Brown planthopper-resistant insecticidal protein sourced from bacillus thuringiensis as well as coding gene and application of brown planthopper-resistant insecticidal protein

Abstract

The invention belongs to the technical field of genetic engineering, and discloses a bacillus thuringiensis-sourced brown planthopper-resistant insecticidal protein and a coding gene and application thereof, the bacillus thuringiensis-sourced brown planthopper-resistant insecticidal protein is an insecticidal protein orf1715, the amino acid sequence is shown as SEQ ID NO: 1, and the nucleotide coding sequence of the gene is shown as SEQ ID NO: 2. The gene is well expressed in an escherichia coli system in a soluble form, and the purified protein has a remarkable specific insecticidal effect on rice pest brown planthopper, so that the gene can be applied to special prevention and control of brown planthopper, and a candidate gene resource is provided for cultivation of a new transgenic insect-resistant rice variety.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the cloning and expression of a novel insecticidal gene orf1715 derived from Bacillus thuringiensis and its insecticidal application. Background Art

[0002] As one of the main food sources for the global population, rice production losses due to pests and diseases are as high as 30%. In recent years, brown planthoppers with piercing-sucking mouthparts have gradually developed into major pests of rice (SAVARY S, WILLOCQUET L, PETHYBRIDGE SJ, et al. 2019. The global burden of pathogens and pests on major food crops. Nature Ecology & Evolution, 3: 430-439.). Brown planthoppers are extremely destructive Hemiptera pests with fast reproduction and strong migration ability. Not only does it suck rice phloem sap through its piercing-sucking mouthparts, causing the plants to turn yellow and wilt, it also spreads a variety of plant diseases, such as rice stripe virus (RSV), rice grassy stunt virus (RGSV) and rice sawtooth leaf dwarf virus (RRSV). Against the backdrop of global warming, it has become more difficult to predict the migration routes and outbreak areas of brown planthoppers. Over-reliance on and extensive use of chemical pesticides have long been the norm in the management of brown planthoppers, resulting in significant resistance of brown planthopper populations to a variety of insecticides, while also posing a threat to biodiversity and food safety. Therefore, it is urgent to develop safe, efficient and sustainable pest management strategies.

[0003] Bacillus thuringiensis is a Gram-positive bacterium belonging to the waxy Bacillus group and is widely distributed in natural environments such as soil, insect corpses, and leaf surfaces. Its insecticidal crystal protein Cry produced in the spore stage has specific high activity against a variety of agricultural pests and plant parasitic nematodes and mites, but is safe for humans and animals and plants (JOUZANI GS, VALIJANIAN E, SHARAFI R 2017. Bacillus thuringiensis: a successful insecticide with new environmental features and tidings. Appl Microbiol Biotechnol, 101: 2691-2711.). In addition, Bt also secretes a variety of other virulence factors such as the insecticidal protein Vip in the vegetative period (RAKESH V, KALIAV K, GHOSHA 2023. Diversity of transgenes insustainable management of insect pests. Transgenic Res, 32: 351-381.). As more and more insecticidal genes are discovered, a new naming system based on structural features was announced in 2020. This revised version retains the basic principle of classification based on sequence similarity in the 1998 naming version, while introducing specific mnemonics to distinguish insecticidal proteins with different folding conformations. Based on this, the insecticidal crystal protein Cry is further divided into six groups: Cry, Tpp, Mpp, App, Gpp, and Xpp. For example, Cry here specifically refers to a three-domain insecticidal protein, and Tpp specifically refers to a type of β-perforin protein with a Toxin_10 domain (CRICKMORE N, BERRYC, PANNEERSELVAM S, et al. 2021. A structure-based nomenclature for Bacillus thuringiensis and other bacteria-derived pesticidal proteins. J Invertebr Pathol, 186: 107438.).Several Cry proteins represented by three-domain Cry proteins have been successfully applied to the research and development of transgenic insect-resistant crops, mainly for the prevention and control of lepidoptera and other pests (LI Y, HALLERMAN EM, WU K, et al. 2020. Insect-Resistant Genetically Engineered Crops in China: Development, Application, and Prospects for Use. Annu Rev Entomol, 65: 273-292.). Currently, many Hemiptera pests, including brown planthoppers and aphids, have developed into the main pests or even the number one pests of important crops, and there are very few effective genes for such pests.

[0004] Some researchers have made efforts to identify insecticidal genes against brown planthoppers. A gut-binding peptide was used to replace the loop structure in the Cry1Ab domain II, and the resulting fusion protein had a half-lethal concentration (LC) of 1.177 mmol / l to brown planthoppers. 50 is 21.54μg / ml (SHAO E, LIN L, CHEN C, et al. 2016. Loop replacements with gut-bindingpeptides in Cry1Ab domain II enhanced toxicity against the brown planthopper, Nilaparvata lugens Sci Rep, 6:20106.); The LC of the binary toxins Mpp64Ba and Mpp64Ca against white-backed planthopper and brown planthopper 50The median lethality of the first single component protein Tpp78Aa1 against brown planthopper and gray planthopper were 15.78 μg / ml and 6.89 μg / ml respectively (WANG Y, LIU Y, ZHANG J, et al. 2018. Cry78Aa, a novel Bacillus thuringiensis insecticidal protein with activity against Laodelphax striatellus and Nilaparvata lugens. J Invertebr Pathol, 158: 1-5.). Subsequently, based on Tpp78Aa1, the same team discovered five genes with similar insecticidal effects on Laodelphax striatellus, namely Tpp78Ab1, Tpp78Ba1, Tpp78Bb1, Tpp78Ca1, and Tpp80Ae1. 50 They are 8.1 μg / ml, 9.723 μg / ml, 8.6 μg / ml, 10.1 μg / ml, and 9.6 μg / ml respectively (CAO B, SUN X, SHU C, et al. 2023. Identification and functional characterization of eight novel tpp family genes from Bacillus thuringiensis. Pest Manag Sci, 79: 4244-4253.). The insecticidal proteins discovered above all belong to the Tpp family insecticidal proteins and contain two conserved domains, Ricin_B_Lectin and Toxin_10. This also shows the great application potential of this type of insecticidal gene in the direction of controlling Hemiptera pests rice planthoppers. However, there is still no new transgenic insect-resistant rice variety that can control brown planthoppers. Therefore, based on the significant degree of damage caused by such pests on rice crops, and to prevent the use of a single gene from causing resistance problems, it is urgent and necessary to discover more new genes for resistance to planthoppers. Summary of the invention

[0005] In view of the shortcomings of the prior art, the first object of the present invention is to provide a new insecticidal protein derived from Bacillus thuringiensis and its encoding gene, the encoding gene of which has very low sequence similarity with the currently reported insecticidal gene sequence, and according to the naming principle, it belongs to a first-class new insecticidal gene. The gene is well expressed in a soluble form in the Escherichia coli system, and the purified protein has a significant insecticidal effect on the rice pest brown planthopper.

[0006] Specifically, the object of the present invention is achieved according to the following technical scheme: an insecticidal protein orf1715 derived from Bacillus thuringiensis, whose amino acid sequence is as follows:

[0007] MKIISKKVMTGLMVAAMGLSIWTPASQAAVPENNRYYTINLKANPNKIWDVANEYTENG

[0008] RAILLYNETHGDNQQFVFFQLDGGTYAIVNKNSGKPITFGPNAWFGHHSYPGILTGDVLQQ

[0009] QSWTGAPAEQWYLRDKGSNNYEVVNQGNGRVASYAGVHRTHGWIDYVDLDEPNPSDSD

[0010] RVFNIANSPSGLSLPTLPATGTRPTAPNYTGGIEQQLPLTSNSVVIGASLIPCIMVNDSQASE

[0011] YTKIHNSPYYVLIKEEYWEQTFSKVIQPGLSETYSYKTGISSVDQQKMTDTLSIQVGGDLG

[0012] LKFGDKSASLKAQITKTLQTEVSTTSTQASEETITQTATGEPGKATGYTQYQLVTKYTLKR

[0013] QDGTTVSNPWVVKNNRITVTRKSS (as shown in SEQ ID NO: 1).

[0014] At the same time, the present invention also provides a gene encoding the above insecticidal protein orf1715. Further preferably, the gene encoding the above insecticidal protein orf1715 is derived from Bacillus thuringiensis, and the nucleotide coding sequence of the gene is as follows:

[0015] atgaaaattatatcaaaaaaagtcatgacagggttaatggtagcagcaatgggtctatctatctggactcctgcaagtcaagcagcagttccaga

[0016] aaacaatcgatattatactattaatttgaaagctaaccccaacaaaatttgggatgtagctaatgaatatactgagaacggtagggcaattcttttat

[0017] ataatgagacacatggtgacaatcaacaatttgtattttttcagcttgatggaggaacatatgcaattgtaaataaaaatagtggaaagcctataac

[0018] atttggtcctaatgcttggtttggtcatcatagttatcctggaatactaacgggtgatgtgctacaacaacagagttggactggagctcctgctgaa

[0019] caatggtatttacgagacaagggaagcaataattatgaggttgtgaatcaaggaaatggaagagttgcatcttatgcgggagtgcataggaca

[0020] cacggatggatatagattatgtagatttagatgaacccaatccttctgattcagatagggtatttaacattgccaattctccaagtggtttatcactacc

[0021] aactttaccagctacaggtactagaccaactgctccaaattatactggaggtattgaacaacaattacctctaacttcaaattctgttgttattggag

[0022] catctttaataccatgtattatggtaaacgatagccaagcgagtgaatatacaaagatacacaattctccatattatgttttgataaaagaggaatat

[0023] tgggagcaaacattttcaaaggttattcaacctggtttgagtgaaacttattcatataaaacaggtataagttctgttgaccaacaaaagatgactg

[0024] atacgctttctattcaggttggaggggatttaggattaaaatttggagataaatctgcatcacttaaagcgcagattacaaaaacattacaaacag

[0025] aagttagtacaactagtacgcaagcatcggaagaaacgattacacaaactgctacaggtgaacccggtaaggcaacaggatatacacaatat

[0026] caactggtgacaaaatatacgctaaagagacaagatggtacaactgtttcaaacccttgggttgtaaaaaataatagaataacagtaacaagaa

[0027] aaagttcataa (as shown in SEQ ID NO: 2).

[0028] It should be noted that the insecticidal protein gene orf1715 was identified from a new strain of Bacillus thuringiensis, which was named Bacillus thuringiensis BMBRJT048. The BMBRJT048 strain was isolated from soil samples collected from Yun'an Garden in Kunming, Yunnan in 2013 by our research group. The strain has been deposited in the China Center for Type Culture Collection on December 31, 2024, with the deposit number CCTCC M 20242967. Optical microscopy and scanning electron microscopy showed that the BMBRJT048 strain can form rhombic parasporal crystals during the spore stage. The genome information of the strain was obtained with the help of second-generation sequencing technology, and the insecticidal gene sequence contained in the BMBRJT048 strain was further predicted by the insecticidal gene prediction analysis pipeline BtToxin_Scanner2 pipeline (Liu et al., 2021) developed by our laboratory. Among them, the orf1715 gene has a 31% sequence identity with the anti-brown planthopper protein Tpp78Aa1 gene and the sequence is complete, so it was selected as the target gene for further functional research.

[0029] The second object of the present invention is to provide a recombinant vector, which contains the gene of the insecticidal protein orf1715. Further preferably, the vector is an Escherichia coli expression vector pET15d. At the same time, the present invention also provides a genetically engineered bacterium, which contains the above-mentioned recombinant vector.

[0030] The cloning method of the insecticidal gene orf1715 of the present invention is: using the genome of the Bacillus thuringiensis strain BMBRJT048 as a template, using the PCR amplification method to obtain a complete insecticidal gene fragment of orf1715. The primer sequence of the PCR amplification includes: orf1715 primer F: GAAGTTGATGCACATATG ATGA AAATTATATCAAAA, orf1715 Primer R: AGCAGCCGGATCCTCGAG TTATGAAC TTTTTCTTGT, wherein the underlined part is the homology arm of the E. coli expression vector pET15d. Further explanation, the endonucleases used for linearization of the E. coli heterologous expression vector pET15d are NdeI and XhoI.

[0031] The third object of the present invention is to provide a product use, namely, the use of the above-mentioned insecticidal protein orf1715, the above-mentioned insecticidal gene orf1715, the above-mentioned recombinant vector or the above-mentioned engineered bacteria in resisting brown planthoppers or preparing products resisting brown planthoppers.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows: through experiments such as cloning of the orf1715 gene, protein expression and biological activity determination, the present invention finds that the novel insecticidal protein orf1715 has significant specific toxic activity against brown planthoppers, has no effect on the growth of dipterans such as Aedes and Culex pipiens, and has no insecticidal activity against Caenorhabditis elegans and Spodoptera frugiperda, and therefore can be applied to the special prevention and control of brown planthoppers, providing candidate gene resources for the cultivation of new transgenic insect-resistant rice varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The spore and paraspore crystal formation of Bacillus thuringiensis strain BMBRJT048 under optical microscope (A) and scanning electron microscope (B).

[0034] Figure 2 The diagram is a schematic diagram of the structural domain of the protein encoded by the insecticidal gene orf1715 of the present invention.

[0035] Figure 3 This is a multiple sequence alignment analysis diagram of the insecticidal gene orf1715 of the present invention and the Tpp insecticidal gene.

[0036] Figure 4 The figure is an agarose electrophoresis analysis diagram of the PCR amplification product of the insecticidal gene orf1715 of the present invention.

[0037] Figure 5 This is a colony PCR identification diagram of the insecticidal gene orf1715 clone of the present invention.

[0038] Figure 6 This is an SDS-PAGE detection diagram of the expression of the insecticidal protein orf1715 of the present invention in Escherichia coli.

[0039] Figure 7 This is a graph showing the concentration of the insecticidal protein orf1715 of the present invention.

[0040] Figure 8 The figure is a graph showing the insecticidal activity test results of the insecticidal protein orf1715 of the present invention against brown planthoppers.

[0041] Fig. 9 This is a graph showing the results of the insecticidal activity assay of the insecticidal protein orf1715 of the present invention against Aedes mosquitoes and Culex pipiens.

[0042] Fig.10 This is a graph showing the insecticidal activity assay results of the insecticidal protein orf1715 of the present invention against the fall armyworm.

[0043] Fig.11 This is a diagram showing the insecticidal activity assay results of the insecticidal protein orf1715 of the present invention against Caenorhabditis elegans. DETAILED DESCRIPTION

[0044] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention. In the examples, unless otherwise specified, the methods involved are conventional methods in the art and can be operated with reference to the general techniques described in the literature in the art. The reagents or instruments used without indicating the manufacturer can be obtained through commercial channels.

[0045] Example 1: Isolation and identification of BMBRJT048 strain

[0046] (1) Remove weeds, stones, plant roots and leaves, etc. mixed in the collected soil samples. Use sodium acetate screening combined with high temperature treatment to isolate the Bt strains in the soil samples. Weigh 1g of soil sample into a liquid Erlenmeyer flask culture medium containing LB+NaAC, and culture it in a shaker at 30°C and 220rpm / min for 4 hours. Under the action of NaAC, the germination of Bacillus thuringiensis will be inhibited, while other strains will not. Then place the flask in an 80°C water bath for 180s to kill other strains. After cooling and shaking, take 100μl of the culture solution and apply it to the LA solid plate. Repeat the application of 3 LA plates for each soil sample and invert and culture it in a constant temperature incubator at 30°C for 24 hours;

[0047] (2) Select colonies with morphology similar to that of Bt, i.e., those that are milky white and nearly circular with slightly diffused outer edges but not very neat, and streak them onto T3 plates. Incubate them upside down in a 30°C constant temperature incubator for 72 h.

[0048] (3) The formation of parasporal crystals was observed under an optical microscope and the strain was further identified as Bacillus thuringiensis BMBRJT048. A small amount of bacteria was picked up on a glass slide using a 10 μl small pipette tip, spread with sterile water, and stained with carbolic acid fuchsin stain. The BMBRJT048 strain was observed under an optical microscope to form spores and produce rhombic parasporal crystals. The morphology of the parasporal crystals was further observed using a scanning electron microscope, as shown in FIG. Figure 1 shown.

[0049] (4) The culture medium formula (1 L) used in the above experimental operation is as follows:

[0050] LB+NaAC liquid medium: peptone 10g, yeast powder 5g, sodium chloride 10g, sodium acetate 34.02g, pH 7.0;

[0051] LA solid medium: peptone 10g, yeast powder 5g, NaCl 5g, agar powder 15-20g, PH7.0;

[0052] T3 solid culture medium: peptone 5g, yeast powder 1.5g, MnCl20.005g, NaH2PO43.9g, Na2HPO48.95g, agar powder 15g-20g, pH 7.0.

[0053] Example 2: Extraction of genomic DNA from BMBRJT048 strain

[0054] (1) Take a small amount of bacterial solution from the glycerol tube and streak it on the LB solid plate, and incubate it at 28°C overnight. Pick a single colony and inoculate it into a PA bottle containing 10ml of liquid LB medium, and activate it overnight at 28°C and 220rpm in a shaking incubator. The next day, transfer it to a new Erlenmeyer flask containing 30ml of liquid LB medium at a ratio of 1% and continue culturing for 4-6h;

[0055] (2) Transfer all bacterial suspension to a 50 ml centrifuge tube, centrifuge at 12,000 rpm for 1 min, and discard the supernatant. Add 1000 μl TE Buffer to wash the bacteria, transfer equal amounts of bacterial suspension to two 2 ml EP tubes, centrifuge at 12,000 rpm for 1 min, and discard the supernatant;

[0056] (3) Add 300μl of solution I (25mM Tris-HCl PH8.0, 10mM EDTA, 50mM Glucose) and 40μl lysozyme (100mg / ml) to each EP tube, mix well, and lyse at 4℃ overnight. The next day, add 3μl RNaseA (10mg / ml) to each tube and treat at 37℃ for 1.5h (note to shake gently several times in the middle). Then add 300μl SDS (10%) and mix gently, and treat in a 55℃ water bath until the bacterial solution is clear. Then add 360μl NaCl (5M) and mix gently, and a white precipitate can be seen. Let stand on ice for 10min, centrifuge at 12,000rpm for 15min, and take the supernatant to a new EP tube;

[0057] (4) Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) and invert the mixture about 200 times. Centrifuge at 12,000 rpm for 15 min and collect the supernatant. Add 2 volumes of pre-cooled anhydrous ethanol and 50 μl of 3 M sodium acetate to the supernatant and place in a -20°C refrigerator for 10 min to precipitate DNA.

[0058] (5) After taking out, centrifuge at 12,000 rpm for 15 min to obtain the precipitate, and wash it with 70% ethanol. Use a gun to remove excess liquid and dry the precipitate. Add 50 μl TE Buffer to each tube to dissolve the precipitate, and check the quality of the extracted DNA by electrophoresis.

[0059] Example 3: Prediction of insecticidal genes in BMBRJT048 strain

[0060] The genome sequence of the BMBRJT048 strain was obtained by next-generation sequencing technology, and the BtToxin_Scanner2 pipeline system developed by our team was used to analyze the potential insecticidal genes in the strain, as shown in Table 1. The gene numbers are named according to the numbering information automatically generated by the insecticidal gene mining system. Among them, two genes, orf1715 and orf1984, have the highest matching degree with the Tpp78 family protein gene with planthopper killing activity, and from the perspective of amino acid sequence length, the orf1715 gene is more complete, so it was selected for further functional research.

[0061] Table 1 Identification and analysis results of insecticidal genes in the BMBRJT048 strain of the present invention

[0062]

[0063] Example 4: Basic physicochemical properties and gene sequence analysis of orf1715 novel insecticidal protein

[0064] (1) The basic physicochemical properties of orf1715 were analyzed using an online prediction method (https: / / web.expasy.org / protparam / ). The orf1715 protein has a total of 387 amino acid residues, a theoretical molecular weight of 42.5 kDa, and an isoelectric point of 6.61. The instability coefficient is 30.31, which is less than 40, indicating that the protein is relatively stable as a whole. The hydrophilicity index is -0.476, which is less than 0, indicating that it is a hydrophilic protein.

[0065] (2) Using blastp to search for homologous sequences of orf1715 of the present invention, the first one ranked was Tpp78Aa1 (accession number ATY50144), with an amino acid sequence identity of 31.36%, which was consistent with the analysis results using the insecticide gene mining pipeline.

[0066] (3) Multiple sequence alignment analysis of orf1715 and other reported planthopper-killing proteins, such as Figure 3 . From the comparison results, the N-terminus (1-190) of orf1715 is highly variable, which also suggests that it may have different insect recognition receptors and insecticidal mechanisms from the reported planthopper-killing proteins. The insecticidal protein sequences used for multiple sequence alignment were obtained from the insecticidal gene naming website (https: / / www.bpprc.org / ), and the multiple sequence alignment analysis was completed using T-Coffee (https: / / tcoffee.crg.eu / apps / tcoffee / index.html) and visualized with the help of ESPript3.0 (https: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi).

[0067] (4) The conserved domains of proteins were annotated using the InterPro database and visualized using IBS1.0 software. Figure 2 As shown in the figure, orf1715 contains two domains, Rincin_B_Lectin located in the N-terminal 31-96 residue region and Toxin_10 located in the C-terminal 222-377 residue region, and contains a signal peptide at the N-terminal 1-28 residues. The Rincin_B_Lectin domain is similar to the lectin domain of the ricin B chain, and the structure presents a cloverleaf conformation, which has the potential to bind to carbohydrates. It contains two conserved motifs QxW and QxF, which may be the key sites for binding to the receptor. Another Toxin_10 domain was originally defined in mosquito-killing crystal proteins. Although the proteins containing this domain have different sequences, they have similar conformations and are related to the perforating toxicity of the toxin.

[0068] Example 5: Cloning of orf1715 insecticidal gene

[0069] The genomic DNA of BMBRJT048 strain obtained in Example 2 was used as a template, primers were designed according to the gene sequence of orf1715, and it was inserted into the Escherichia coli expression vector pET15d using seamless cloning technology. Figure 4 As shown: the target band was successfully amplified at a higher annealing temperature of 52-60°C using a high-fidelity enzyme, wherein M represents Trans 2KPlusIIDNAMarker (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and lanes 1-4 correspond to the target product bands amplified at annealing temperatures of 52°C, 56.6°C, 59°C, and 60°C, respectively.

[0070] Table 2 Amplification system

[0071]

[0072] Table 3 Amplification procedures

[0073]

[0074] Table 4 Amplification primers

[0075] orf1715-F GAAGTTGATGCACATATGATGAAAATTATATCAAAA orf1715-R AGCAGCCGGATCCTCGAGTTATGAACTTTTTCTTGT

[0076] The seamless cloning technology is described in detail as follows:

[0077] (1) Linearize the vector using NdeI and XhoI and purify by gel recovery;

[0078] (2) Use primers to perform PCR amplification of the target fragment, and use electrophoresis to detect whether the amplified band size is correct. If there are any impurities, gel recovery and purification are required;

[0079] (3) The forward and reverse primers each carry an 18 nt target fragment-specific primer sequence and an 18 nt sequence homologous to the end of the vector;

[0080] (4) The recombinase was purchased from Ezmax Single One-Step Cloning kit (Cat. No. 24305) from Tolo Biotech. The reaction system for connecting the exogenous DNA fragment to the linearized vector is shown in Table 4. The reaction system was prepared on ice and then placed at 37°C for 30 min. After the reaction was completed, the reaction was terminated by placing on ice for 5 min.

[0081] Table 5 Recombination reaction system

[0082] 5×BufferforEzmaxOne-StepCloning 4μl Linearized vector Minimum 40ng Foreign DNA fragments Minimum 20ng Ezmax recombinant enzyme 2μl <![CDATA[ddH2O]]> Up to 20 μl

[0083] (5) Add the ligation product to 100 μl DH5α competent cells, place on ice for 30 min, heat shock at 42°C for 90 s, and ice bath for 2 min. Then add 800 μl LB medium and resume culture in a shaker at 37°C for 40 min. Centrifuge at 5,000 rpm for 5 min, remove the supernatant, blow the remaining 100 μl of bacterial solution evenly and spread it on an LB plate containing ampicillin antibiotics, and invert and culture overnight at 37°C;

[0084] (6) The next day, colony PCR was performed to verify the clones. Figure 5 As shown, No. 1 and No. 6 are positive transformants. M represents Trans 2K PlusII DNA Marker, lanes 1-6 correspond to 6 different transformants, lane 7 is a negative control, and lane 8 is a positive control. The positive clones were sent for sequencing to ensure that they completely matched the target fragment. Finally, the successfully constructed strain was stored in a glycerol tube, and the recombinant plasmid was extracted for subsequent protein expression.

[0085] Example 6: Expression and purification of orf1715 insecticidal protein

[0086] (1) The recombinant plasmid successfully constructed in Example 3 was transformed into BL21 (DE3) competent cells and cultured overnight in 10 ml LB liquid medium (with ampicillin added at a final concentration of 100 μg / ml) at 37° C. and 220 rpm until OD 600 The next day, the cells were transferred to 1,000 ml LB liquid medium (with ampicillin added at a final concentration of 100 μg / ml) at a ratio of 1:100 (v / v) until OD 600 It takes about 4-6 hours to reach 0.6-0.8. Take one milliliter of bacterial solution and centrifuge it at 12,000 rpm for 1 minute to obtain the precipitate. Add 40ul of sterile water and 10μl of 5× loading buffer to the precipitate and mix well. This will be used as the pre-induction sample.

[0087] The temperature was then adjusted to 16°C and the rotation speed was 150 rpm. After the temperature of the culture medium in the shake flask dropped, isopropyl-β-D-thiogalactoside (IPTG) was added at a final concentration of 0.1 mmol to induce the reaction for 12-16 hours.

[0088] (2) After induction, collect the cells using a 4°C centrifuge at 12,000 rpm for 3 min. Resuspend the cells in lysis buffer (20 mM Tris-HCl, pH 8.0). Disrupt the cells using a high-pressure cell disruptor (JNBIO, Inc., China) and centrifuge at 12,000 rpm for 45 min. Take 40 μl of the supernatant and add 10 μl of 5× loading buffer to mix, and use it as the post-induction supernatant sample for later use. Take a small amount of the precipitate, add 40 ul of sterile water and 10 μl of 5× loading buffer to mix, and use it as the post-induction precipitate sample for later use.

[0089] (3) Purify the protein by nickel column affinity chromatography. Take out the supernatant and pass it through the nickel column repeatedly for 3 times. Use lysis buffer to wash away non-specifically bound impurities. Add 10 ml Elution Buffer (20 mM Tris-HCl pH 8.0, 500 mM imidazole) to elute the target protein from the nickel column. Take 40 μl of the eluted protein and add 10 μl 5× loading buffer to mix well for later use. Finally, use dialysis technology (dialysis fluid: 20 mM Tris-HCl pH 8.0) to remove the imidazole in the eluted protein, use an ultrafiltration tube for concentration, and divide the protein into 1.5 ml EP tubes and freeze them in a -80°C refrigerator.

[0090] (4) The pre-induction sample and the post-induction precipitate sample were placed in a boiling water bath and boiled for 10 min, and centrifuged at 12,000 rpm for 10 min. The pre-induction sample, post-induction supernatant sample, post-induction precipitate sample, and eluted protein sample were taken for SDS-PAGE analysis. Figure 6 , orf1715 protein exists in the supernatant in a soluble form and can be obtained by high-quality elution and purification. Where M is the 26614 protein marker, and lanes 1-4 correspond to the sample before induction, the supernatant sample after induction, the precipitate sample after induction, and the eluted protein sample, respectively. Figure 7 , simultaneously spot BSA protein standard solutions with concentration gradients of 5, 2.5, 1.25, 0.625, and 0.3125 mg / ml on a piece of gel, scan the gel image with an optical density scanner, analyze the grayscale of the corresponding protein bands with Quantity One software, make a BSA standard curve and calculate the concentration of the protein to be tested.

[0091] Example 7: Determination of the biological activity of orf1715 insecticidal protein against brown planthoppers

[0092] The brown planthoppers used in the experiment were raised indoors at a temperature of 27°C, a humidity of 60%, and a photoperiod of 16L:8D. The third-instar brown planthoppers were taken and the double-layer film feeding method was used for the experiment (for details, refer to: Liu Yonglei, 2015. Mining and functional verification of new genes for efficient insecticide of Bacillus thuringiensis against gray planthoppers. [Doctoral dissertation]. Nanjing Agricultural University). The specific description is: use a hole opener (d = 20cm) to punch holes in a transparent plastic bottle (bottle height 69mm, diameter 42mm), and use 3M medical breathable tape to seal the holes from the inside for the test insects to breathe, and add 1ml of 1% water agar to the bottom of the bottle to maintain the humidity in the bottle; gently spread a layer of paraffin film to cover the bottle mouth, drop 180μl of the sample to be tested (a mixture of protein and artificial feed in a volume of 1:2) in the center of the film, and cover it with a layer of paraffin film again to make a feed naan; add 20 third-instar brown planthopper nymphs to each bottle, and treat each sample three times. Place the test insects in an incubator at 28°C, with a photoperiod of 16L:8D and a humidity of 70%. Observe the feeding and survival of the test insects every day, change the feed every 2 days, and count the number of dead brown planthoppers after 5 days and calculate the mortality rate. Figure 8 As shown in the figure, the orf1715 protein concentration used in the experiment was 30 μg / ml, the protein buffer was used as the negative control, and the same concentration of Tpp78Aa1 was used as the positive control. Compared with the group treated with the simple addition of buffer, orf1715 had a significant lethal effect on brown planthoppers, with an average mortality rate of 38.48% after 120 hours of treatment. As shown in Table 6, the LC50 of the half-lethal concentration was further tested to be 46.56 μg / ml.

[0093] Table 6 LC of orf1715 protein of the present invention against brown planthopper 50

[0094]

[0095] Example 8: Determination of the biological activity of orf1715 against mosquitoes, fall armyworms and Caenorhabditis elegans

[0096] Mosquitoes: Culex pipiens and Aedes mosquitoes used in the experiment were provided by Kono Biotech. Experimental method: Use a 9 cm deep-well plastic culture dish, add 500 μl of protein sample to each culture dish, and add purified water to 5 ml. Use protein buffer (Na2CO3-NaHCO3buffer, 20 mM) as a negative control. Three replicates were set for each sample, and 20 4th-instar mosquito larvae were added to each treatment. After 48 hours, the number of surviving and dead insects was counted. As shown in the figure, compared with the control treatment, orf1715 did not affect Aedes mosquitoes ( Fig. 9 A) and Culex pipiens ( Fig. 9 B) survival.

[0097] Fall armyworm: Insects and artificial feed were purchased from Jiyuan Baiyun Industrial Co., Ltd. in Henan Province. The purchased eggs were placed in an incubator for incubation at a temperature of 27±1°C and a humidity of about (65±5)%. A 24-well plate was used for the bioassay experiment. 15g of artificial feed was added with 3ml of protein sample and mixed thoroughly. The sample was dispensed into a 24-well plate and one newly hatched larva was added to each well. Three replicates were set for each sample. Protein buffer (Na2CO3-NaHCO3buffer, 20mM) was used as a negative control, and Cry1Ac (protein / feed, 5μg / g) was used as a positive control. Fig.10 As shown, orf1715 had no toxic activity against Spodoptera frugiperda compared with the control group.

[0098] Caenorhabditis elegans: The nematodes used in the experiment were obtained by indoor culture in the applicant's team. The methods for culturing Caenorhabditis elegans and determining the biological activity were referred to Bischof et al, 2006 (Bischof LJ, Huffman DL, Aroian RV. Assays for toxicity studies in C. elegans with Bt crystal proteins. Methods Mol Biol. 2006; 351: 139-54. doi: 10.1385 / 1-59745-151-7: 139. PMID: 16988432.). The biological activity assay was carried out in a 96-well plate, with 20-30 L4 instar larvae added to each well, and 3 replicates for each sample. The protein samples used in the experiment were dissolved in 20 mM HEPES (PH 7.4). The protein buffer was used as the negative control, and the Cry5Ba / BMB171 crystal cell mixture was used as the positive control, in which the Cry5Ba protein concentration was 50 μg / ml. Fig.11 As shown, no nematicidal activity was found for orf1715 at concentrations of 50, 100, 300, and 500 μg / ml compared with the control group.

Claims

1. An insecticidal protein orf1715 derived from Bacillus thuringiensis, whose amino acid sequence is shown in SEQ ID NO:

1.

2. A gene encoding the insecticidal protein orf1715 according to claim 1.

3. The insecticidal protein gene according to claim 1, characterized in that: The coding sequence of the gene is shown in SEQ ID NO:

2.

4. A recombinant vector, characterized in that: A gene comprising the insecticidal protein orf1715 according to claim 2.

5. A genetically engineered bacterium, characterized in that: Comprising the recombinant vector according to claim 4.

6. Use of the insecticidal protein according to claim 1, the gene according to claim 2 or 3, the recombinant vector according to claim 4 or the engineered bacteria according to claim 5 in resisting brown planthoppers or preparing products resisting brown planthoppers.

7. A Bacillus thuringiensis BMBRJT048, whose strain deposit number is CCTCC M 20242967.

8. The Bacillus thuringiensis BMBRJT048 according to claim 1, characterized in that The strain contains the insecticidal gene orf1715, and the coding sequence of the insecticidal gene orf1715 is shown in SEQ ID NO:2.

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