An insecticidal protein against Nilaparvata lugens, its encoding gene and application, which are derived from Bacillus thuringiensis

Abstract

The present invention belongs to the technical field of genetic engineering, and discloses an insecticidal protein against brown planthopper derived from Bacillus thuringiensis, its coding gene and application. The insecticidal protein is orf1715, and its amino acid sequence is shown in SEQ ID NO: 1, and the nucleotide coding sequence of the gene is shown in SEQ ID NO: 2. This gene is well-expressed in a soluble form in the Escherichia coli system, and the purified protein has a significant specific insecticidal effect on the rice pest brown planthopper. Therefore, it can be applied to the special prevention and control of brown planthopper, providing candidate gene resources for the cultivation of new transgenic insect-resistant rice varieties.

Description

Technical Field

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

[0002] As one of the main food sources for the global population, rice suffers a yield loss of up to 30% due to pests and diseases. In recent years, the brown planthopper (BPH) with a piercing-sucking mouthpart has gradually become the main pest of rice (SAVARY S, WILLOCQUET L, PETHYBRIDGE S J, et al. 2019. The global burden of pathogens and pests on major food crops. Nature Ecology & Evolution, 3: 430 - 439.). The BPH is a highly destructive hemipteran pest with a fast reproduction rate and strong migration ability. It not only sucks the phloem sap of rice through its piercing-sucking mouthpart, causing the plants to turn yellow and wither, but also spreads various plant diseases, such as rice stripe virus (RSV), rice grassy stunt virus (RGSV) and rice ragged stunt virus (RRSV). Against the backdrop of global warming, it has become more difficult to predict the migration routes and outbreak areas of the BPH. For a long time, in the control of the BPH, over-reliance on and extensive use of chemical pesticides have become the norm, resulting in significant resistance of the BPH population to various insecticides and posing threats to biodiversity and food safety. Therefore, it is urgent to develop safe, efficient and sustainable pest control strategies.

[0003] Bacillus thuringiensis is a Gram-positive bacterium belonging to the Bacillus cereus group and is widely distributed in natural environments such as soil, insect corpses, and leaf surfaces. The insecticidal crystal protein Cry produced during its spore stage has specific high activity against a variety of agricultural pests, plant parasitic nematodes, and mites, but is safe for humans, animals, and plants (JOUZANI G S, 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 various other virulence factors such as vegetative insecticidal protein Vip during the vegetative stage (RAKESH V, KALIAV K, GHOSHA 2023. Diversity of transgenes in sustainable management of insect pests. Transgenic Res, 32: 351-381.). With the discovery of more and more insecticidal genes, a new nomenclature system based on structural characteristics was published in 2020. This revised version retains the basic principle of classification based on sequence similarity in the 1998 nomenclature version, and at the same time introduces specific mnemonic symbols 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, Xpp. For example, here Cry specifically refers to the three-domain type of insecticidal protein, and Tpp specifically refers to a class of β-pore-forming proteins with the 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.).Multiple Cry proteins, represented by the three-domain Cry protein, have been successfully applied to the development of transgenic insect-resistant crops, mainly for controlling pests such as Lepidoptera (LI Y, HALLERMAN E M, 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, various Hemiptera pests, including the brown planthopper and aphids, have developed into the main pests or even the number one pests of important crops, and there are very few effective genes against such pests.

[0004] Some researchers have made efforts in identifying insecticidal genes against the brown planthopper. Replacing the loop structure in domain II of Cry1Ab with an intestinal-binding polypeptide, the resulting fusion protein had a median lethal concentration LC 50 of 21.54 μg / ml against the brown planthopper (SHAO E, LIN L, CHEN C, et al. 2016. Loop replacements with gut-binding peptides in Cry1Ab domain II enhanced toxicity against the brown planthopper, Nilaparvata lugens Sci Rep, 6:20106.); the binary toxins Mpp64Ba and Mpp64Ca had an LC 50They were 3.15 μg / ml and 2.14 μg / ml, respectively (LIU Y, WANG Y, SHU C, et al. 2018. Cry64Ba and Cry64Ca, Two ETX / MTX2-Type Bacillus thuringiensis Insecticidal Proteins Active against Hemipteran Pests. Appl Environ Microbiol, 84.); the median lethal concentrations of the first single-component protein Tpp78Aa1 against Nilaparvata lugens and Laodelphax striatellus 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 successively discovered 5 genes with similar insecticidal effects against Laodelphax striatellus, namely Tpp78Ab1, Tpp78Ba1, Tpp78Bb1, Tpp78Ca1, and Tpp80Ae1, and their LC 50 values against Laodelphax striatellus were 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 of insecticidal proteins and contain two conserved domains, Ricin_B_Lectin and Toxin_10. This also indicates the great application potential of such insecticidal genes in the control of the hemipteran pest Nilaparvata lugens. However, no new transgenic insect-resistant rice varieties capable of controlling Nilaparvata lugens have emerged so far. Therefore, based on the significant damage of this pest to rice crops and to prevent the emergence of resistance problems caused by the use of a single gene, it is urgent and necessary to discover more new genes resistant to Nilaparvata lugens. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the first object of the present invention is to provide a novel insecticidal protein derived from Bacillus thuringiensis and its encoding gene. The encoding gene has a very low sequence similarity with the currently reported insecticidal gene sequences and belongs to a first-class novel insecticidal gene according to the naming principle. This 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 Nilaparvata lugens.

[0006] Specifically, the object of the present invention is achieved according to the following technical solution: An insecticidal protein orf1715 derived from Bacillus thuringiensis, and its 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] Meanwhile, the present invention also provides a gene encoding the above-mentioned insecticidal protein orf1715. Further preferably, the gene encoding the above-mentioned 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] cacggatggatagattatgtagatttagatgaacccaatccttctgattcagatagggtatttaacattgccaattctccaagtggtttatcactacc

[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 the soil sample collected from Yun'an Garden, Kunming, Yunnan in 2013 by our research group. This strain has been deposited in the China Center for Type Culture Collection, with the deposit date of December 31, 2024 and the deposit number of CCTCC M 20242967. Through the observation of optical microscope and scanning electron microscope, it was found that the BMBRJT048 strain could form rhomboid parasporal crystals during the spore stage. And the genomic information of this strain was obtained by using the next-generation sequencing technology. Further, the insecticidal gene sequences contained in the BMBRJT048 strain were predicted by the insecticidal gene prediction and analysis pipeline BtToxin_Scanner2 pipeline (Liu et al., 2021) developed by our laboratory. Among them, the orf1715 gene has 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 insecticidal protein orf1715. Further preferably, the vector is the Escherichia coli expression vector pET15d. Meanwhile, 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 as follows: using the genome of Bacillus thuringiensis strain BMBRJT048 as a template, and adopting the PCR amplification method to obtain a complete insecticidal gene fragment of orf1715. The primer sequences for the PCR amplification include: orf1715 primer F: GAAGTTGATGCACATATG ATGA AAATTATATCAAAA, orf1715 primer R: AGCAGCCGGATCCTCGAG TTATGAAC TTTTTCTTGT, where the underlined part is the homologous arm of the Escherichia coli expression vector pET15d. Further explanation, the endonucleases for linearizing the Escherichia coli heterologous expression vector pET15d are NdeI and XhoI.

[0031] The third object of the present invention is to provide a product use, that is, the application of the above-mentioned insecticidal protein orf1715, the above-mentioned insecticidal gene orf1715, the above-mentioned recombinant vector or the above-mentioned engineered bacterium in controlling Nilaparvata lugens or preparing products for controlling Nilaparvata lugens.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows: through experiments such as cloning, protein expression and bioactivity determination of the orf1715 gene, the present invention finds that the novel insecticidal protein orf1715 has significant specific toxicity against Nilaparvata lugens, has no effect on the growth of dipteran mosquitoes such as Aedes and Culex, and has no insecticidal activity against Caenorhabditis elegans and Spodoptera frugiperda. Therefore, it can be applied to the special prevention and control of Nilaparvata lugens, providing candidate gene resources for the cultivation of new transgenic insect-resistant rice varieties. Description of the Drawings

[0033] Figure 1 Shows the spore and parasporal crystal formation of Bacillus thuringiensis strain BMBRJT048 under an optical microscope (A) and a scanning electron microscope (B).

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

[0035] Figure 3 Is a multiple sequence alignment analysis diagram of the insecticidal gene orf1715 of the present invention and Tpp-like insecticidal genes.

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

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

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

[0039] Figure 7 This is the concentration determination map of the insecticidal protein orf1715 of the present invention.

[0040] Figure 8 This is the map of the determination result of the insecticidal activity of the insecticidal protein orf1715 of the present invention against Nilaparvata lugens.

[0041] Figure 9 This is the map of the determination result of the insecticidal activity of the insecticidal protein orf1715 of the present invention against Aedes mosquitoes and Culex mosquitoes.

[0042] Figure 10 This is the map of the determination result of the insecticidal activity of the insecticidal protein orf1715 of the present invention against Spodoptera frugiperda.

[0043] Figure 11 This is the map of the determination result of the insecticidal activity of the insecticidal protein orf1715 of the present invention against Caenorhabditis elegans. Detailed implementation manners

[0044] To better understand the technical content of the present invention, specific examples are provided below to further elaborate on the present invention in detail. Unless otherwise specified in the examples, the methods involved are conventional methods in the art, and operations can be performed by referring to the general techniques described in the literature in the art. Reagents or instruments without indicating the manufacturer can be obtained through commercial channels.

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

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

[0047] (2) Pick colonies with a morphology similar to Bt and streak them onto a T3 plate, i.e., those milky white, approximately circular colonies with a slightly diffused outer edge but not very neat. Incubate them upside down in a constant temperature incubator at 30 °C for 72 h;

[0048] (3) Observe the formation of parasporal crystals through an optical microscope, further identify them as Bt strains, and name them Bacillus thuringiensis BMBRJT048. Use a 10 μl pipette tip to pick up a small amount of bacteria onto a glass slide, smear it with sterile water, stain it with carbol fuchsin staining solution, and observe under an optical microscope that the BMBRJT048 strain can form spores and produce rhomboid parasporal crystals. Further observe the morphology of the formed parasporal crystals using a scanning electron microscope, as Figure 1 shown.

[0049] (4) The medium formulations (1 L) used in the above experimental operations are as follows:

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

[0051] LA solid medium: 10 g of peptone, 5 g of yeast extract, 5 g of NaCl, 15 - 20 g of agar powder, pH 7.0;

[0052] T3 solid medium: 5 g of peptone, 1.5 g of yeast extract, 0.005 g of MnCl2, 3.9 g of NaH2PO4, 8.95 g of Na2HPO4, 15 g - 20 g of agar powder, pH 7.0.

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

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

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

[0056] (3) Add 300 μl of Solution I (25 mM Tris-HCl, pH 8.0, 10 mM EDTA, 50 mM Glucose) and 40 μl of lysozyme (100 mg / ml) to each EP tube, mix well, and incubate at 4°C overnight for lysis. The next day, add 3 μl of RNase A (10 mg / ml) to each tube and incubate at 37°C for 1.5 h (gently shake and mix several times during the process). Then add 300 μl of SDS (10%) and mix gently, and incubate in a water bath at 55°C until the bacterial solution becomes clear. Then add 360 μl of NaCl (5 M) and mix gently, and a white precipitate can be seen. Let it stand on ice for 10 min, centrifuge at 12,000 rpm at low temperature for 15 min, and transfer the supernatant to a new EP tube;

[0057] (4) Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), invert about 200 times up and down, centrifuge at 12,000 rpm for 15 min, and take the supernatant. Add 2 volumes of pre-cooled absolute ethanol and 50 μl of 3 M sodium acetate to the obtained supernatant and let it stand in a -20°C refrigerator for 10 min for DNA precipitation.

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

[0059] Example 3: Prediction of Insecticidal Genes in Strain BMBRJT048

[0060] Obtain the genomic sequence of strain BMBRJT048 through next-generation sequencing technology, and use the BtToxin_Scanner2 pipeline system developed by our team 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. From the perspective of the amino acid sequence length, the orf1715 gene is more complete, so it is selected for further functional research.

[0061] Table 1 Identification and Analysis Results of Insecticidal Genes in Strain BMBRJT048 of the Present Invention

[0062]

[0063] Example 4: Analysis of the Basic Physicochemical Properties and Gene Sequence of the Novel Insecticidal Protein orf1715

[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] Using the genomic DNA of strain BMBRJT048 obtained in Example 2 as a template, primers were designed according to the gene sequence of orf1715 and inserted into the Escherichia coli expression vector pET15d using seamless cloning technology. As Figure 4 shown: Using a high-fidelity enzyme, the target band was successfully amplified at a relatively high annealing temperature of 52 - 60 °C. Here, M represents Trans 2KPlus II DNA Marker (purchased from Beijing TransGen Biotech 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 Program

[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) The vector was linearized using NdeI and XhoI and purified by gel extraction.

[0078] (2) PCR amplification of the target fragment was performed using primers, and the size of the amplified band was detected by electrophoresis. If there were additional bands, gel extraction and purification were required.

[0079] (3) Each of the forward and reverse primers carried an 18-nt target fragment-specific primer sequence and an 18-nt sequence homologous to the vector end.

[0080] (4) The recombinase was purchased from Ezmax Single One-Step Cloning kit of Tolo Harbour Biotechnology Company (product number #24305). The reaction system for ligating the foreign DNA fragment to the linearized vector is shown in Table 4. The reaction system was prepared on ice and then incubated at 37 °C for 30 min. After the reaction, it was placed on ice for 5 min to terminate the reaction.

[0081] Table 5 Recombination Reaction System

[0082] 5× Buffer for Ezmax One-Step Cloning 4 μl Linearized vector Minimum 40 ng Exogenous DNA fragment Minimum 20 ng Ezmax recombinase 2 μl <![CDATA[ddH2O]]> Up to 20 μl

[0083] (5) Add the ligation product to 100 μl of DH5α competent cells, place on ice for 30 min, heat shock at 42°C for 90 s, and then incubate on ice for 2 min. Subsequently, add 800 μl of LB medium, and recover the culture in a shaker at 37°C for 40 min. Centrifuge at 5,000 rpm for 5 min, discard the supernatant, resuspend the remaining approximately 100 μl of bacterial solution, and spread it evenly on an LB plate containing ampicillin antibiotic. Incubate the plate upside down at 37°C overnight;

[0084] (6) The next day, verify the clones by colony PCR. As Figure 5 shown, No. 1 and No. 6 are positive transformants. Here, M represents Trans 2K Plus II DNA Marker, lanes 1 - 6 correspond to 6 different transformants respectively, lane 7 is the negative control, and lane 8 is the positive control. Send the positive clones for sequencing to ensure complete matching with the target fragment. Finally, store the successfully constructed strain in a glycerol tube and extract the recombinant plasmid for subsequent protein expression.

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

[0086] (1) Transform the successfully constructed recombinant plasmid in Example 3 into BL21(DE3) competent cells, add it to 10 ml of LB liquid medium and culture overnight (added with ampicillin at a final concentration of 100 μg / ml), at a temperature of 37°C and a rotation speed of 220 rpm until the OD 600 reaches about 0.6. The next day, transfer it to 1,000 ml of LB liquid medium for scale-up culture at a ratio of 1:100 (v / v) (added with ampicillin at a final concentration of 100 μg / ml) until the OD 600 reaches 0.6 - 0.8, which takes about 4 - 6 h. Take 1 ml of the bacterial solution, centrifuge at 12,000 rpm for 1 min to obtain the precipitate, add 40 μl of sterile water and 10 μl of 5×loading buffer to the precipitate and mix well for use as a sample before induction.

[0087] Subsequently, adjust the temperature to 16°C and the rotation speed to 150 rpm. After waiting for the temperature of the medium in the shake flask to drop, add isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.1 mmol for induction reaction for 12 - 16 h.

[0088] (2) After the induction was completed, the cells were collected using a 4°C centrifuge at 12,000 rpm for 3 min. The cell pellet was resuspended in lysis buffer (20 mM Tris-HCl, pH 8.0). The cells were disrupted using a high-pressure cell disruptor (JNBIO, Inc., China) and then centrifuged at 12,000 rpm for 45 min. 40 μl of the supernatant was taken and mixed with 10 μl of 5× loading buffer as the induced supernatant sample for standby. A small amount of the precipitate was taken, mixed with 40 μl of sterile water and 10 μl of 5× loading buffer as the induced precipitate sample for standby.

[0089] (3) The protein was purified according to the nickel column affinity chromatography method. The supernatant was taken out and passed through the nickel column repeatedly 3 times. Non-specifically bound impurities were washed away using lysis buffer. 10 ml of Elution Buffer (20 mM Tris-HCl pH 8.0, 500 mM imidazole) was added to elute the target protein from the nickel column. 40 μl of the eluted protein was taken and mixed with 10 μl of 5× loading buffer for standby. Finally, dialysis technology (dialysis buffer: 20 mM Tris-HCl pH 8.0) was used to remove the imidazole in the eluted protein, and ultrafiltration tubes were used for concentration. The protein was aliquoted into 1.5 ml EP tubes and stored at -80°C in the refrigerator.

[0090] (4) The above samples before induction and the induced precipitate sample were placed in a boiling water bath for 10 min and then centrifuged at 12,000 rpm for 10 min. Samples before induction, induced supernatant sample, induced precipitate sample, and eluted protein sample were subjected to SDS-PAGE analysis. As Figure 6 , the orf1715 protein existed in the supernatant in a soluble form and could be eluted and purified with high quality. Among them, M was the 26614 protein Marker, and lanes 1-4 corresponded to the sample before induction, the induced supernatant sample, the induced precipitate sample, and the eluted protein sample respectively. As Figure 7 , BSA protein standard solutions with concentration gradients of 5, 2.5, 1.25, 0.625, and 0.3125 mg / ml were spotted on the same gel at the same time. After scanning the gel using an optical density scanner and analyzing the gray scale of the corresponding protein bands in Quantity One software, a BSA standard curve was made and the concentration of the protein to be measured was calculated.

[0091] Example 7: Bioactivity assay of orf1715 insecticidal protein against Nilaparvata lugens

[0092] The Nilaparvata lugens used in the experiment was reared indoors at a temperature of 27°C, a humidity of 60%, and a light cycle of 16L:8D. Third-instar Nilaparvata lugens were taken and the double-layer membrane feeding method was used to conduct the experiment (for detailed reference: Liu Yonglei, 2015. Exploration and functional verification of new genes with high insecticidal activity against Laodelphax striatellus by Bacillus thuringiensis. [Doctoral dissertation]. Nanjing Agricultural University). The specific description is as follows: An opener (d = 20 cm) was used to punch holes in a transparent plastic bottle (bottle height 69 mm, diameter 42 mm), and 3M medical breathable tape was used to seal the holes from the inside for the test insects to breathe. 1 ml of 1% water agar was added to the bottom of the bottle to maintain the humidity inside the bottle; a layer of paraffin film was gently stretched and covered over the bottle mouth, and 180 μl of the test sample (a mixture of protein and artificial feed in a volume ratio of 1:2) was dropped in the center of the film, and then another layer of paraffin film was covered to make a feed pouch; 20 third-instar Nilaparvata lugens nymphs were added to each bottle, and three replicates were set for each sample. It was placed in an incubator at 28°C, a photoperiod of 16L:8D, and a humidity of 70%. The feeding and survival of the test insects were observed every day, the feed was changed every 2 days, and the number of dead Nilaparvata lugens was counted after 5 days and the mortality rate was calculated. As Figure 8 shown, the concentration of orf1715 protein used in the experiment was 30 μg / ml, protein buffer was used as the negative control, and Tpp78Aa1 at the same concentration was used as the positive control. Compared with the treatment group with only buffer added, orf1715 had an obvious lethal effect on Nilaparvata lugens, and the average mortality rate after 120 h of treatment was 38.48%. As shown in Table 6, its median lethal concentration LC50 was further tested to be 46.56 μg / ml.

[0093] Table 6 Median lethal concentration LC of orf1715 protein of the present invention against Nilaparvata lugens 50

[0094]

[0095] Example 8: Bioactivity determination of orf1715 against mosquitoes, Spodoptera frugiperda, and Caenorhabditis elegans

[0096] Mosquitoes: The Culex and Aedes mosquitoes used in the experiment were provided by Keno Biosciences. Experimental method: A 9-cm deep-well plastic culture dish was used, and 500 μl of protein sample was added to each culture dish, and pure water was added to make up to 5 ml. Protein buffer (Na2CO3-NaHCO3 buffer, 20 mM) was used as the negative control. Three replicates were set for each sample, and 20 fourth-instar mosquito larvae were added to each treatment. After 48 h, the number of surviving and dead insects was counted. As shown in the figure, compared with the control treatment, orf1715 did not affect the survival of Aedes mosquitoes ( Figure 9 A) and Culex mosquitoes ( Figure 9 B).

[0097] Spodoptera frugiperda: Insects and artificial feed were purchased from Jiyuan Baiyun Industry Co., Ltd., Henan Province. The obtained eggs were hatched in an incubator at a temperature of 27 ± 1°C and a humidity of about (65 ± 5)%. The bioassay experiment was carried out using 24-well plates. 15 g of artificial feed was added with 3 ml of protein sample and thoroughly mixed, and then dispensed into 24-well plates. One newly hatched larva was added to each well, and 3 replicates were set for each sample. Protein buffer (Na2CO3-NaHCO3 buffer, 20 mM) was used as the negative control, and Cry1Ac (protein / feed, 5 μg / g) was used as the positive control. As Figure 10 shown, compared with the control group, orf1715 had no toxic activity against Spodoptera frugiperda.

[0098] Caenorhabditis elegans: The nematodes used in the experiment were obtained from the indoor culture of the applicant's team. The culture and bioactivity assay methods of Caenorhabditis elegans refer 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 bioactivity assay was carried out in 96-well plates. 20-30 L4-stage larvae were added to each well, and 3 replicates were set for each sample. The protein samples used in the experiment were dissolved in 20 mM HEPES (pH 7.4). Protein buffer was used as the negative control, and the Cry5Ba / BMB171 crystal cell mixture was used as the positive control, in which the concentration of Cry5Ba protein was 50 μg / ml. As Figure 11 shown, compared with the control group, no nematicidal activity of orf1715 was found at concentrations of 50, 100, 300, and 500 μg / ml.

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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