Truncated Vip3Af protein and application thereof

Abstract

The invention discloses a truncated Vip3Af protein and application thereof.The transgenic plant obtained by genetic transformation of the truncated Vip3Af protein can express the Vip3Af protein in a high-dose mode, meanwhile, the phenotype of the transgenic plant is not remarkably different from that of conventional non-transgenic corn, the purpose of reducing the toxicity of the Vip3Af protein to plant cells is achieved, and the transgenic plant can be applied to the field of plant growth. And the insecticidal activity of the Vip3Af protein is not different from that of the original Vip3Af protein. Meanwhile, a section of polypeptide is fused at the N terminal of the truncated Vip3Af protein, and the Vip3Af protein transcribed and expressed in plant cells is guided into chloroplast, so that the toxicity of the Vip3Af protein to the plant cells is further reduced, and a transgenic plant with higher expression quantity of the Vip3Af protein, better insect-resistant activity and no influence on plant phenotype can be obtained. The problems that in the prior art, Vip3Af protein is high in toxicity and low in expression quantity are solved.

Description

(I) Technical field

[0001] The present invention relates to a truncated Vip3Af protein and an application thereof, in particular to a truncated Vip3Af protein and a method for reducing the toxicity of the Vip3Af protein to transgenic plant cells, and an application of the truncated Vip3Af protein in preventing and controlling lepidopteran pests. (II) Background technology

[0002] Pests cause billions of dollars in losses to global agricultural production every year. Pest control currently relies mainly on the use of chemical pesticides and the promotion of genetically modified crops. The residues of chemical pesticides can have adverse effects on human health and the environment. The use of biotechnology to cultivate genetically modified insect-resistant crops containing insect-resistant genes can significantly reduce the use of chemical pesticides and effectively protect crops from pests. There are many types of insecticidal proteins. The most commonly used ones are insecticidal crystal proteins from Bacillus thuringiensis (Bt), such as Cry1A, Cry1F, etc.; and Bt vegetative insecticidal proteins, such as Vip3, etc. (Estruch, Warren et al. 1996, Adang, Crickmore et al. 2014). Bt insecticidal proteins have been used in genetically modified plants for more than 20 years. In 2017, the global application area of ​​insect-resistant genetically modified crops exceeded 70 million hectares (ISAAA 2018). Insecticidal crystal proteins are the earliest discovered and most widely used Bt insecticidal proteins.

[0003] Vip3A proteins have high insecticidal activity against Noctuidae pests (e.g., fall armyworm, cotton bollworm, etc.) of the Lepidoptera, but low or no insecticidal activity against Pyralidae pests (e.g., corn borer) (Chakroun, Banyuls et al. 2016). The most diverse and commonly used Vip3Aa proteins (e.g., Vip3Aa1, Vip3Aa20, Vip3Aa45, etc.) have no insecticidal activity against Pyralidae pests (e.g., corn borer, striped stem borer, etc.).

[0004] High-dose expression of exogenous insecticidal proteins in plant cells often causes a certain degree of cytotoxicity. During the research process, the researchers found that when Vip3Af protein was expressed at high doses in crops such as rice and corn, albinism was common in transformed seedlings, the leaves lost their green color, and they could not survive; while the transformed seedlings without albinism often expressed insecticidal proteins at medium or even low doses. Such transformed seedlings could not achieve the expected insect-resistant effect, could not fully solve the insect-resistant problem, and had the risk of pest resistance in actual production applications. Chinese patent CN103045628B discloses a method for reducing plant cell toxicity, that is, by gene fusion, a polypeptide is connected to the N-terminus of the Vip3 protein through a connecting peptide, thereby achieving the effect of reducing plant cell toxicity. This invention is characterized in that the full-length Vip3 protein is used to reduce plant cell toxicity while retaining the insecticidal activity of the original Vip3 protein; however, this method must introduce a new polypeptide, such as green fluorescent protein GFP. In production practice, sometimes additional proteins are not needed, and the introduction of additional proteins may cause other problems, such as the impact of the newly introduced protein on other non-target organisms.

[0005] Although the N-terminal sequence of Vip3 protein is highly conserved and has an important influence on solubility, hydrolysis and insecticidal activity, and is an important region for Vip3 protein to form insecticidal activity, the effect of N-terminal modification on insecticidal activity is highly uncertain. At present, the specific insecticidal mechanism of Vip3A protein has not been clearly elucidated, and its binding receptor on the surface of insect midgut cells is also unclear. The N-terminal sequence of Vip3A protein is highly conserved. For example, Zack et al. found that the insecticidal activity of the new protein obtained by exchanging the N-terminal (20kDa) sequence of Vip3Ab1 and Vip3Bc1 has been completely lost (Zack, Sopko et al. 2017); BANYULS et al. found that mutating the 34th amino acid of Vip3Af protein from methionine to leucine can improve the insecticidal activity against fall armyworm and cotton leafworm (S. littoralis) (Banyuls, Quan et al. 2021). Deleting the first 27 N-terminal amino acids from Vip3Aa can inactivate the protein and completely lose its solubility; after deleting the first 33 amino acids, it has no killing effect on Spodoptera litura, Plutella xylostella and Eariasvitella (Bhalla, Dalal et al. 2005); while deleting the first 39 N-terminal amino acids will have different effects on its toxicity to S. litura and Chilo partellus (Selvapandiyan, Arora et al. 2001). In general, existing studies have shown that the N-terminus of Vip3 protein has an important influence on its insecticidal activity, but the specific mechanism has not yet been clarified. Deleting, adding or mutating it may change the insecticidal activity of Vip3 protein.

[0006] Since the N-terminal sequence of Vip3Af protein is highly conserved, it has an important influence on its insecticidal activity. Any change may weaken or even eliminate the insecticidal protein activity. At the same time, it is currently impossible to predict whether deleting the N-terminal sequence of Vip3Af protein can reduce plant cell toxicity. Therefore, how to reduce the toxicity of exogenous insecticidal proteins to plant cells and maintain high expression levels is an important problem faced by researchers. (III) Summary of the invention

[0007] The present invention provides a truncated Vip3Af protein obtained by modifying the N-terminal sequence of the Vip3Af protein, a method for reducing the toxicity of the Vip3Af protein to transgenic plant cells, and an application thereof in preventing and controlling lepidopteran pests. The coding gene of the protein is introduced into plant cells. The transgenic plants obtained by genetic transformation can express the Vip3Af protein at a high dose, and the phenotype of the transgenic plants is not significantly different from that of conventional non-transgenic corn, thereby achieving the purpose of reducing the toxicity of the Vip3Af protein to plant cells, and the insecticidal activity is no different from that of the original Vip3Af protein. At the same time, we fuse a polypeptide to the N-terminus of the truncated Vip3Af protein, guide the Vip3Af protein transcribed and expressed in the plant cells to the chloroplast, further reduce the toxicity of the Vip3Af protein to the plant cells, and obtain transgenic plants with higher expression of the Vip3Af protein, better insect-resistant activity, and unaffected plant phenotype. The problem of high toxicity and low expression of the Vip3Af protein in the prior art is solved.

[0008] The technical solution adopted by the present invention is:

[0009] In a first aspect, the present invention provides a truncated Vip3Af protein, wherein the truncated Vip3Af protein is obtained by partially deleting amino acids 2 to 32 at the N-terminus of the original Vip3Af protein amino acid sequence shown in SEQ ID NO: 1, and retaining the remaining amino acids.

[0010] Since the first amino acid of the original Vip3Af protein (amino acid sequence as shown in SEQ ID NO: 1) is methionine encoded by the start codon and cannot be deleted, all truncated Vip3Af proteins provided by the present invention retain the first amino acid methionine encoded by the start codon.

[0011] Furthermore, the truncated Vip3Af protein is obtained by deleting one of positions 2-10, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17 or 2-18, 2-32 at the N-terminus of the amino acid sequence shown in SEQ ID NO:1.

[0012] Furthermore, the amino acid sequence of the truncated Vip3Af protein is shown in one of SEQ ID NO:2-SEQ ID NO:11, which is obtained by deleting one of the following sites at the N-terminus of the amino acid sequence shown in SEQ ID NO:1:

[0013] (1) Deleting the amino acids from position 2 to position 10 of the N-terminus (NKNNTKLST) to obtain a truncated Vip3Af protein named Vip3Af_T10, whose amino acid sequence is shown in SEQ ID NO: 2 and nucleotide sequence is shown in SEQ ID NO: 13;

[0014] (2) Deleting the amino acids from position 2 to position 11 of the N-terminus (NKNNTKLSTR) to obtain a truncated Vip3Af protein named Vip3Af_R11, whose amino acid sequence is shown in SEQ ID NO:3 and nucleotide sequence is shown in SEQ ID NO:14;

[0015] (3) deleting the amino acids 2 to 12 at the N-terminus (NKNNTKLSTRA) to obtain a truncated Vip3Af protein named Vip3Af_A12, whose amino acid sequence is shown in SEQ ID NO:4 and nucleotide sequence is shown in SEQ ID NO:15;

[0016] (4) deleting the amino acids from position 2 to position 13 of the N-terminus (NKNNTKLSTRAL) to obtain a truncated Vip3Af protein named Vip3Af_L13, whose amino acid sequence is shown in SEQ ID NO:5 and nucleotide sequence is shown in SEQ ID NO:16;

[0017] (5) Deleting the amino acids from position 2 to position 14 of the N-terminus (NKNNTKLSTRALP) to obtain a truncated Vip3Af protein named Vip3Af_P14, whose amino acid sequence is shown in SEQ ID NO: 6 and nucleotide sequence is shown in SEQ ID NO: 17;

[0018] (6) Deleting the amino acids from position 2 to position 15 of the N-terminus (NKNNTKLSTRALPS) to obtain a truncated Vip3Af protein named Vip3Af_S15, whose amino acid sequence is shown in SEQ ID NO: 7 and nucleotide sequence is shown in SEQ ID NO: 18;

[0019] (7) deleting the amino acids from position 2 to position 6 of the N-terminus (NKNNTKLSTRALPSF) to obtain a truncated Vip3Af protein named Vip3Af_F16, whose amino acid sequence is shown in SEQ ID NO: 8 and nucleotide sequence is shown in SEQ ID NO: 19;

[0020] (8) deleting the amino acids from position 2 to position 17 at the N-terminus (NKNNTKLSTRALPSFI) to obtain a truncated Vip3Af protein named Vip3Af_I17, whose amino acid sequence is shown in SEQ ID NO:9 and nucleotide sequence is shown in SEQ ID NO:20;

[0021] (9) deleting the amino acids from position 2 to position 18 of the N-terminus (NKNNTKLSTRALPSFID) to obtain a truncated Vip3Af protein named Vip3Af_D18, whose amino acid sequence is shown in SEQ ID NO: 10 and nucleotide sequence is shown in SEQ ID NO: 21;

[0022] (10) The amino acids from position 2 to position 32 of the N-terminus were deleted (NKNNTKLSTRALPSFIDYFNGIYGFATGIKD) to obtain a truncated Vip3Af protein named Vip3Af_D32, whose amino acid sequence is shown in SEQ ID NO:11 and nucleotide sequence is shown in SEQ ID NO:22.

[0023] In a second aspect, the present invention provides a method for reducing the toxicity of Vip3Af protein to plant cells, wherein the method comprises expressing a truncated Vip3Af protein in a plant genome, wherein the amino acid sequence of the truncated Vip3Af protein is shown in one of the above SEQ ID NOs: 2 to 11.

[0024] In a third aspect, the present invention provides a method for reducing the toxicity of Vip3Af protein to plant cells while increasing the expression level, wherein the method is to guide the truncated Vip3Af protein into the chloroplasts of plant cells via a guide peptide.

[0025] Furthermore, the guide peptide is connected to the N-terminus of the truncated Vip3Af protein and expressed in fusion in plant cells.

[0026] After the truncated Vip3Af protein is transcribed and translated in plant cells, it is transported to the chloroplasts of plant cells through polypeptides to obtain transgenic transformed plants with higher expression levels and lower toxicity. The polypeptide (such as guide peptide / transit peptide / signal peptide) includes the small subunit and the first intron (SSU-CTP) of the DNA coding region of corn ribulose 1,5-bisphosphate carboxylase, corn acetolactate synthase 1 transit peptide (AHAS), etc. The polypeptide is spliced ​​to the N-terminus of the Vip3Af truncated protein through gene fusion, so that the two are fused and expressed in plant cells, and the aforementioned process of guiding the Vip3 protein to the chloroplast can be achieved. The purpose of guiding the Vip3 protein to the chloroplast is to accumulate the Vip3 protein and obtain a transgenic plant with a higher expression level of the Vip3 protein. Therefore, no matter which polypeptide is used, as long as any polypeptide that can guide the Vip3 protein to the chloroplasts of plant cells is fused and expressed with the Vip3 protein, the transgenic plant with a higher Vip3 expression level and lower toxicity described in the present invention can be obtained, which should be regarded as the content of the present invention.

[0027] Furthermore, the polypeptide is preferably the small subunit and the first intron SSU-CTP of the maize ribulose 1,5-bisphosphate carboxylase DNA coding region, and the nucleotide sequence is shown in SEQ ID NO:24.

[0028] In a fourth aspect, the present invention also provides an expression cassette comprising a gene encoding a truncated Vip3Af protein. The gene expression cassette comprises a promoter, a truncated Vip3Af protein encoding gene and a terminator. The promoter includes, but is not limited to, promoters commonly used in genetic engineering such as the maize ubiquitin promoter pZmUbi and the cauliflower mosaic virus 35s promoter pCaMV 35s, preferably the promoter is pZmUbi (nucleotide sequence as shown in SEQ ID NO: 23); the terminator includes, but is not limited to, the terminator tNOS of the Agrobacterium tumefaciens nicotinic acid synthase and the terminator tCaMV 35s of the cauliflower mosaic virus 35s RNA, preferably the terminator is tNOS (nucleotide sequence as shown in SEQ ID NO: 25). It should be noted that the truncated Vip3Af protein of the present invention can be encoded by different nucleotide sequences. In particular, the usage frequencies of different codons may be different in different species, and different nucleotide sequences may be used according to the changes in the usage scenarios. For example, the GC content is low in the prokaryotic organism Escherichia coli, while the GC content is high in plants such as rice and corn. Therefore, as long as the truncated Vip3Af protein disclosed in the present invention can be encoded, even if the nucleotide sequence is not included in the present invention, it should be regarded as the content of the present invention.

[0029] Furthermore, the expression cassette also includes a polypeptide sequence that guides the truncated Vip3 protein to the chloroplast, and the polypeptide sequence includes but is not limited to the small subunit and the first intron (SSU-CTP) of the corn ribulose 1,5-bisphosphate carboxylase DNA coding region, and the corn acetolactate synthase 1 transit peptide (AHAS). Preferably, the guiding peptide is the small subunit and the first intron SSU-CTP of the corn ribulose 1,5-bisphosphate carboxylase DNA coding region (the nucleotide sequence is shown in SEQ ID NO: 24).

[0030] In a fifth aspect, the present invention provides a transformation vector for expressing a truncated Vip3Af protein. The transformation vector comprises a gene expression cassette encoding a truncated Vip3Af protein according to the present invention. The basic vector of the transformation vector is a conventional plant genetic transformation vector, including but not limited to pCambia1300, pCambia1301, pCambia2301, etc. There is no limitation on the relative order and insertion position of the gene expression cassette in the transformation vector, and any transformation vector comprising the gene expression cassette according to the present invention shall be regarded as the content of the present invention.

[0031] Furthermore, the transformation vector also includes a glyphosate selection marker gene G10-2 expression cassette, wherein the G10-2 expression cassette is constructed by constructing an expression cassette with a marker gene G10-epsps (SEQ ID NO: 26) that can confer glyphosate resistance selection, and its expression is promoted by the yellow leaf curl virus promoter pCmYLCV (SEQ ID NO: 27), and its expression is terminated by the original t35s terminator on pCambia1300.

[0032] The transgenic plants of the present invention can be obtained by plant genetic transformation methods known to those skilled in the art. The transformation vector containing the gene expression cassette of the present invention is introduced into plant cells (corn (Ishida, Saito et al. 1996), soybean (Zeng, Vadnais et al. 2004), rice (Hiei, Komari et al. 1997)) by plant genetic transformation methods such as gene gun method and Agrobacterium transformation method, and integrated into the plant genome, so that the plant cells can express the truncated Vip3A protein of the present invention. Through the transgenic plants obtained by the genetic transformation method, researchers can screen transgenic plants that express the truncated Vip3Af protein of the present invention at high doses. Such transgenic plants have two characteristics: (1) transgenic plants have high insecticidal activity against pests; (2) the phenotype of transgenic plants is no different from that of conventional non-transgenic corn.

[0033] In a sixth aspect, the present invention provides an application of the truncated Vip3Af protein in the preparation of transgenic insect-resistant plants for controlling lepidopteran pests, wherein the application is to transfer the gene expression cassette encoding the truncated Vip3Af protein into plants to cultivate transgenic plants resistant to lepidopteran pests.

[0034] The pests of the present invention include lepidopteran pests, and the lepidopteran pests include at least one of the following pests: fall armyworm (Spodoptera frugiperda), beet armyworm (Spodoptera exigua), fall armyworm (Spodoptera litura), armyworm (Mythimna separata), cotton bollworm (Helicoverpa armigera), cutworm (Agrotis ipsilon), corn borer (Ostrinia furnacalis), rice leaf roller (Cnaphalocrocismedinalis), striped stem borer (Chilo suppressalis), peach borer (Dichocrocis punctiferalis), bean pod borer (Maruca vitrata), diamondback moth (Plutella xylostella), gray armyworm (Spodoptera mauritia), and two-spotted armyworm (Athetis lepigone).

[0035] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0036] (1) The present invention provides a truncated Vip3Af protein with high insecticidal activity. Since the modification of Vip3Af protein is highly uncertain, any deletion, addition or mutation may lead to unpredictable changes in the insecticidal activity of Vip3Af protein. Therefore, the prior art cannot predict whether the Vip3 protein can retain its insecticidal activity after deleting part of the N-terminal sequence. The present invention has conducted in-depth research on the N-terminal sequence of Vip3Af protein, and through comparative verification, a series of truncated Vip3Af proteins with high insecticidal activity and broad insecticidal spectrum have been obtained, which can kill a variety of important Lepidoptera pests such as fall armyworm, beet armyworm, oriental armyworm, corn borer, and striped stem borer.

[0037] (2) The present invention provides a method for reducing the toxicity of insecticidal proteins to transgenic plant cells. The prior art mainly uses methods such as gene fusion, which require the introduction of additional polypeptides or proteins into plant cells to achieve the purpose of reducing cytotoxicity. By using the truncated Vip3 protein described in the present invention, the goal of reducing cytotoxicity in plant cells can be achieved without the introduction of additional proteins or polypeptides. The present invention obtains an N-terminally truncated Vip3Af protein that retains insecticidal activity through screening, and introduces its encoding gene into the plant genome to verify its effect on plant cells, creatively obtaining a Vip3Af truncated protein that has reduced toxicity to plant cells and has high insecticidal activity.

[0038] (3) The present invention provides a method for obtaining transgenic plants with higher Vip3 protein expression levels. Transgenic plants expressing Vip3 protein are obtained using existing technologies, wherein transformants with high expression of Vip3 protein will show albinism, dwarfing and even death; while the expression level of Vip3 protein in the transgenic plant tissues screened is often relatively low. By the method provided by the present invention, the Vip3 protein expressed in plant cells is guided to the chloroplasts so that its expression level is accumulated. Since the truncated Vip3Af protein with reduced toxicity is used, even if the expression level in the cells accumulates to a high level, the phenotype of the transgenic plant is not significantly different from that of conventional non-transgenic corn. By the method of the present invention, transgenic transformants with extremely high Vip3 protein expression and no phenotype difference from conventional non-transgenic corn can be created. Researchers can use the method described in the present invention to create a new generation of transgenic plants with extremely high insect-resistant protein expression, improve the insect-resistant efficiency of transgenic plants, slow down the occurrence of pest resistance to transgenic plants, and extend the service life of transgenic insect-resistant plants. (IV) Specific implementation methods

[0039] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0040] The molecular biology and biochemical methods used in the following examples of the present invention are all known techniques. They are described in Current Protocols in Molecular Biology, edited by Ausubel and published by John Wiley and Sons, and Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al. and published by Cold Spring Harbor Laboratory Press (2001), 3 rd ED. and other literature have detailed descriptions.

[0041] Example 1. Design of Vip3Af truncated protein and preparation of recombinant protein

[0042] 1. Design of Vip3Af truncated protein

[0043] The original Vip3Af protein consists of 788 amino acids, and its amino acid sequence is shown in SEQ ID NO: 1, and its nucleotide sequence is shown in SEQ ID NO: 12. The present invention designed ten Vip3Af proteins with different truncated lengths to verify their insecticidal activity and effects on plant cell toxicity. Since the protein still requires a start codon, all truncated proteins retain the first amino acid methionine. The amino acids at positions 2 to 10 of the N-terminus (NKNNTKLST) were deleted to obtain a truncated Vip3Af protein named Vip3Af_T10, whose amino acid sequence is shown in SEQ ID NO:2, and whose nucleotide sequence is shown in SEQ ID NO:13; the amino acids at positions 2 to 11 of the N-terminus (NKNNTKLSTR) were deleted to obtain a truncated Vip3Af protein named Vip3Af_R11, whose amino acid sequence is shown in SEQ ID NO:3, and whose nucleotide sequence is shown in SEQ ID NO:14; the amino acids at positions 2 to 12 of the N-terminus (NKNNTKLSTRA) were deleted to obtain a truncated Vip3Af protein named Vip3Af_A12, whose amino acid sequence is shown in SEQ ID NO:4, and whose nucleotide sequence is shown in SEQ ID NO:15; the amino acids at positions 2 to 13 of the N-terminus (NKNNTKLSTRAL) were deleted to obtain a truncated Vip3Af protein named Vip3Af_L13, whose amino acid sequence is shown in SEQ ID NO:5, and the nucleotide sequence is shown in SEQ ID NO:16; the amino acids at positions 2 to 14 of the N-terminus (NKNNTKLSTRALP) are deleted to obtain a truncated Vip3Af protein named Vip3Af_P14, whose amino acid sequence is shown in SEQ ID NO:6, and the nucleotide sequence is shown in SEQ ID NO:17; the amino acids at positions 2 to 15 of the N-terminus (NKNNTKLSTRALPS) are deleted to obtain a truncated Vip3Af protein named Vip3Af_S15, whose amino acid sequence is shown in SEQ ID NO:7, and the nucleotide sequence is shown in SEQ ID NO:18; the amino acids at positions 2 to 16 of the N-terminus (NKNNTKLSTRALPSF) are deleted to obtain a truncated Vip3Af protein named Vip3Af_F16, whose amino acid sequence is shown in SEQ ID NO:8, and the nucleotide sequence is shown in SEQ ID NO:19; the amino acids from position 2 to position 17 of the N-terminus (NKNNTKLSTRALPSFI) were deleted to obtain a truncated Vip3Af protein named Vip3Af_I17, the amino acid sequence of which is shown in SEQ ID NO:9, and the nucleotide sequence is shown in SEQ ID NO:20;The amino acids at positions 2 to 18 of the N-terminus (NKNNTKLSTRALPSFID) were deleted to obtain a truncated Vip3Af protein named Vip3Af_D18, whose amino acid sequence is shown in SEQ ID NO:10 and whose nucleotide sequence is shown in SEQ ID NO:21; the amino acids at positions 2 to 32 of the N-terminus (NKNNTKLSTRALPSFIDYFNGIYGFATGIKD) were deleted to obtain a truncated Vip3Af protein named Vip3Af_D32, whose amino acid sequence is shown in SEQ ID NO:11 and whose nucleotide sequence is shown in SEQ ID NO:22. The coding genes involved in the present invention were all obtained by artificial gene synthesis by Shanghai Shenggong Company. ;

[0044] SEQ ID NO:1 Vip3Af amino acid sequence

[0045] 。

[0046] 2、Preparation of protein composition

[0047] The recombinant protein is expressed and prepared using the Escherichia coli pET expression system. The target gene is introduced into the pET expression vector, and then the expression vector is transferred into a suitable expression strain, and the expression of the target gene is induced under certain conditions to prepare the recombinant protein. In this embodiment, regardless of the original Vip3Af protein or the truncated Vip3Af protein, the preparation process is completely the same, and the original Vip3Af protein is used as an example for explanation in this embodiment.

[0048] The preparation process of the original Vip3Af protein is as follows: the Vip3Af gene is cloned between the two restriction endonuclease sites of BamHI and SacI of the expression vector pet28a (Novagen, 69015-3, USA) to obtain the recombinant plasmid pet28a-Vip3Af; the recombinant plasmid is introduced into the Escherichia coli BL21 (DE3) cell line by heat shock transformation, and cultured overnight at 37°C on LB solid medium containing 50 mg / L kanamycin to screen for monoclonal positive colonies; the monoclonal positive colonies are inoculated into LB liquid medium containing 50 mg / L kanamycin, and after overnight culture at 37°C, 0.2 ml of the bacterial solution is subcultured and inoculated into a conical flask containing 200 ml of LB liquid culture medium containing 50 mg / L kanamycin, and cultured at 37°C with shaking until the OD 600=0.6, then IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to a final concentration of 0.5 mM, cooled in a 4°C refrigerator for 10 min, and then transferred to a 16°C constant temperature horizontal shaking incubator and cultured at a constant speed of 100 rpm for 16 hours to induce the expression of the recombinant protein; after the expression was completed, the culture solution was centrifuged at 5000 rcf for 15 minutes, the supernatant culture solution was discarded, the cell pellet was collected, and 50 ml of 20 mM phosphate buffer (PBS, pH = 7.4 ) resuspend the cells; then place the resuspended solution in ice water to cool sufficiently, and then use an ultrasonic disruptor (Ningbo Xinzhi Biotechnology Co., Ltd., model JY92-IIN) to crush the cells (probe diameter 10 mm, crushing at 650 W, 50% machine display power for 15 min, working for 3 s and resting for 6 s), after centrifugation at 12000 rpm for 10 min, collect the supernatant and precipitate respectively, and use polyacrylamide gel electrophoresis (SDS-PAGE) to verify the band size of the target protein, and store the protein samples containing the correct target band in a -80 ℃ refrigerator for later use. The Vip3Af recombinant protein was present in the supernatant after ultrasonic disruption. The supernatant containing the Vip3Af protein was purified by His-tag (Qiagen, Ni-NTA superflow, Lot No. 160049084). The purification steps were carried out according to the instructions of the reagent, as follows: (1) 1 ml of Ni-NTA resin was filled into the column and allowed to stand until stratification; (2) The preservation solution was discharged and the resin was washed with 8 to 10 column volumes of Lysis Buffer until it was completely balanced; (3) The sample was added to the column, sealed, and gently shaken (or the column was laid flat), and allowed to stand at room temperature (or 4°C, depending on the protein situation) for 30 minutes; (4) The column was placed on an iron stand and the supernatant was released at a flow rate of 1 ml / min; (5) The column was washed with 5 column volumes of Wash buffer at a flow rate of 1 ml / min to wash away the impurities; (6) The column was eluted with 5 column volumes of Elution buffer at a flow rate of 1 ml / min, and the eluate was stored. Finally, the absorbance of the eluate at A280 was measured using a Mona eva3100 detector (Cat. No. GD30101) to obtain the concentration of the target protein in the recombinant protein. The protein concentration was adjusted to 2.5 mg / ml using Carbonate buffer (50 mM Na2CO3, pH = 10) to obtain the Vip3Af recombinant protein solution.

[0049] The formula of Lysis buffer in the protein purification process is: 20mM NaH2PO4, 300mM NaCl, 10mM imidazole, and the pH is adjusted to 8.0 with NaOH; the formula of Wash buffer is: 20mM NaH2PO4, 300mM NaCl, 20mM imidazole, and the pH is adjusted to 8.0 with NaOH; the formula of Elution buffer is 20mM NaH2PO4, 300mM NaCl, 250mM imidazole, and the pH is adjusted to 8.0 with NaOH.

[0050] The LB liquid culture medium is composed of: 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride, the solvent is double distilled water, and it is sterilized at 120°C and high temperature and high pressure for 15 minutes; the LB solid culture medium is prepared by adding 15 g / L agar to the LB liquid culture medium, sterilizing it at high temperature and high pressure, and then inverting it in a sterilized culture dish to cool it.

[0051] Vip3Af_T10, Vip3Af_R11, Vip3Af_A12, Vip3Af_L13, Vip3Af_P14, Vip3Af_S15, Vip3Af_F16, Vip3Af_I17, Vip3Af_D18, and Vip3Af_D32 proteins were prepared by the same method and stored in a -80°C refrigerator for future use.

[0052] Example 2: Determination of biological activity of recombinant protein against Lepidoptera insects

[0053] Experimental subjects: Insects within 2 hours of birth were selected as test insects, including fall armyworm, beet armyworm, cotton bollworm, corn borer, armyworm, and striped stem borer.

[0054] Treatment group: The recombinant protein prepared by the method of Example 1 was subjected to a preliminary test of biological activity (1 ng / cm 2 , 10ng / cm 2 , 100ng / cm 2 、1000ng / cm 2 、10000ng / cm 2 Five concentration groups); according to the preliminary experimental results, the lowest concentration at which all the test insects died was set as the upper limit concentration, and 6 concentration gradients were set (the concentration ranges are shown in Tables 1 to 6 below, diluted with PBS buffer at pH = 7.4), and the concentration interval with 50% lethality to insects was determined. The number of insects tested in each concentration group was 80.

[0055] Negative control group: The preparation method of the negative control is the same as that of Example 1, the only difference being that the plasmid transferred into the E. coli expression strain is a pet28a empty vector without any inserted DNA sequence.

[0056] Determination method: The lethal concentration LC of recombinant protein to insects was determined by surface tiling method. 50 .

[0057] After the artificial insect feed is cooked, it is spread flat on a 24-well culture plate (each well has a diameter of 1.5 cm) and the feed is waited for to solidify. One 24-well plate is used for each concentration, and 20 wells are randomly set up in each 24-well plate as the experimental group and 4 as the negative control group. 200 microliters of recombinant protein is spread on the surface of the feed in each well of the experimental group, and 200 microliters of negative control protein is spread on the surface of the feed in each well of the negative control group. After the entire plate is spread, it is placed on a shaker at 120 rpm and shaken at room temperature (25-30°C) until the liquid dries. Two test insects are placed in each well and sealed with a breathable sealing film. At least 120 test insects are tested for each test concentration. They are kept in a light-proof incubator at 28°C for 7 days, and the insect mortality rate is counted to obtain the corresponding lethal concentration LC. 50 , the results are shown in Table 7 below.

[0058] Table 1 Concentration gradient of recombinant proteins for biological activity determination against newly hatched larvae of Spodoptera frugiperda (ng / cm 2 )

[0059] Recombinant protein Concentration 1 Concentration 2 Concentration 3 Concentration 4 Concentration 5 Concentration 6 Vip3 0.5 2 5 25 100 250 Vip3Af_T10 0.5 2 5 25 100 250 Vip3Af_R11 0.5 2 5 25 100 250 Vip3Af_A12 0.5 2 5 25 100 250 Vip3Af_L13 0.5 2 5 25 100 250 Vip3Af_P14 0.5 2 5 25 100 250 Vip3Af_S15 0.5 2 5 25 100 250 Vip3Af_F16 0.5 2 5 25 100 250 Vip3Af_I17 0.5 2 5 25 100 250 Vip3Af_D18 0.5 2 5 25 100 250 Vip3Af_D32 0.5 2 5 25 100 250

[0060] Table 2 Concentration gradient of recombinant proteins for biological activity determination against newly hatched larvae of Spodoptera exigua (ng / cm 2 )

[0061] Recombinant protein Concentration 1 Concentration 2 Concentration 3 Concentration 4 Concentration 5 Concentration 6 Vip3 2 5 25 50 250 500 Vip3Af_T10 2 5 25 50 250 500 Vip3Af_R11 2 5 25 50 250 500 Vip3Af_A12 2 5 25 50 250 500 Vip3Af_L13 2 5 25 50 250 500 Vip3Af_P14 2 5 25 50 250 500 Vip3Af_S15 2 5 25 50 250 500 Vip3Af_F16 2 5 25 50 250 500 Vip3Af_I17 2 5 25 50 250 500 Vip3Af_D18 2 5 25 50 250 500 Vip3Af_D32 2 5 25 50 250 500

[0062] Table 3 Concentration gradient of recombinant proteins for biological activity determination on newly hatched larvae of cotton bollworm (ng / cm 2 )

[0063]

[0064]

[0065] Table 4 Concentration gradient of recombinant proteins for biological activity determination on newly hatched larvae of armyworm (ng / cm 2 )

[0066] Recombinant protein Concentration 1 Concentration 2 Concentration 3 Concentration 4 Concentration 5 Concentration 6 Vip3 50 100 250 500 2000 5000 Vip3Af_T10 50 100 250 500 2000 5000 Vip3Af_R11 50 100 250 500 2000 5000 Vip3Af_A12 50 100 250 500 2000 5000 Vip3Af_L13 50 100 250 500 2000 5000 Vip3Af_P14 50 100 250 500 2000 5000 Vip3Af_S15 50 100 250 500 2000 5000 Vip3Af_F16 50 100 250 500 2000 5000 Vip3Af_I17 50 100 250 500 2000 5000 Vip3Af_D18 50 100 250 500 2000 5000 Vip3Af_D32 50 100 250 500 2000 5000

[0067] Table 5 Concentration gradient of recombinant protein for biological activity determination on newly hatched larvae of corn borer (ng / cm 2 )

[0068] Recombinant protein Concentration 1 Concentration 2 Concentration 3 Concentration 4 Concentration 5 Concentration 6 Vip3 25 50 100 250 500 2000 Vip3Af_T10 25 50 100 250 500 2000 Vip3Af_R11 25 50 100 250 500 2000 Vip3Af_A12 25 50 100 250 500 2000 Vip3Af_L13 25 50 100 250 500 2000 Vip3Af_P14 25 50 100 250 500 2000 Vip3Af_S15 25 50 100 250 500 2000 Vip3Af_F16 25 50 100 250 500 2000 Vip3Af_I17 25 50 100 250 500 2000 Vip3Af_D18 25 50 100 250 500 2000 Vip3Af_D32 25 50 100 250 500 2000

[0069] Table 6 Concentration gradient of recombinant protein for biological activity determination on newly hatched larvae of Chilo suppressalis (ng / cm 2 )

[0070] Recombinant protein Concentration 1 Concentration 2 Concentration 3 Concentration 4 Concentration 5 Concentration 6 Vip3 25 50 100 250 500 2000 Vip3Af_T10 25 50 100 250 500 2000 Vip3Af_R11 25 50 100 250 500 2000 Vip3Af_A12 25 50 100 250 500 2000 Vip3Af_L13 25 50 100 250 500 2000 Vip3Af_P14 25 50 100 250 500 2000 Vip3Af_S15 25 50 100 250 500 2000 Vip3Af_F16 25 50 100 250 500 2000 Vip3Af_I17 25 50 100 250 500 2000 Vip3Af_D18 25 50 100 250 500 2000 Vip3Af_D32 25 50 100 250 500 2000

[0071] The biological activity of newly hatched larvae of Lepidoptera insects was determined according to the above experimental method and concentration gradient. The results are shown in Table 7 below.

[0072] Table 7 The median lethal concentration (LC) of the recombinant protein to newly hatched larvae of important lepidopteran pests 50 (ng / cm 2 )

[0073] Recombinant protein Fall Armyworm Corn borer Beet Armyworm cotton bollworm Sticky insects Borer borer Vip3 16.7 100.6 84.5 9.2 378 256 Vip3Af_T10 >5000 >5000 128.9 12.7 >5000 >5000 Vip3Af_R11 33.1 158.8 117.1 10.9 1249 505 Vip3Af_A12 16.3 92.7 90.4 7.9 502 277 Vip3Af_L13 17.1 103.9 121.7 15.2 818 493 Vip3Af_P14 15.5 96.1 88.2 7.6 431 249 Vip3Af_S15 72.8 >5000 157.0 16.9 1735 >5000 Vip3Af_F16 92.4 >5000 161.8 19.2 >5000 >5000 Vip3Af_I17 >5000 >5000 >5000 17.7 >5000 >5000 Vip3Af_D18 >5000 >5000 >5000 30.6 >5000 >5000 Vip3Af_D32 >5000 >5000 >5000 >5000 >5000 >5000

[0074] The following conclusions can be drawn from the above results of biological activity test on newly hatched larvae of Lepidoptera insects:

[0075] (1) The insecticidal activities of the truncated proteins obtained by deleting the N-terminal sequence of Vip3Af proteins of different lengths were very different. Compared with the original Vip3Af protein, Vip3Af_T10 retained some of its insecticidal activities against Spodoptera exigua and Helicoverpa armigera, but lost its insecticidal activities against Spodoptera frugiperda, Ostrinia furnacalis, Armyworm, and Chilo suppressalis. Vip3Af_R11, Vip3Af_A12, Vip3Af_L13, and Vip3Af_P14 proteins all retained a certain degree of insecticidal activity: among them, the insecticidal activity of Vip3Af_A12 and Vip3Af_P14 against six insects was close to that of the original Vip3Af, the activity of Vip3Af_R11 against fall armyworm, corn borer, armyworm, and striped stem borer was lower than that of Vip3Af, the activity of Vip3Af_R12 against fall armyworm and cotton bollworm was improved, and the activity of Vip3Af_L13 against cotton bollworm, armyworm, and striped stem borer was lower than that of Vip3Af, and the activity of Vip3Af_R14 against fall armyworm and cotton bollworm was improved. The insecticidal activity of Vip3Af_S15, Vip3Af_F16, Vip3Af_I17, Vip3Af_D18, and Vip3Af_D32 was partially lost: their insecticidal activity against corn borer and striped stem borer dropped sharply and was almost inactive; Vip3Af_S15 and Vip3Af_F16 retained some insecticidal activity against fall armyworm, beet armyworm, and cotton bollworm, but their performance was significantly lower than that of the original Vip3Af; Vip3Af_I17 and Vip3Af_D18 only had partial insecticidal activity against cotton bollworm, and were completely inactive against other tested insects; Vip3Af_D32 completely lost its activity against all insects.

[0076] (2) The modification of the N-terminal sequence of the Vip3Af protein is unpredictable. It can be inferred from the experimental results that not all truncated proteins can retain insecticidal activity. Some may only retain partial activity, and some almost completely lose activity. This may be related to the hydrolysis of the Vip3Af protein in the insect body, the formation of the tetrameric structure, and the receptor binding on the midgut epidermal cells. It is impossible to predict the changes in the insecticidal activity of the truncated Vip3 protein in advance; at the same time, the insecticidal activity of the same protein to different test insect pairs is different, and the change in insecticidal activity after truncation is also unpredictable.

[0077] Example 3: Construction of recombinant expression vector

[0078] In this embodiment, the recombinant expression vector is constructed based on the pCambia1300 (NCBI sequence number AF234296) vector. The readers of the present invention can achieve the same effect on other plant genetic transformation vectors according to the method provided in this embodiment, such as pCambia1301, pCambia2301, etc. The promoter used in the expression frame of the insect-resistant gene can be any constitutive promoter, such as the cauliflower mosaic virus 35S promoter PCAMV35S, the maize ubiquitin promoter pZMUBI, etc., without specific range restrictions; the choice of terminator is not limited, for example, the commonly used Agrobacterium NOS terminator tNOS, the cauliflower mosaic virus 35S terminator T35S, etc. can all be used as the terminator of the insect-resistant gene.

[0079] The coding genes involved in the present invention are all artificially synthesized by Shanghai Bioengineering Corporation. The nucleotide sequence encoding gene Vip3Af is SEQ ID NO:12, the nucleotide sequence encoding gene Vip3Af_T10 is SEQ ID NO:13, the nucleotide sequence encoding gene Vip3Af_R11 is SEQ ID NO:14, the nucleotide sequence encoding gene Vip3Af_A12 is SEQ ID NO:15, the nucleotide sequence encoding gene Vip3Af_L13 is SEQ ID NO:16, the nucleotide sequence encoding gene Vip3Af_P14 is SEQ ID NO:17, the nucleotide sequence encoding gene Vip3Af_S15 is SEQ ID NO:18, the nucleotide sequence encoding gene Vip3Af_F16 is SEQ ID NO:19, the nucleotide sequence encoding gene Vip3Af_I17 is SEQ ID NO:20, the nucleotide sequence encoding gene Vip3Af_D18 is SEQ ID NO:21, and the nucleotide sequence encoding gene Vip3Af_D32 is SEQ ID NO:22. The promoters are all maize ubiquitin promoter pZmUbi (nucleotide sequence as shown in SEQ ID NO: 23), the guiding peptide is preferably the small subunit and the first intron SSU-CTP of the maize ribulose 1,5-bisphosphate carboxylase DNA coding region (nucleotide sequence as shown in SEQ ID NO: 24), and the terminators are all Agrobacterium tumefaciens nicotinic acid synthase terminator tNOS (nucleotide sequence as shown in SEQ ID NO: 25).

[0080] In this embodiment, the construction of vector T10 containing Vip3Af_T10 gene is described as an example. The remaining vectors (vector AF containing Vip3Af gene, vector R11 containing Vip3Af_R11 gene, vector A12 containing Vip3Af_A12 gene, vector L13 containing Vip3Af_L13 gene, vector P14 containing Vip3Af_P14 gene, vector S15 containing Vip3Af_S15 gene, vector F16 containing Vip3Af_F16 gene, vector I17 containing Vip3Af_I17 gene, vector D18 containing Vip3Af_D18 gene, vector D32 containing Vip3Af_D32 gene) are constructed in the same manner as the T10 vector, with the only difference being the different coding genes. The specific method is as follows.

[0081] (1) Vector fragment: In order to use glyphosate to screen positive tissues during plant tissue culture, pCambia1300 was double-digested with restriction endonucleases KpnI and XhoI (purchased from ThermoFisher) to remove the original hygromycin selection gene expression cassette in the 1300 vector. After incubation at 37°C for 1 hour, the vector fragment 1300-FV with a size of approximately 6.8 kb was recovered.

[0082] (2) Insect-resistant gene Vip3Af_T10 expression cassette: The maize ubiquitin promoter pZmUbi, the guide peptide SSU-CTP, the coding gene Vip3Af_T10, and the Agrobacterium tumefaciens nicotinic acid synthase terminator tNOS were connected to form an expression cassette. The total length of the expression cassette Ubi-T10-nos was 5.1 kb and was synthesized by Shanghai Bioengineering Co., Ltd. PCR amplification was performed using the primers in Table 8, and the fragments of the corresponding size were recovered by agarose gel electrophoresis for subsequent vector construction.

[0083] (3) Glyphosate selection marker gene G10-2 expression cassette: The G10-2 expression cassette is an expression cassette constructed with the marker gene G10-epsps (SEQ ID NO: 26) that can confer glyphosate resistance selection, and its expression is promoted by the yellow leaf curl virus promoter pCmYLCV (SEQ ID NO: 27), and its expression is terminated by the original t35s terminator on pCambia1300. The total length of the fragment cmp-g102 is 1.9 kb and was synthesized by Shanghai Bioengineering Co., Ltd. PCR amplification was performed using the primers in Table 8, and the corresponding size fragment was recovered by agarose gel electrophoresis for subsequent vector construction.

[0084] (4) Construction of transformation vector: Seamless cloning by homologous recombination (e.g., Nanjing Novozyme MultiS kit, product number C113-01) connect the insect-resistant gene expression cassette in step (2) and the G10-2 expression cassette in step (3) between the HindIII and XhoI sites of the 1300-FV vector in step (1). According to the instructions, mix the seamless cloning reaction solution, incubate at 37°C for 30 minutes, and quickly place it on ice to cool for more than 2 minutes.

[0085] Table 8 Primer sequences for PCR reaction and corresponding amplified fragment lengths

[0086]

[0087] Amplification can be performed using conventional PCR reactions. For example, high-fidelity enzyme 2×Phanta Max Master Mix (Novozyme, catalog number P515-01) is selected and the reaction system is performed according to the instructions.

[0088] Table 9 PCR reaction program

[0089]

[0090]

[0091] The PCR products were recovered by agarose gel electrophoresis and gel cutting, and the concentration of the recovered products was determined.

[0092] Table 10 Seamless cloning reaction solution

[0093]

[0094] (5) Transformation of recombinant expression vector: Escherichia coli TG1 competent cells (Shanghai Angyu Biotechnology Co., Ltd., catalog number G6017) were thawed on ice, and the reaction solution after seamless cloning reaction was added. The cells were placed in an ice bath for 25 min, followed by heat shock in a 42°C water bath for 45 s. The cells were then quickly cooled on ice for 2 min, and 1 ml of LB culture medium was added. The cells were cultured at 37°C with shaking at 200 rpm for 1 h. The cells were then plated on LB solid medium containing 50 mg / L kanamycin. After overnight culture at 37°C, a single clone was selected for sequencing and identification.

[0095] (6) Identification and extraction of recombinant expression vector: The single clone colony in step (5) was selected for sequencing identification, and the glycerol strain of the correctly identified clone was stored in a -80°C refrigerator. Extract and identify the correctly cloned recombinant expression vector and transform Escherichia coli TG1 competent cells by heat shock method. The operation steps are as follows: TG1 competent cells are placed in ice to melt (or placed in the palm of your hand or at room temperature for a while, and quickly inserted into ice when the bacteria are in an ice-water mixed state), add 10 μL of recombinant expression vector, and gently mix it by hand on the bottom of the EP tube, and let it stand on ice for 25 minutes; heat shock in a 42°C water bath for 45 seconds, quickly put it back on ice and let it stand for 2 minutes; add 700 μL of LB sterile culture medium without antibiotics to the centrifuge tube, mix it and recover it at 37°C, 200rpm for 60 minutes; centrifuge at 5000rpm for one minute to collect the bacteria, retain about 100 μL of supernatant, gently blow and resuspend the bacteria and spread it on LB culture medium containing 50 mg / L kanamycin antibiotics; put the plate upside down in a 37°C incubator for overnight culture. Pick a monoclonal plaque and shake it overnight in LB liquid culture medium at 37°C shaker at 220rpm. Use AxyPrep Plasmid DNA Mini Kit to extract plasmid. The operation method is as follows: take 1-4ml of bacterial solution cultured overnight in LB medium, centrifuge at 12,000rpm for 1min, and discard the supernatant; add 250μL Buffer S1 to suspend the bacterial precipitate, the suspension should be uniform, and no small bacterial lumps should be left; add 250μL Buffer S2, gently and fully turn upside down 4-6 times to mix evenly to fully lyse the bacteria until a transparent solution is formed. This step should not exceed 5min; add 350μL Buffer S3, gently and fully turn upside down 6-8 times to mix, centrifuge at 12,000rpm for 10min; take the supernatant from the previous step and transfer it to the preparation tube (placed in a 2ml centrifuge tube), centrifuge at 12,000rpm for 1min, and discard the filtrate; put the preparation tube back into the centrifuge tube, add 500μL Buffer W1, centrifuge at 12,000rpm for 1min, and discard the filtrate; put the preparation tube back into the centrifuge tube, add 700μL Buffer W2, centrifuge at 12,000rpm for 1min, discard the filtrate; wash once again with 700μL Buffer W2 in the same way, discard the filtrate; put the preparation tube back into a 2ml centrifuge tube, centrifuge at 12,000rpm for 1min; move the preparation tube into a new 1.5ml centrifuge tube, add 60-80μL Eluent or deionized water to the center of the preparation tube membrane, and let it stand at room temperature for 1min. Centrifuge at 12,000rpm for 1min. The plasmid of the recombinant expression vector is obtained, namely the transformation vector T10, which is stored in a -20℃ refrigerator for future use.

[0096] Following the same steps as above, only the insect-resistant gene Vip3Af_T10 is replaced with Vip3Af, Vip3Af_R11, Vip3Af_A12, Vip3Af_L13, Vip3Af_P14, Vip3Af_S15, Vip3Af_F16, Vip3Af_I17, Vip3Af_D18 or Vip3Af_D32 to construct recombinant expression vectors AF, R11, A12, L13, P14, S15, F16, I17, D18, and D32, respectively.

[0097] By removing the SSU-CTP in the AF vector (only synthesizing pZmUbi-Vip3Af-tNos during gene synthesis) and keeping the other steps unchanged, the control group vector AFOri without the SSU-CTP sequence can be obtained; by analogy, the recombinant vectors R11ori, A12ori, L13ori, P14ori, S15ori, F16ori, I17ori, D18ori, and D32ori without the SSU-CTP sequence and expressing the Vip3Af_R11, Vip3Af_A12, Vip3Af_L13, Vip3Af_P14, Vip3Af_S15, Vip3Af_F16, Vip3Af_I17, Vip3Af_D18, or Vip3Af_D32 genes can be obtained.

[0098] It should be pointed out that this embodiment is only an example of a method for constructing a transformation vector. Obviously, there is more than one transformation vector that can achieve the intended purpose of the present invention. Researchers in this field can easily realize the construction of the transformation vector through existing technologies. Researchers in this field can freely select promoters and terminators according to the actual needs of specific research, as long as the expression of the Vip3A protein described in the present invention can be achieved in the cell. In this embodiment, the use of the glyphosate screening marker gene G10-2 is only to provide a screening method. Researchers in this field can replace the screening marker gene (such as neomycin phosphotransferase gene (nptⅡ), hygromycin phosphotransferase gene (hpt), chloramphenicol phosphotransferase gene (cat) and streptomycin phosphotransferase gene (spt), Agrobacterium glyphosate-resistant epsps gene (CP4-epsps), glufosinate-resistant gene (pat), etc.) according to actual needs. The screening marker gene has no effect on the insecticidal activity of the Vip3A mutant protein. Therefore, any expression cassette or transformation vector containing the gene encoding the mutant protein described in the present invention should be regarded as the content of the present invention.

[0099] Example 4: Transformation of Agrobacterium with recombinant expression vector

[0100] The recombinant expression vectors constructed in Example 3 were respectively transformed into competent cells of Agrobacterium tumefaciens LBA4404 strain (Shanghai Angyu Biotechnology Co., Ltd., Product No. G6033) by electroporation transformation (2200V) to prepare Agrobacterium tumefaciens strains for corn genetic transformation.

[0101] The method is as follows: the plasmid obtained in Example 3 is transformed into competent cells of Agrobacterium LBA4404 by electroporation, and the cells are evenly plated on a YEP solid medium containing 50 mg / L kanamycin, 25 mg / L rifampicin and 15 mg / L tetracycline, and cultured at 28°C for 48 to 72 hours. After a single clone grows, a single colony is picked and transferred to a YEP liquid medium containing 50 mg / L kanamycin, 25 mg / L rifampicin and 15 mg / L tetracycline. After culture at 28°C for 48 to 72 hours, an Agrobacterium culture solution (OD 600 =1.5~2.0), extract the plasmid and sequence it, and store the positive cloned bacteria correctly in a -80℃ refrigerator for later use.

[0102] YEP liquid culture medium consists of 10 g / L peptone, 10 g / L yeast extract, 5 g / L NaCl, the solvent is water, and it is sterilized at high temperature and high pressure. If a solid culture medium is prepared, add 15 g / L agar.

[0103] Example 5: Obtaining transgenic corn

[0104] The genetic transformation of corn was mediated by Agrobacterium, specifically according to the method and medium formula reported by Frame et al. (Plant Physiol, 2002, 129: 13-22), and glyphosate was used as the screening agent. The steps were as follows: corn ears were taken 8 to 10 days after pollination, and immature embryos with a size of 1.0 to 1.5 mm were collected. The Agrobacterium solution (OD 600 ≈1.0) and immature embryos were co-cultured at 22°C for 3-5 days. The cultured immature embryos were transferred to a callus induction medium containing a final concentration of 200 mg / L timentin antibiotic (Shanghai Yisheng Biological Company, Product No. 60230ES07), and cultured in the dark at 28°C for 10-14 days to kill Agrobacterium. All callus tissues after induction culture were transferred to a screening medium containing a final concentration of 2mM glyphosate and cultured in the dark at 28°C for 2-3 weeks. The surviving embryonic tissue was transferred to a regeneration medium, cultured in the dark at 28°C for 10-14 days, and then transferred to a fresh regeneration medium and cultured in the light at 26°C for 10-14 days. Select fully developed plants and place them on a rooting medium, culture them in the light at 26°C until the roots are fully developed, and transplant the rooted regenerated seedlings into a greenhouse for growth and reproduction.

[0105] The transformants transformed by the recombinant expression vector of Example 3 are named according to their vector numbers, for example, the transformed seedlings obtained by the transformation of the recombinant expression vector AF are named AF-1, AF-2, AF-3, etc., and the other transformants are named by analogy. It should be pointed out that the transgenic transformed seedlings obtained by the recombinant expression vectors AF, R11, A12, L13, P14, S15, F16, I17, D18, and D32, and different strains obtained by the same recombinant expression vector, will show albinism to varying degrees during the tissue culture stage. Some seedlings can grow normally, while some seedlings show leaf loss of green and plant withering; and the albino transformed seedlings cannot survive and will not be transplanted to the greenhouse to continue growing.

[0106] Example 6: Analysis of the expression level of Vip3 truncated protein in transgenic crop transformants

[0107] The recombinant transformation vectors T10ori, R11ori, A12ori, L13ori, P14ori, S15ori, F16ori, I17ori, D18ori, D32ori and the control group AFori in Example 3 were transferred into Agrobacterium for plant infection, and transformed seedlings were obtained after tissue culture. The transformed seedlings that did not express the guide peptide obtained in Example 5 were transferred to the greenhouse for continued growth after survival. The expression level of Vip3 protein was measured when the corn grew to the 5-6 leaf stage. The measurement method is as follows. Take 50 mg of fresh corn leaf tissue, take 3 samples of each leaf tissue as a repeat, grind with liquid nitrogen, add 500 microliters of PBS buffer (pH 7.4, containing 1% protease inhibitor cocktail (MCE Company, item number HY-K0010)), mix thoroughly, centrifuge at 12000rpm for 5 minutes, and dilute the supernatant 500 times with PBS buffer for enzyme-linked immunosorbent assay (ELISA) measurement. The Vip3A ELISA quantitative kit (product number AA1641) of Shanghai Youlong Biological Company was used for detection, and the operation was performed according to the product instructions. The negative control sample was a non-transgenic corn leaf tissue of a similar growth period, and the positive control sample was a positive protein sample provided in the ELISA kit. The measured expression level of the Vip3A mutant protein in the T0 generation transgenic corn transformant was between 0.2-45.6 micrograms per gram of fresh leaf tissue (μg / g-fwt). The Vip3 protein expression levels of all the transformed seedlings obtained were statistically analyzed, and the results are shown in Table 11 below.

[0108] Table 11Albino rate of T0 generation transformed seedlings and average expression of Vip3 protein in transplanted seedlings

[0109]

[0110] The results showed that the expression of Vip3 protein in different transformant strains that survived transplantation was not much different, but the proportion of albino seedlings in transformants expressing different Vip3 proteins was quite different. Different proportions of albino seedlings still appeared in the transformant seedlings expressing AFori, T10ori, R11ori, A12ori, and L13ori, indicating that these proteins still have different degrees of toxicity to plants. No albino seedlings were found in P14ori, S15ori, F16ori, I17ori, D18ori, and D32ori, indicating that their toxicity to corn cells has decreased or even has no toxicity. However, judging from the expression level of Vip3 protein, AFori, T10ori, R11ori, A12ori, and L13ori reached 4.7, 4.9, 5.5, 7.4, and 7.6 μg / g-fwt, respectively; and even if the toxicity-reduced proteins P14ori, S15ori, F16ori, I17ori, D18ori, and D32ori were used, their protein expression levels were only 8.8, 9.3, 9.2, 8.9, 9.1, and 8.7 μg / g-fwt. This may be due to the reduced toxicity of these proteins, which allowed the transformants with relatively high expression of Vip3 protein to survive.

[0111] Example 7: Analysis of the expression level of Vip3 protein in transgenic crop transformants containing a guide peptide

[0112] The recombinant transformation vectors AF, R11, A12, L13, P14, S15, F16, I17, D18, and D32 in Example 3 were transferred into Agrobacterium for plant infection, and transformed seedlings were obtained after tissue culture. The transformed seedlings containing the expression guide peptide obtained in Example 5 were transferred to the greenhouse for continued growth after survival. The expression level of Vip3 protein was determined when the corn grew to the 5-6 leaf stage. The determination method is as follows. Take 50 mg of fresh corn leaf tissue, take 3 samples of each leaf tissue as a repeat, grind with liquid nitrogen, add 500 microliters of PBS buffer (pH 7.4, containing 1% protease inhibitor cocktail (MCE company, item number HY-K0010)), mix thoroughly, centrifuge at 12000rpm for 5min, and dilute the supernatant 500 times with PBS buffer for enzyme-linked immunosorbent assay (ELISA). Use the Vip3A ELISA quantitative kit (product number AA1641) of Shanghai Youlong Biological Company for detection, and refer to the product instruction manual for operation. The negative control sample was a non-transgenic corn leaf tissue of a similar growth period, and the positive control sample was a positive protein sample provided in the ELISA kit. The measured expression of the Vip3A mutant protein in the T0 generation transgenic corn transformants was between 0.2-45.6 micrograms per gram of fresh leaf tissue (μg / g-fwt). The expression of the Vip3 protein in all the transformed seedlings obtained was statistically analyzed, and the results are shown in Table 12 below.

[0113] Table 12 Albino rate of T0 generation transformed seedlings and average expression of Vip3 protein in transplanted seedlings

[0114]

[0115] The results showed that there were large differences in the expression of Vip3 protein in different transformant strains that survived transplantation. In the transformant AF expressing the original Vip3Af protein, 35 of the 127 tissue culture seedlings showed albino status and ultimately did not survive; after the remaining 92 transformants were transplanted to the greenhouse and survived, the average expression of their Vip3Af protein was determined to be 5.3 micrograms per gram of fresh leaves (μg / g-fwt). In the transformant T10 expressing the Vip3Af_T10 protein, the proportion of albino seedlings also reached 26.1%, and the average expression of the Vip3Af_T10 protein in the remaining surviving transplanted seedlings was 4.9 micrograms per gram of fresh leaves (μg / g-fwt). In the transformant R11 expressing the Vip3Af_R11 protein, the proportion of albino seedlings reached 24.3%, and the average expression level of the Vip3Af_R11 protein in the remaining surviving transplanted seedlings was 5.1 micrograms per gram of fresh leaves (μg / g-fwt). In the transformant A12 expressing the Vip3Af_A12 protein, the proportion of albino seedlings decreased to 14.6%, and the average expression level of the Vip3Af_A12 protein in the remaining surviving transplanted seedlings also increased to 13.1 micrograms per gram of fresh leaves (μg / g-fwt). This shows that compared with Vip3Af, Vip3Af_T10, and Vip3Af_R11 proteins, Vip3Af_A12 has lower toxicity to plant cells, but still has a certain degree of cytotoxicity (some albino seedlings are still present). In the transformants L13, P14, S15, F16, I17, D18, and D32 expressing Vip3Af_L13, Vip3Af_P14, Vip3Af_S15, Vip3Af_F16, Vip3Af_I17, Vip3Af_D18, and Vip3Af_D32, no albino seedlings were found, and their average expression levels of Vip3 proteins were significantly higher than those of transformants AF, T10, and R11, indicating that these seven proteins have almost no toxicity to plant cells, and the plants can still grow normally even when the expression level of Vip3 proteins is relatively high. To put it another way, transformants expressing Vip3Af_L13, Vip3Af_P14, Vip3Af_S15, Vip3Af_F16, Vip3Af_I17, Vip3Af_D18, and Vip3Af_D32 proteins can be used to screen transgenic seedlings with higher Vip3 protein expression levels, and the phenotypes of these transformed seedlings are not significantly different from those of conventional non-transgenic seedlings in the same growth period.

[0116] It should be pointed out that the expression of Vip3A mutant protein can also be measured in other crop transformation seedlings by the same method described in this embodiment, such as transgenic rice, transgenic soybean, and transgenic cotton, by simply replacing the plant sample from the corn leaves in this embodiment with corresponding plant tissues (such as leaf tissues of transgenic rice). This embodiment only takes corn as an example to illustrate the effects of different truncated proteins on plant cells. In fact, the phenomenon of whitening of transformed seedlings will occur in transgenic rice, transgenic soybean, and transgenic cotton with the Vip3 gene. Researchers can screen, observe, and quantitatively measure the transformed seedlings by the method provided in this embodiment, and analyze the effects of Vip3 truncated proteins on the phenotypes of transgenic plants.

[0117] Example 8: Determination of insect-resistant biological activity of transgenic insect-resistant corn transformants

[0118] The transgenic plant transformants obtained by the method described in Example 5 were tested for insect-resistant protein expression using the methods of Examples 6 and 7, and transplanted to a greenhouse. The biological activity was tested after the plants were able to grow stably (corn entered the V6-8 leaf stage). The test insects in this example were fall armyworm, corn borer, beet armyworm, armyworm, cotton bollworm, and striped stem borer.

[0119] In this embodiment, the method of the bioassay experiment is described by taking the newly hatched larvae of fall armyworm as an example. Other insects can be tested in the same way, and the test insects only need to be replaced by the newly hatched larvae of fall armyworm with the corresponding newly hatched larvae. Take 5-6cm fresh plant leaf tissue, place it in a culture dish with a diameter of 7.5cm, pad it with a moist filter paper, access 10 fall armyworm larvae within 2h of initial hatching, cover the lid and seal the culture dish with a sealing film to prevent the larvae from escaping. Each transformant is set with 5 repeated treatment groups. Non-transgenic plants of the same growth period are used as negative controls. After 5 days, the number of surviving larvae in each experimental group is counted, and the mortality rate of the larvae is calculated. According to the mortality data obtained by statistics.

[0120] The biological activity of the transplanted seedlings that survived in Examples 6 and 7 was determined. The insecticidal activity of each transformant strain against fall armyworm and oriental armyworm was determined. The results are shown in Tables 13 and 14 below. Since the expression of Vip3 protein varies greatly among different transformants, we classified and counted the transformants according to the expression of Vip3, which were 0.2-2.0, 2.0-5.0, 5.0-10.0, 10.0-20.0 and greater than 20.0 micrograms per gram of fresh weight leaf (μg / g-fwt). The statistical results show that for fall armyworm, the four types of transformants, T10, I17, D18, and D32, have almost no insecticidal activity, and the insecticidal activity of S15 and F16 transformants is significantly lower than that of AF, while the insecticidal activity of R11, A12, L13, and P14 is close to that of AF. For oriental armyworm, the transformants T10, S15, F16, I17, D18, and D32 had almost no insecticidal activity, the insecticidal activity of R11 transformant was significantly lower than that of AF, while the insecticidal activities of A12, P14, and AF were relatively close.

[0121] It should be pointed out that this embodiment describes the determination of the biological activity of transgenic corn against important corn pests. The data of this embodiment show that the multiple Vip3Af truncated proteins described in the present invention have the characteristics of reducing plant cell toxicity and can maintain insecticidal activity against specific insects. However, for specific situations, researchers can choose different Vip3Af truncated proteins according to their own research purposes. For example, if you want to control the expression of Vip3Af protein in the leaves of transformed seedlings to about 10.0 μg / g-fwt, and at the same time achieve the purpose of resisting fall armyworm and oriental armyworm, you can use A12, P14 and other proteins; if you want to obtain Vip3Af expression exceeding 15.0 μg / g-fwt, and only need high resistance to fall armyworm (for example, the most common pest in the tropical region of southwest my country is fall armyworm), then you can use A12, L13, P14, S15, F16 and other proteins. In summary, according to the use areas, transformed varieties and target pests of different plant transformed seedlings, any of the Vip3Af truncated proteins described in the present invention can be selected to achieve the research objectives of researchers.

[0122] Table 13 Average mortality of transformed seedlings with different Vip3 protein expression levels against fall armyworm

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[0124] Table 14 Average mortality of transformed seedlings with different Vip3 protein expression levels against Oriental armyworm

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[0126] Finally, the above are only some embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there may be many extensions and expansions. For example, researchers in this field can select suitable Vip3Af truncated proteins for genetic transformation according to their own research needs, and achieve the purpose of reducing the toxicity of Vip3 proteins to plant cells in crops such as soybeans, cotton, rapeseed, cowpeas, and sunflowers, while maintaining the insect-resistant properties of transformed seedlings. The truncated Vip3Af protein described in the present invention can be used to prepare transgenic insect-resistant plants, and can also be used to prepare new pesticides for preventing and controlling the damage of lepidopteran pests. All extensions that can be directly derived or associated with the content disclosed by a person of ordinary skill in the art from the present invention should be considered to be within the scope of protection of the present invention.

Claims

1. A truncated Vip3Af protein, characterized in that The truncated Vip3Af protein is obtained by partially deleting the amino acids from positions 1 to 32 at the N-terminus of the original Vip3Af protein.

2. The truncated Vip3Af protein according to claim 1, characterized in that The amino acid sequence of the truncated Vip3Af protein is shown in one of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:

11.

3. The truncated Vip3Af protein according to claim 1, characterized in that The nucleotide sequence of the gene encoding the truncated Vip3Af protein is shown in one of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO:

22.

4. A method for reducing the toxicity of Vip3 protein to plant cells, characterized in that: The method comprises expressing the truncated Vip3Af protein according to claim 1 in a plant genome.

5. A method for reducing the toxicity of Vip3 protein to plant cells and increasing the expression level, characterized in that: The method comprises guiding the truncated Vip3Af protein according to claim 1 into the chloroplast of the plant cell via a guide peptide.

6. The method according to claim 5, characterized in that The guide peptide is connected to the N-terminus of the truncated Vip3Af protein and expressed in fusion in plant cells.

7. An expression cassette comprising the gene encoding the truncated Vip3Af protein of claim 1.

8. A transformation vector for expressing the truncated Vip3Af protein according to claim 1.

9. Use of the truncated Vip3Af protein according to claim 1 in preparing transgenic insect-resistant plants for controlling lepidopteran pests.

10. The use according to claim 9, characterized in that The lepidopteran pests include at least one of the following pests: fall armyworm, beet armyworm, fall armyworm, armyworm, cotton bollworm, cutworm, corn borer, rice leaf roller, striped stem borer, peach borer, bean pod borer, diamondback moth, black armyworm, and two-spotted armyworm.

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