s and PpMVP 2s sequences as the core. Upstream components included a signal peptide and a SUMO protein peptide, while downstream components included a linker and a GNA protein (expression vector design as follows). Figure 4 (As shown). The designed amino acid sequence of the rMVP1s protein is shown in Sequence 9, and the amino acid sequence of the rMVP2s protein is shown in Sequence 11. The DNA sequences of the two recombinant proteins in this example were obtained by codon optimization design from *E. coli*, and their sequences are shown in Sequence 10 and Sequence 12, respectively. Restriction endonucleases NcoI and NotI were added to both ends of the DNA and assembled into the expression vector pET28a to obtain the expression vector. In addition, the above method was used to construct vectors to express recombinant spider silk proteins rCtx-4 and rCs1A, whose amino acid sequences are shown in Sequences 13 and 15, and DNA sequences are shown in Sequences 14 and 16; and NbVP2, whose amino acid sequence is shown in Sequence 17, and DNA sequence is shown in Sequence 18. As a comparison, the spider venom protein HxTx-Hv1h was also prepared in this example using the same method, and its fusion protein amino acid sequence is shown in Sequence 24, and its nucleotide sequence is shown in Sequence 25.

[0072] Sequence 9 (amino acid sequence of rMVP1s fusion protein):

[0073] MNDLNDFLKTISLSFGFFLLLLSLPTVAEADVTNGTGGGSGHHHHHHSDSEVNQEAKPEVKPEVKPETHI

[0074] NLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHR

[0075] EQIGGGGAMDRRSCLRRGASCDARPHDCCEYSACRCNLWGTNCRCQRAGLLQRLGGGGGSAAADNILYS

[0076] GETLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWATNTGGLSRSCFLSMQTDGNLVVYNPSNKPIWAS

[0077] NTGGQNGNYVCILQKDRNVVIYGTDRWATG

[0078] Sequence 10 (rMVP1s fusion protein DNA sequence):

[0079] atgAACgacctgaacgacttcctgaaaaccatctctctgtctttcggtttcttcctgctgctgtctctg

[0080] ccgaccgttgctgaagctgacgttaccaacggtaccggtggtggttctggtcatcaccatcaccatcac [[ID=]14]

[0081] TCCGATTCGGAAGTCAACCAGGAAGCGAAGCCGGAAGTTAAACCAGAGGTGAAACCAGAAACCCACATT <00UU179>AATCTGAAAGTGTCCGATGGGTCTTCCGAAATCTTCTTTAAAATTAAGAAGACCACTCCGCTGCGTCGT

[0083] CTGATGGAAGCCTTCGCCAAACGCCAGGGGAAGGAGATGGATAGCCTGCGTTTCCTGTATGACGGTATT

[0084] CGCATCCAAGCGGATCAAACCCCGGAAGATCTGGATATGGAAGACAATGATATCATCGAGGCCCATCGT

[0085] GAGCAAATTGGCGGTggcggcgccatGGATCGTCGCTCCTGCCTGCGCCGTGGTGCTTCTTGTGACGCT

[0086] CGCCCTCACGATTGCTGCGAATACTCTGCATGTCGCTGCAACCTGTGGGGCACCAACTGTCGTTGTCAG

[0087] Note: There seems to be an error in the tag "UU" in line 17 which should probably be "00". I translated it as it is but this might need to be corrected in the original text.CGTGCTGGTCTGCTGCAACGTCTGGGCGGCGGCGGCGGCAGCGCGGCGGCGGATAACATTCTGTACAGC

[0088] GGTGAAACGCTGTCGACCGGTGAGTTCCTGAACTATGGCAGCTTTGTCTTCATCATGCAGGAAGACTGC

[0089] AACCTGGTGTTGTATGATGTGGATAAACCTATCTGGGCAACCAACACGGGCGGCCTGTCGCGTAGTTGC

[0090] TTTCTGTCTATGCAGACCGATGGGAACTTAGTGGTCTACAATCCGTCTAATAAGCCAATTTGGGCCTCT

[0091] AACACCGGCGGCCAGAACGGGAACTACGTTTGTATTTTACAGAAAGATCGCAATGTGGTCATTTATGGC

[0092] ACGGATCGCTGGGCCACGGGC

[0093] Sequence 11 (Amino acid sequence of rMVP2s fusion protein):

[0094] MNDLNDFLKTISLSFGFFLLLSLPTVAEADVTNGTGGGSGHHHHHHSDSEVNQEAKPEVKPEVKPETHI

[0095] NLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHR

[0096] EQIGGGGAMDRIVASKKRSCIRRGGSCDARPGDCCYHSSCRCNLWGTNCRCMRQGLLQRWIQGKRGGGG

[0097] SAAADNILYSGETLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWATNTGGLSRSCFLSMQTDGNLVVY

[0098] NPSNKPIWASNTGGQNGNYVCILQKDRNVVIYGTDRWATG

[0099] Sequence 12 (rMVP2s fusion protein DNA sequence):

[0100] atgAACgacctgaacgacttcctgaaaaccatctctctgtctttcggtttcttcctgctgctgtctctg

[0101] ccgaccgttgctgaagctgacgttaccaacggtaccggtggtggttctggtcatcaccatcaccatcac

[0102] TCCGATTCGGAAGTCAACCAGGAAGCGAAGCCGGAAGTTAAACCAGAGGTGAAACCAGAAACCCACATT

[0103] AATCTGAAAGTGTCCGATGGGTCTTCCGAAATCTTCTTTAAAATTAAGAAGACCACTCCGCTGCGTCGT

[0104] CTGATGGAAGCCTTCGCCAAACGCCAGGGGAAGGAGATGGATAGCCTGCGTTTCCTGTATGACGGTATT

[0105] CGCATCCAAGCGGATCAAACCCCGGAAGATCTGGATATGGAAGACAATGATATCATCGAGGCCCATCGT

[0106] GAGCAAATTGGCGGTggcggcgccatGGATCGTATCGTAGCCTCTAAAAAACGTTCTTGCATTCGTCGT

[0107] GGCGGTTCTTGCGATGCGCGTCCGGGTGATTGCTGCTACCACAGCTCCTGCCGTTGCAACCTGTGGGGC

[0108] ACCAACTGCCGCTGTATGCGTCAGGGTCTGCTGCAACGCTGGATTCAGGGTAAACGCGGCGGCGGCGGC

[0109] AGCGCGGCGGCGGATAACATTCTGTACAGCGGTGAAACGCTGTCGACCGGTGAGTTCCTGAACTATGGC

[0110] AGCTTTGTCTTCATCATGCAGGAAGACTGCAACCTGGTGTTGTATGATGTGGATAAACCTATCTGGGCA

[0111] ACCAACACGGGCGGCCTGTCGCGTAGTTGCTTTCTGTCTATGCAGACCGATGGGAACTTAGTGGTCTAC

[0112] AATCCGTCTAATAAGCCAATTTGGGCCTCTAACACCGGCGGCCAGAACGGGAACTACGTTTGTATTTTA

[0113] CAGAAAGATCGCAATGTGGTCATTTATGGCACGGATCGCTGGGCCACGGGC

[0114] Sequence 13 (Amino acid sequence of rCtx-4 fusion protein):

[0115] MNDLNDFLKTISLSFGFFLLLSLPTVAEADVTNGTGGGSGHHHHHHSDSEVNQEAKPEVKPEVKPETHI

[0116] NLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHR

[0117] EQIGGMGKCGDINAPCQSDCDCCGYSVTCDCYWSKDCKCRESNFAAGMALRKAFCKNKIGGGGSAAADN

[0118] ILYSGETLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWATNTGGLSRSCFLSMQTDGNLVVYNPSNKP

[0119] IWASNTGGQNGNYVCILQKDRNVVIYGTDRWATG

[0120] Sequence 14 (DNA sequence of rCtx-4 fusion protein):

[0121] atgAACgacctgaacgacttcctgaaaaccatctctctgtctttcggtttcttcctgctgctgtctctg

[0122] ccgaccgttgctgaagctgacgttaccaacggtaccggtggtggttctggtcatcaccatcaccatcac

[0123] TCCGATTCGGAAGTCAACCAGGAAGCGAAGCCGGAAGTTAAACCAGAGGTGAAACCAGAAACCCACATT

[0124] AATCTGAAAGTGTCCGATGGGTCTTCCGAAATCTTCTTTAAAATTAAGAAGACCACTCCGCTGCGTCGT

[0125] CTGATGGAAGCCTTCGCCAAACGCCAGGGGAAGGAGATGGATAGCCTGCGTTTCCTGTATGACGGTATT

[0126] CGCATCCAAGCGGATCAAACCCCGGAAGATCTGGATATGGAAGACAATGATATCATCGAGGCCCATCGT

[0127] GAGCAAATTGGCGGtatgGGTAAATGCGGTGACATTAATGCGCCGTGTCAGAGCGATTGCGATTGCTGT

[0128] GGCTATAGTGTGACCTGTGATTGCTACTGGTCGAAAGATTGCAAATGTCGTGAATCCAACTTTGCCGCC

[0129] GGCATGGCGCTGCGCAAAGCATTTTGCAAGAACAAGATCGGCGGCGGCGGCAGCGCGGCGGCGGATAAC

[0130] ATTCTGTACAGCGGTGAAACGCTGTCGACCGGTGAGTTCCTGAACTATGGCAGCTTTGTCTTCATCATG

[0131] CAGGAAGACTGCAACCTGGTGTTGTATGATGTGGATAAACCTATCTGGGCAACCAACACGGGCGGCCTG

[0132] TCGCGTAGTTGCTTTCTGTCTATGCAGACCGATGGGAACTTAGTGGTCTACAATCCGTCTAATAAGCCA

[0133] ATTTGGGCCTCTAACACCGGCGGCCAGAACGGGAACTACGTTTGTATTTTACAGAAAGATCGCAATGTG

[0134] GTCATTTATGGCACGGATCGCTGGGCCACGGGC

[0135] Sequence 15 (Amino acid sequence of rCs1A fusion protein)

[0136] MNDLNDFLKTISLSFGFFLLLSLPTVAEADVTNGTGGGSGHHHHHHSDSEVNQEAKPEVKPEVKPETHI

[0137] NLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHR

[0138] EQIGGMGCVGLNKYCMATPCCKPFHCRGIMFLYFCAKSGGGGSAAADNILYSGETLSTGEFLNYGSFVF

[0139] IMQEDCNLVLYDVDKPIWATNTGGLSRSCFLSMQTDGNLVVYNPSNKPIWASNTGGQNGNYVCILQKDR

[0140] NVVIYGTDRWATG [[ID=​​​​​​​​​TCCGATTCGGAAGTCAACCAGGAAGCGAAGCCGGAAGTTAAACCAGAGGTGAAACCAGAAACCCACATT

[0145] AATCTGAAAGTGTCCGATGGGTCTTCCGAAATCTTCTTTAAAATTAAGAAGACCACTCCGCTGCGTCGT

[0146] CTGATGGAAGCCTTCGCCAAACGCCAGGGGAAGGAGATGGATAGCCTGCGTTTCCTGTATGACGGTATT

[0147] CGCATCCAAGCGGATCAAACCCCGGAAGATCTGGATATGGAAGACAATGATATCATCGAGGCCCATCGT

[0148] GAGCAAATTGGCGGtatgGGCTGTGTGGGTCTGAACAAGTACTGCATGGCGACCCCGTGCTGCAAACCG

[0149] TTTCATTGCCGCGGGATTATGTTTCTGTATTTCTGTGCCAAAAGTGGCGGCGGCGGCAGCGCGGCGGCG

[0150] GATAACATTCTGTACAGCGGTGAAACGCTGTCGACCGGTGAGTTCCTGAACTATGGCAGCTTTGTCTTC

[0151] ATCATGCAGGAAGACTGCAACCTGGTGTTGTATGATGTGGATAAACCTATCTGGGCAACCAACACGGGC

[0152] GGCCTGTCGCGTAGTTGCTTTCTGTCTATGCAGACCGATGGGAACTTAGTGGTCTACAATCCGTCTAAT

[0153] AAGCCAATTTGGGCCTCTAACACCGGCGGCCAGAACGGGAACTACGTTTGTATTTTACAGAAAGATCGC

[0154] AATGTGGTCATTTATGGCACGGATCGCTGGGCCACGGGC

[0155] Sequence 17 (Amino acid sequence of NbVP2 fusion protein)

[0156] MNDLNDFLKTISLSFGFFLLLSLPTVAEADVTNGTGGGSGHHHHHHSDSEVNQEAKPEVKPEVKPETHI

[0157] NLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHR

[0158] EQIGGGGAMDMERLVPFEIILGLFLLSALPASLEPLPTGQSVLLQQLLEGREDSLQTFEEEAYETCRRY

[0159] VALHDAGRAVASQAFQAGSSREATSLMQADEEEPPQGANLARDIRRPPSRARTLSYRGQGEEGDFGGSH

[0160] DATRIVTSKKRSCIRRGGSCDARPSDCCYHSACRCNLWGTNCRCMRMGLLRRWINGKRGGGGSAAADNI

[0161] LYSGETLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWATNTGGLSRSCFLSMQTDGNLVVYNPSNKPI

[0162] WASNTGGQNGNYVCILQKDRNVVIYGTDRWATG

[0163] Sequence 18 (DNA sequence of NbVP2 fusion protein)

[0164] atgAACgacctgaacgacttcctgaaaaccatctctctgtctttcggtttcttcctgctgctgtctctg

[0165] ccgaccgttgctgaagctgacgttaccaacggtaccggtggtggttctggtcatcaccatcaccatcac

[0166] TCCGATTCGGAAGTCAACCAGGAAGCGAAGCCGGAAGTTAAACCAGAGGTGAAACCAGAAACCCACATT

[0167] AATCTGAAAGTGTCCGATGGGTCTTCCGAAATCTTCTTTAAAATTAAGAAGACCACTCCGCTGCGTCGT

[0168] CTGATGGAAGCCTTCGCCAAACGCCAGGGGAAGGAGATGGATAGCCTGCGTTTCCTGTATGACGGTATT

[0169] CGCATCCAAGCGGATCAAACCCCGGAAGATCTGGATATGGAAGACAATGATATCATCGAGGCCCATCGT

[0170] GAGCAAATTGGCGGTggcggcgccatGGATATGGAACGTCTGGTCCCGTTCGAAATCATCCTGGGCCTG

[0171] TTTCTGCTGAGCGCTCTGCCAGCCTCTCTGGAACCTCTGCCGACCGGTCAGTCTGTTCTGCTGCAACAG

[0172] CTGCTGGAAGGCCGCGAAGATAGCCTGCAAACCTTCGAAGAGGAAGCGTATGAAACGTGCCGTCGTTAC

[0173] GTAGCTCTGCACGACGCAGGCCGTGCAGTTGCGAGCCAGGCTTTCCAAGCGGGCAGCTCTCGCGAAGCA

[0174] ACTTCCCTGATGCAGGCCGATGAAGAAGAACCGCCGCAGGGTGCTAACCTGGCACGTGACATTCGTCGT

[0175] CCGCCTTCCCGTGCTCGTACCCTGTCTTACCGTGGTCAAGGTGAGGAGGGCGATTTTGGTGGTAGCCAC

[0176] GATGCGACCCGTATCGTGACGAGCAAAAAACGTAGCTGCATCCGTCGTGGTGGCTCTTGCGATGCACGT

[0177] CCGAGCGACTGCTGCTATCACTCTGCGTGTCGTTGCAACCTGTGGGGCACCAACTGTCGTTGTATGCGT

[0178] ATGGGCCTGCTGCGCCGTTGGATTAACGGTAAACGCGGCGGCGGCGGCAGCGCGGCGGCGGATAACATT

[0179] CTGTACAGCGGTGAAACGCTGTCGACCGGTGAGTTCCTGAACTATGGCAGCTTTGTCTTCATCATGCAG

[0180] GAAGACTGCAACCTGGTGTTGTATGATGTGGATAAACCTATCTGGGCAACCAACACGGGCGGCCTGTCG

[0181] CGTAGTTGCTTTCTGTCTATGCAGACCGATGGGAACTTAGTGGTCTACAATCCGTCTAATAAGCCAATT

[0182] TGGGCCTCTAACACCGGCGGCCAGAACGGGAACTACGTTTGTATTTTACAGAAAGATCGCAATGTGGTC

[0183] ATTTATGGCACGGATCGCTGGGCCACGGGC

[0184] Sequence 19 (G1M5 signal peptide):

[0185] MNDLNDFLKTISLSFGFFLLLSLPTVAEADVTNGTGGGSGHHHHHH [[ID=2?]]

[0186] Sequence 20 (SUMO peptide):

[0187] SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGI

[0188] RIQADQTPEDLDMEDNDIIEAHREQIGGGGAMD

[0189] sSequence 21 (SUMO peptide):

[0190] It should be noted that there seems to be a typo in the original text where "[[ID=2?]]" is likely incorrect and should be "". Also, "

[0189] s " seems to have an extra "s" which might be a mistake.SDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGI

[0191] RIQADQTPEDLDMEDNDIIEAHREQIGGM

[0192] Sequence 22 (GNA):

[0193] DNILYSGETLSTGEFLNYGSFVFIMQEDCNLVLYDVDKPIWATNTGGLSRSCFLSMQTDGNLVVYNPSN

[0194] KPIWASNTGGQNGNYVCILQKDRNVVIYGTDRWATG

[0195] Sequence 23 (linker):

[0196] GGGGSAAA.

[0197] HxTx-Hv1h originates from Hadronyche versuta

[0198] Sequence 24 (amino acid sequence of HxTx-Hv1h fusion protein):

[0199] MNDLNDFLKTISLSFGFFLLLLSLPTVAEADVTNGTGGGSGHHHHHHSDSEVNQEAKPEVKPEVKPETHI

[0200] NLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHR

[0201] EQIGGMGSQYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRAGGGGSAAADNILYSGETLSTGE

[0202] FLNYGSFVFIMQEDCNLVLYDVDKPIWATNTGGLSRSCFLSMQTDGNLVVYNPSNKPIWASNTGGQNGN

[0203] YVCILQKDRNVVIYGTDRWATG

[0204] Sequence 25 (HxTx-Hv1h fusion protein DNA sequence):

[0205] atgAACgacctgaacgacttcctgaaaaccatctctctgtctttcggtttcttcctgctgctgtctctg

[0206] ccgaccgttgctgaagctgacgttaccaacggtaccggtggtggttctggtcatcaccatcaccatcac

[0207] TCCGATTCGGAAGTCAACCAGGAAGCGAAGCCGGAAGTTAAACCAGAGGTGAAACCAGAAACCCACATT

[0208] AATCTGAAAGTGTCCGATGGGTCTTCCGAAATCTTCTTTAAAATTAAGAAGACCACTCCGCTGCGTCGT

[0209] CTGATGGAAGCCTTCGCCAAACGCCAGGGGAAGGAGATGGATAGCCTGCGTTTCCTGTATGACGGTATT

[0210] CGCATCCAAGCGGATCAAACCCCGGAAGATCTGGATATGGAAGACAATGATATCATCGAGGCCCATCGT

[0211] GAGCAAATTGGCGGtatgGGTAGCcaatacTGTGTTCCGGTCGATCAGCCGTGCAGCCTGAATACCCAG

[0212] CCATGCTGTGACGATGCCACGTGCACCCAGGAGCGTAACGAAAACGGCCATACTGTGTATTACTGTAGA

[0213] GCAGGCGGCGGCGGCAGCGCGGCGGCGGATAACATTCTGTACAGCGGTGAAACGCTGTCGACCGGTGAG

[0214] TTCCTGAACTATGGCAGCTTTGTCTTCATCATGCAGGAAGACTGCAACCTGGTGTTGTATGATGTGGAT

[0215] AAACCTATCTGGGCAACCAACACGGGCGGCCTGTCGCGTAGTTGCTTTCTGTCTATGCAGACCGATGGG

[0216] AACTTAGTGGTCTACAATCCGTCTAATAAGCCAATTTGGGCCTCTAACACCGGCGGCCAGAACGGGAAC

[0217] TACGTTTGTATTTTACAGAAAGATCGCAATGTGGTCATTTATGGCACGGATCGCTGGGCCACGGGC

[0218] Example 3: Induction of Expression and Post-processing

[0219] All expression vectors were transformed into BL21(DE3) strain and plated on K-antibody plates. Single colonies from the plates were inoculated into LB medium containing K-antibody and cultured overnight at 37°C and 220 rpm in a shaker. The overnight culture was then dilute 1:100 in 400 ml of LB medium and cultured at 37°C and 220 rpm until the OD600 reached between 0.8 and 1.0. Subsequently, the culture was induced with 0.1 mM IPTG and expressed overnight at 16°C and 220 rpm. The overnight expressed bacterial culture was transferred to centrifuge tubes to collect *E. coli* cells. The cells were resuspended in 15 mL of 20 mM Tris-HCl, and the cells were disrupted using an ultrasonic homogenizer. The supernatant and precipitate were separated by centrifugation, and the protein expression results were determined by SDS-PAGE (e.g., [missing information]). Figure 5 (As shown in Part A). After confirming that rMVP1s, rMVP2s, and other proteins were correctly expressed in the supernatant, the supernatant was digested overnight at 4°C with SUMOylase (Sangon Biotech), and the results were determined by SDS-PAGE. The protein length after SUMOylase treatment was approximately 17 kDa (e.g., ...). Figure 5 As shown in Part B), the enzyme digest can be used directly for mite function testing. BL21(DE3) cells without exogenous protein expression plasmids were treated in the same way, and the supernatant was used for overnight lysis as a negative control sample.

[0220] Example 4 Functional Test

[0221] The toxicity activity of the SUMO enzyme-treated supernatant against adult female Tetranychus two-spotted mites was tested. The test method followed the People's Republic of China Agricultural Industry Standard NT / T 1154.13-2008 (Guidelines for Indoor Bioassay of Pesticides Part 13; Leaf Disc Spray Method), and the specific method is as follows:

[0222] The tested mite was the two-spotted spider mite, obtained from the State Key Laboratory of Green Pesticides at South China Agricultural University. Clean, flat, appropriately sized, and uniformly aged kidney bean leaves were prepared and laid flat in a petri dish containing clean filter paper, with the underside facing up. The filter paper was moistened with water. Twenty female adult two-spotted spider mites were then inoculated onto each leaf using a paintbrush. The leaves were then sprayed with mist until the leaf surface and mite bodies were just covered with droplets. The leaves were left to dry naturally in the environment. A shallow layer of water was then added to the petri dish to surround the leaves to prevent the mites from escaping, and the dish was covered. After treatment, the mites were placed in an incubator at a temperature of (25±1℃), humidity of approximately 60%, and a photoperiod of L∶D=(16∶8)h for rearing and observation. Mortality was assessed after 72 hours. During examination, the mites were considered dead if their legs did not move or they showed no reaction when gently touched with a paintbrush.

[0223] Each treatment was repeated three times, with the control sample treated with the same amounts of solvent and emulsifier. Data processing included calculating mortality and corrected mortality rates using the Abbott formula, and analyzing and comparing the toxicological activity of the agents. Mortality rate (%) = (Number of dead insects / Total number of insects) × 100. Corrected mortality rate (%) = (Treatment mortality rate - Control mortality rate) / (1 - Control mortality rate) × 100.

[0224] The experimental results are shown in the table below:

[0225] Table 1. Results of virulence activity tests against female adult two-spotted spider mites.

[0226]

[0227] As the results showed, various predatory mite proteins and spider venom proteins exhibited good acaricidal activity. Among them, rMVP2s showed excellent acaricidal activity, with a corrected mortality rate of 98.25% within 24 hours and 100% within 48 hours. Ctx-4 also showed good acaricidal activity, reaching 100% within 48 hours as well.

[0228] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A predatory mite toxin protein or spider toxin protein, characterized in that, The amino acid sequence of the predatory mite toxin protein or spider toxin protein is selected from SEQ ID NO. 1-8.

2. The predatory mite toxin protein or spider toxin protein according to claim 1, characterized in that, The amino acid sequence of the predatory mite toxin protein or spider toxin protein is selected from SEQ ID NO. 1-8.

3. The predatory mite toxin protein or spider toxin protein according to claim 2, characterized in that, The amino acid sequence of the predatory mite toxin protein is SEQ ID NO. 3 or SEQ ID NO.

4.

4. The predatory mite toxin protein or spider toxin protein according to claim 2, characterized in that, The amino acid sequence of the spider toxin protein is SEQ ID NO.

5.

5. A recombinant predatory mite toxin protein or spider toxin protein, characterized in that, The recombinant predatory mite toxin protein or spider toxin protein comprises a predatory mite toxin protein or spider toxin protein as shown in any one of SEQ ID NO. 1-8, or a predatory mite toxin protein or spider toxin protein as shown in a sequence having more than 95% identity with any one of SEQ ID NO. 1-8, and a snowdrop lectin protein; Optionally, the recombinant predatory mite toxin protein or spider toxin protein further comprises a linker.

6. The recombinant predatory mite toxin protein or spider toxin protein of claim 5, wherein, The recombinant predatory mite toxin protein or spider toxin protein comprises a predatory mite toxin protein or spider toxin protein as shown in any one of SEQ ID NO. 1-8, and a snowdrop lectin protein; Optionally, the recombinant predatory mite toxin protein or spider toxin protein further comprises a linker.

7. The recombinant predatory mite toxin protein or spider toxin protein of claim 5 or 6, wherein, The recombinant predatory mite toxin protein or spider toxin protein further comprises a coding sequence of a signal peptide and / or a SUMO peptide sequence.

8. The recombinant predatory mite toxin protein or spider toxin protein of claim 7, wherein the amino acid sequence of the SUMO peptide is SEQ ID NO. 20 or SEQ ID NO.

21.

9. The recombinant predatory mite toxin protein or spider toxin protein of claim 7 or 8, wherein, The amino acid sequence of the snowdrop lectin protein is SEQ ID NO.

22.

10. A recombinant predatory mite toxin protein or spider toxin protein gene, characterized in that, The recombinant predatory mite toxin protein or spider toxin protein gene comprises a coding sequence of the predatory mite toxin protein or spider toxin protein according to any one of claims 1-4, or the recombinant predatory mite toxin protein or spider toxin protein according to any one of claims 5-9.

11. The recombinant predatory mite toxin protein or spider toxin protein gene of claim 10, wherein, The recombinant predatory mite toxin protein or spider toxin protein gene further comprises a coding sequence of a signal peptide and / or a coding sequence of a SUMO peptide.

12. The recombinant predatory mite toxin protein or spider toxin protein gene of claim 11, wherein, The amino acid sequence of the SUMO peptide is SEQ ID NO. 20 or SEQ ID NO.

21.

13. The recombinant predatory mite toxin protein or spider toxin protein gene of claim 10, wherein, The nucleotide sequence of the recombinant predatory mite toxin protein or spider toxin protein gene is selected from SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16 or SEQ ID NO.

18.

14. Vector, characterized in that, The vector comprises the recombinant predatory mite toxin protein or spider toxin protein gene according to any one of claims 10-13.

15. A host cell characterized in that, The host cell comprises the vector according to claim 14.

16. A method for producing a predatory mite toxin protein or a spider toxin protein, characterized by, The step of culturing the host cell according to claim 15.

17. Use of the predatory mite toxin protein or spider toxin protein according to any one of claims 1-4, or the recombinant predatory mite toxin protein or spider toxin protein according to any one of claims 5-9 in the preparation of a pesticide for inhibiting red spider mites.

18. The use according to claim 17, characterized in that, The red spider mite is Tetranychus urticae.

19. A pesticide for inhibiting red spider mites, characterized by, The pesticide comprises the predatory mite toxin protein or spider toxin protein according to any one of claims 1-4, or the recombinant predatory mite toxin protein or spider toxin protein according to any one of claims 5-9.

20. The pesticide of claim 19, wherein, The red spider is Tetranychus urticae.

21. The pesticide according to claim 19 or 20, characterized in that, The pesticide further comprises a pharmaceutically acceptable adjuvant.

22. The pesticide of claim 21, wherein, The pesticide is an aqueous preparation, an oily preparation or a solid preparation.

Images