Cry53-like insecticidal protein and application thereof in control of diptera pests

Abstract

The invention provides a Cry53-like insecticidal protein and application thereof in prevention and treatment of diptera pests, and belongs to the technical field of biopesticides, the Cry53-like insecticidal protein is separated from bacillus thuringiensis, and the amino acid sequence of the Cry53-like insecticidal protein is shown as SEQ ID NO.2. The invention further provides a preparation method of the Cry53-like insecticidal protein. Compared with the reported Cry insecticidal protein, the Cry53-like insecticidal protein has the advantages that the highest homology is only 38%, the Cry53-like insecticidal protein is determined to be a novel insecticidal protein, the Cry53-like insecticidal protein has advantages in prevention and treatment of diptera pests, the killing effect of the Cry53-like insecticidal protein on bradysia odoriphaga is obviously superior to that of other same-type Cry insecticidal proteins, and the Cry53-like insecticidal protein is a novel insecticidal protein. Therefore, the gene has application prospects in the fields of biopesticide creation, transgenic insect-resistant crop cultivation and the like.

Description

Technical Field

[0001] This invention relates to the field of biopesticide technology, specifically to a Cry53-like insecticidal protein, its preparation method, and its application in controlling dipteran pests. Background Technology

[0002] Leek late-eye mushroom mosquito ( Bradysia odoriphaga This insect belongs to the genus *Tectus* of the family Tectusidae in the order Diptera. It primarily damages lily family vegetables such as leeks, scallions, and shallots, and is one of the major pests of allium vegetables. It is distributed throughout China and causes serious economic losses to vegetable production. Long-term reliance on large amounts of chemical pesticides easily leads to pesticide residues and increased pesticide resistance in pests. Therefore, it is necessary to find alternative methods of biological control.

[0003] Bacillus thuringiensis (Bt) Bacillus thuringiensis Bt is the safest and most widely used insecticidal bacterium to date. The main insecticidal active substances in Bt are crystalline proteins such as Cry, Cyt, and Vip. Among them, there are 78 types of Cry proteins, which are toxic to Lepidoptera, Coleoptera, Hemiptera, Diptera, and nematodes (Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH). Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and MolecularBiology Reviews, 1998, 62(3):775-806; Liu G, Lai S, Shu C, Wang P, Deng C,Peng Q, Lereclus D, Wang X, Huang D, Zhang J, Song F. Complete genomesequence of Bacillus thuringiensis subsp. kurstaki strain HD73. Genome Announcements, 2017, 1(2): e0008013.). In recent years, the production of biopesticides using insecticidal proteins as raw materials or the encoding of insecticidal crystal proteins has become increasingly important. cry The transfer of genes into a few important crops to control agricultural pests has become a research hotspot.

[0004] Currently, most research focuses on using Cry proteins to control lepidopteran pests, with less research on dipteran pests. Among these, only Cry39 and Cry40 proteins (CN201610537583, CN201610537585) are found in 2016 to control the leek leafminer. To obtain more effective biopesticides and delay the development of pesticide resistance in pests, the search for novel insecticidal proteins continues. Summary of the Invention

[0005] In view of this, the present invention provides a novel Cry53-like insecticidal protein, its preparation method and its application in the control of dipteran pests, aiming to enrich the types of insecticidal proteins for controlling dipteran pests and provide insecticidal proteins with better killing effect on dipteran pests.

[0006] In a first aspect, the present invention provides a novel Cry53-like insecticidal protein, specifically which can be: i) Proteins with amino acid sequences as shown in SEQ ID NO.2; ii) or a protein that has at least 70% sequence identity and function with the protein in i); preferably, the sequence identity is 95% or more, and more preferably, the sequence identity is 98% or more.

[0007] Secondly, the present invention provides the application of Cry53-like insecticidal protein in the preparation of products for killing dipteran pests, preferably, the dipteran pest is the leek gnat mosquito.

[0008] Thirdly, this invention provides a biopesticide with Cry53-like insecticidal protein as one of its active ingredients. It is understood that this biopesticide may contain other insecticidal active ingredients, as well as carriers, adjuvants, etc., required for its preparation.

[0009] Thirdly, the present invention provides biomaterials related to Cry53-like insecticidal proteins, comprising at least the following: (1) Nucleic acid molecules encoding Cry53-like insecticidal proteins, such as the sequence of the nucleic acid molecule as shown in SEQ ID NO.1 in the sequence listing; (2) A recombinant vector containing the nucleic acid molecule shown in (1), wherein the basic vector includes, but is not limited to, pET-Nus32T vector, pET21b vector, etc.; (3) Recombinant bacteria containing the nucleic acid molecule shown in (1) or the recombinant vector shown in (2), wherein the host bacteria include, but are not limited to, Escherichia coli, as long as they can support the expression of Cry53-like insecticidal protein.

[0010] Fourthly, this invention provides the application of Cry53-like insecticidal proteins or nucleic acid molecules encoding Cry53-like insecticidal proteins in the construction of insect-resistant transgenic plants, providing a new approach for the control of dipteran pests in crops.

[0011] Fifthly, the present invention provides a method for preparing Cry53-like insecticidal proteins, comprising the following steps: The nucleic acid molecule encoding the Cry53-like insecticidal protein was inserted into the pET-Nus32T vector, and the resulting recombinant vector was transformed into Escherichia coli BL21(DE3) cells to express the recombinant Escherichia coli. The expression product was then purified to obtain the final product.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention discovers a Cry53-like insecticidal protein from Bacillus thuringiensis, which exhibits a homology of only 38% with previously reported Cry insecticidal proteins, making it a novel insecticidal protein. Furthermore, compared to existing Cry insecticidal proteins, the Cry53-like protein demonstrates advantages against dipteran pests, and its control effect on the leek leafminer is significantly superior to other similar Cry insecticidal proteins. Attached Figure Description

[0013] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0014] Figure 1 The amino acid sequence alignment results of the Cry53-like insecticidal protein and the Cry53Aa1 protein provided by this invention are shown in the figure. Figure 2 In Example 1 cry53 Figure showing the detection results of PCR amplification of -like genes; Figure 3 This is a verification diagram of the recombinant bacterial expression results in Example 1. Lane 0 represents uninduced whole bacteria, lane 1 represents clone 1 induced, lane 2 represents clone 2 induced, and lane M represents the protein marker. Figure 4 The image shows the detection results of the target protein before and after purification by Ni-IDA column in Example 1. Lane 0 is the precipitate after disruption, lane 1 is the supernatant after disruption, lane 2 is the supernatant effluent after purification by Ni-IDA column, lane 3 is the elution buffer, lane 4 is the residual liquid, and lane M is the protein marker. Detailed Implementation

[0015] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention.

[0017] To address the technical problem of the lack of highly effective insecticidal proteins for controlling the leek-eye fungus gnatus in existing technologies, this invention has discovered a novel insecticidal protein from Bacillus thuringiensis. cry53 The -like gene (as shown in SEQ ID NO.1 in the sequence listing) has an ORF size of 2,049 bp, encoding a protein of 682 amino acids (i.e., Cry53-like insecticidal protein, as shown in SEQ ID NO.2 in the sequence listing). It is further clarified that this gene can be used for the creation of biopesticides, the construction of engineered strains, and the cultivation of transgenic insect-resistant crops, thereby being applied to the biological control of agricultural pests.

[0018] In the prior art, the four-level nomenclature system for Bacillus thuringiensis insecticidal proteins classifies them based on amino acid sequence homology. Proteins with homology less than 45% are classified into different levels, namely the first level, such as Cry1 and Cry2 proteins; those with homology between 45% and 78% are classified into the second level, such as Cry1A and Cry1B proteins; those with homology between 78% and 95% are classified into the third level, such as Cry1Aa and Cry1Ab proteins; and those with homology greater than 95% belong to the fourth level, such as Cry1Aa1 and Cry1Aa2 proteins. Figure 1 As shown, the Cry53-like insecticidal protein provided by this invention has the highest homology with the previously reported Cry53Aa1 protein, which is only 38%, belonging to the first classification level, and can be identified as a novel insecticidal protein.

[0019] This invention provides a method for preparing Cry53-like insecticidal proteins, specifically by cloning the novel protein using PCR technology. cry53 The -like gene was inserted into the pET-Nus32T vector, and the resulting recombinant vector was transformed into Escherichia coli BL21(DE3) cells for expression. Finally, the recombinant Cry53-like insecticidal protein was obtained after purification.

[0020] This invention also provides the application of Cry53-like insecticidal protein in killing *Fungiida leekii* larvae. This is in contrast to the previously reported insecticidal activities against *Fungiida leekii* larvae, namely Cry39 and Cry40 proteins (LC...). 50The LC50 of the cry53-like insecticidal protein provided by this invention was 89.42 μg / mL and 126.83 μg / mL, respectively. 50 The concentration was 64.86 μg / mL, indicating that its effect was superior.

[0021] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0022] Example 1 This example demonstrates the preparation of Cry53-like insecticidal proteins using an E. coli expression system, specifically including the following steps: (1) Extraction of genomic DNA from Bacillus thuringiensis strain.

[0023] Extraction was performed using the Bacterial Genomic DNA Extraction Kit from Tiangen Biotech (Beijing) Co., Ltd. The specific procedure was as follows: A single colony of *Bacillus thuringiensis* was inoculated into LB liquid medium and cultured overnight at 30°C and 220 rpm until saturation. 1-5 mL of the bacterial suspension was centrifuged at 12000 rpm for 1 minute, and the supernatant was completely discarded, collecting the bacterial precipitate. 200 μL of buffer GA (with added proteinase K) was added, and the bacterial cells were vortexed vigorously to resuspend them. 20 μL of lysozyme (10 mg / mL) was added, vortexed to mix, and incubated at 37°C for 30-60 minutes. 220 μL of buffer GB was added, vortexed for 15 seconds, and incubated in a 70°C water bath for 10 minutes until the solution became clear. 220 μL of anhydrous ethanol was added, and vortexed thoroughly to mix. All the solution and flocculent precipitate were added to an adsorption column CB3, centrifuged at 12000 rpm for 30 seconds, and the waste liquid was discarded. 500 μL of buffer GD was added to the adsorption column CB3, and centrifuged at 12000 rpm for 1 minute. Centrifuge at 12000 rpm for 30 seconds and discard the waste liquid; add 600 μL of wash buffer PW, centrifuge at 12000 rpm for 30 seconds and discard the waste liquid; put the adsorption column CB3 back into the empty collection tube, centrifuge at 12000 rpm for 2 minutes to completely remove residual ethanol; transfer the adsorption column into a clean 1.5 mL centrifuge tube, add 50-100 μL of elution buffer TE in the middle of the adsorption membrane, let stand at room temperature for 5 minutes, centrifuge at 12000 rpm for 2 minutes, and the liquid in the tube is the genomic DNA.

[0024] (2) cry53 PCR amplification of -like genes.

[0025] Using the genomic DNA obtained in step (1) as a template, PCR amplification was performed using primers Cry53-F and Cry53-R (as shown in SEQ ID NO. 3-4 in the sequence listing, respectively). The amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ for 30 s, 54℃ for 30 s, 72℃ for 2 min, for a total of 32 cycles; 72℃ for 10 min. After purification of the gel extraction product, it was ligated into the pET21b vector. The recombinant plasmid was introduced into E. coli DH5α competent cells. Transformants were cultured overnight at 37℃ on LB agar plates containing Amp (100 ug / ml), X-Gal (64 ug / ml), and IPTG (0.2 mM). White colonies were randomly picked and cultured in LB liquid medium containing Amp. PCR detection was performed on cells with bands (see...). Figure 2 The bacterial culture was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing.

[0026] Sequencing analysis cry53 The -like gene is 2049 bp in length (as shown in SEQ ID NO.1 in the sequence listing) and encodes 682 amino acids (as shown in SEQ ID NO.2 in the sequence listing).

[0027] (3) cry53 Expression and purification of -like genes.

[0028] Expression vector construction: The sequence was amplified using primers Cry53A-F and Cry53A-R (as shown in SEQ ID NO.5-6 in the sequence listing, respectively). After gel purification, the sequence was inserted into pET-Nus32T (purchased from Jiangsu Pubo Biotechnology Co., Ltd.) by BamHI / EcoRI restriction enzyme digestion, with the N-terminus His+NusA-tag.

[0029] Plasmid transformation: 1 μL of the constructed plasmid was added to 100 μL of Rosetta(DE3) competent bacteria and placed on ice for 20 min; heat-shocked at 42℃ for 90 sec, and then immediately placed on ice for 3 min; 500 μL of LB medium was added; cultured at 37℃ for 2 h; 200 μL of the bacterial culture was spread on LB plates containing 50 μg / mL ampicillin and incubated upside down at 37℃ overnight.

[0030] Protein expression: Single colonies were picked from plates and inoculated into fresh LB liquid medium (containing 50 mg / L ampicillin) and cultured at 37°C for approximately 6 hours. When the OD600 reached approximately 0.6, IPTG was added to a final concentration of 0.5 mM to induce Cry53A-pET-NUS32T expression. After induction at 37°C for two hours, the bacterial cells were collected. The protein expression of the processed bacterial cells was detected by 12% SDS-PAGE electrophoresis, and further amplification and purification were performed.

[0031] Protein purification: Transfer 1% of the culture to 3000 ml of bacterial culture and culture until OD=0.6. Induce with 0.2 mM IPTG at 37℃ for 2 hours. Collect cells by centrifugation (6000 rpm, 5 min) and discard the supernatant. Resuspend the cell pellet in 100 mL of ice-cold Buffer D (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.1% Triton-100). Lyse the cells by sonication (600W-800W, 30 min). Centrifuge (10000 rpm, 15 min) and pass the supernatant through a Ni-IDA column. Wash the Ni-IDA medium with 200 ml of Buffer E (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 0.1% Triton X-100). Buffer D1 (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.1% Triton X-100) Wash the Ni-IDA medium with Triton-100; wash the Ni-IDA medium with 20 ml Buffer D2 (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.1% Triton-100, 20 mM imidazole); elute the Ni-IDA medium with an appropriate amount of Buffer C (20 mM Tris-HCl pH 8.0, 50 mM NaCl, 0.1% Triton X-100, 250 mM imidazole). Use the supernatant for 12% SDS-PAGE electrophoresis to detect protein expression.

[0032] Dialysis: Dialyze the eluted target protein to 20 mM Tris, 50 mM NaCl, pH=8.0, three times. Centrifuge after dialysis and collect the supernatant.

[0033] Results and Analysis: Protein expression detection revealed a clear expression band above 116KD, which is comparable to the theoretical molecular weight of Cry53-like insecticidal protein (133KD). Figure 3 The same size target protein was also detected in the purified supernatant. Figure 4 ).

[0034] Example 2 This example uses the immersion method to determine the insecticidal activity of Cry53-like insecticidal protein against the larvae of the leek-eating fungus gnatus. The specific procedure is as follows: The concentration of purified protein was determined using a BCA protein concentration assay kit, and serial dilutions were performed to prepare a series of concentrations: 400 mg / L, 200 mg / L, 150 mg / L, 100 mg / L, 80 mg / L, 50 mg / L, 25 mg / L, and 10 mg / L. 2 ml of each dilution was placed in centrifuge tubes, with five replicates for each concentration. Approximately 2 cm of leek stems were immersed in the centrifuge tubes for 3 min, and then placed in sterile 90 mm petri dishes lined with water agar and double-layered filter paper. The supernatant of Rosetta(DE3) after ultrasonic disruption (i.e., without Cry53-like insecticidal proteins) served as a negative control. Twenty healthy second-instar larvae of the leek maggot were inoculated into each dish and reared in a light incubator with a temperature of 24±1℃, humidity of 75-85%, and a photoperiod of light:dark = 14:10. The mortality of the leek maggots was investigated and recorded 72 hours after inoculation, and the mortality rate of leek maggots under different bacterial concentrations was determined.

[0035] Based on the above experimental results, the LC50 of Cry53-like against second-instar leek maggots was calculated using POLO Plus software. 50 It was 64.86 μg / mL.

[0036] In summary, this invention has successfully extracted a novel insecticidal protein from Bacillus thuringiensis, which has a superior killing effect on the leek leeks venom flea and is expected to be used to control dipteran pests such as the leek leeks venom flea.

[0037] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A Cry53-like insecticidal protein, the amino acid sequence of which is shown in SEQ ID NO.

2.

2. The application of the Cry53-like insecticidal protein as described in claim 1 in the preparation of products for killing dipteran pests.

3. The application according to claim 2, characterized in that, The aforementioned dipteran pest is the leek-eyed fungus mosquito.

4. A biological pesticide, characterized in that, The Cry53-like insecticidal protein described in claim 1 is one of the active ingredients.

5. A nucleic acid molecule encoding the Cry53-like insecticidal protein of claim 1.

6. A recombinant vector containing the nucleic acid molecule of claim 5.

7. Recombinant bacteria containing the nucleic acid molecule of claim 5 or the recombinant vector of claim 6.

8. The use of the Cry53-like insecticidal protein as described in claim 1 or the nucleic acid molecule as described in claim 5 in the construction of insect-resistant transgenic plants.

9. A method for preparing the Cry53-like insecticidal protein of claim 1, characterized in that, Includes the following steps: The nucleic acid molecule encoding the Cry53-like insecticidal protein was inserted into the pET-Nus32T vector, and the resulting recombinant vector was transformed into Escherichia coli BL21(DE3) cells to express the recombinant Escherichia coli. The expression product was then purified to obtain the final product.

10. The method according to claim 9, characterized in that, The sequence of the nucleic acid molecule is shown in SEQ ID NO.1.

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