Novel insecticidal protein as well as coding gene and application thereof

Abstract

The invention discloses a novel insecticidal protein as well as a coding gene and application thereof, and belongs to the technical field of biological control. The invention provides a novel insecticidal protein Rknp1. The amino acid sequence of the novel insecticidal protein Rknp1 is as shown in SEQ ID NO. 1. A biological activity experiment result shows that the insecticidal protein Rknp1 provided by the invention can be used for efficiently preventing and treating pests such as nematodes, culex and the like, the killing effect of the object ear bean root-knot nematode is particularly remarkable, and LC50 is 109.87 mu g / mL; the killing rate of 300 [mu] g / mL insecticidal protein on nematodes and culex is 90% or above. The insecticidal protein Rknp1 provided by the invention can be used for prevention and control of pests, provides a new gene resource for biological prevention and control of pests such as plant nematodes and cultivation of pest-resistant transgenic plants, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control, and particularly to a novel insecticidal protein, its coding gene, and applications thereof. Background Art

[0002] Plant root-knot nematodes (Meloidogyne) are one of the important plant-parasitic nematodes in global agriculture. Due to their wide host range, ability to damage various crops, high temperature tolerance, strong pathogenicity, etc., they have attracted wide attention. These nematodes can infect a variety of cash crops, including vegetables, fruits, etc., and can even overcome some crops carrying nematode-resistant genes (such as tomatoes with the Mi resistance gene and peppers with the Me resistance gene), causing huge economic losses to agricultural production.

[0003] The harms of root-knot nematodes are mainly manifested in the following aspects: 1. Parasitic effect: During the parasitic stage, nematodes can cause root nodules to form on host plants, seriously affecting the normal functions of plant roots. 2. Affecting plant growth: The parasitism of root-knot nematodes can trigger abnormal root proliferation, thus significantly reducing the plant's ability to absorb water and nutrients. 3. Wide spread: Root-knot nematodes are spread through the soil. After their eggs are released on plant roots, they pose a potential threat to subsequent planted crops.

[0004] Currently, chemical agents are still the main means for controlling root-knot nematodes. However, with the gradual ban on highly toxic chemical agents (such as ethoprophos, carbofuran, etc.), the current chemical agents mainly include spirotetramat, fosthiazate, and fluopyram. However, the use of chemical agents is often accompanied by problems such as environmental pollution and increased drug resistance. For Meloidogyne enterolobii, there is currently no highly effective insecticidal protein applied to biological control or the development of transgenic plants. Therefore, the development of highly virulent insecticidal genes can not only enrich the insecticidal gene resources but also provide new gene sources for transgenic crops and engineering strains. Summary of the Invention

[0005] The object of the present invention is to provide a novel insecticidal protein, its coding gene, and applications thereof to solve the problems existing in the above-mentioned prior art. The insecticidal protein Rknp1 provided by the present invention can effectively control pests such as root-knot nematodes and Culex mosquitoes, with a killing rate of over 90%. The present invention reports for the first time that the insecticidal protein has a particularly significant killing effect on Meloidogyne enterolobii, and the LC 50 is 109.87 μg / mL.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an insecticidal protein, and the amino acid sequence of the insecticidal protein is as shown in SEQ ID NO.1.

[0008] The present invention provides a coding gene for the insecticidal protein, and the nucleotide sequence of the coding gene is as shown in SEQ ID NO.2.

[0009] The present invention provides a recombinant vector containing the coding gene.

[0010] Preferably, the recombinant vector is the pET30a(+) vector into which the coding gene has been introduced.

[0011] The present invention provides an engineered bacterium containing the recombinant vector.

[0012] Preferably, the starting bacterium of the engineered bacterium is Bacillus thuringirnsis or Escherichia coli.

[0013] The present invention also provides an application of the insecticidal protein, the coding gene, the recombinant vector or the engineered bacterium in the preparation of a product for controlling pests.

[0014] Preferably, the pest is Meloidogyne enterolobii or Culex quinquefasciatus.

[0015] The present invention also provides a product for controlling pests, and the only active ingredient of the product is the insecticidal protein.

[0016] Preferably, in the product, the concentration of the insecticidal protein is not less than 200 μg / mL.

[0017] The present invention also provides an application of the insecticidal protein or the product in controlling pests.

[0018] Preferably, the pest is Meloidogyne enterolobii or Culex quinquefasciatus.

[0019] The present invention discloses the following technical effects:

[0020] The present invention provides a novel insecticidal protein Rknp1. The results of bioactivity experiments show that the insecticidal protein Rknp1 provided by the present invention can effectively control pests such as nematodes and mosquitoes, and has a particularly significant killing effect on Meloidogyne enterolobii, with an LC 50 of 109.87 μg / mL; the killing rate of the insecticidal protein at 300 μg / mL against nematodes and mosquitoes is more than 90%. The insecticidal protein Rknp1 provided by the present invention can be used for pest control, provides a new gene resource for the biological control of plant nematodes and other pests and the cultivation of pest-resistant transgenic plants, and has broad application prospects. Description of the Drawings

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0022] Figure 1 It is the SDS-PAGE electrophoresis result diagram of the protein expressed by the Rknp1 gene in Escherichia coli BL21(DE3); wherein: the M lane is Marker, the lane 1 is the pET-30a(+) empty vector (negative control), the lane 2 is the pET30a(+)-Rknp1 without induction (positive control), the lane 3 is the pET30a(+)-Rknp1 after induction with IPTG, the lane 4 is the flow-through liquid after passing through the column, and the lane 5 is the purified Rknp1 protein;

[0023] Figure 2 It is the SDS-PAGE electrophoresis result diagram of the Rknp1 protein in the desalted protein solution; wherein, the M lane is Marker, and the lane 1 is the Rknp1 protein in the desalted protein solution. Detailed implementation manners

[0024] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0027] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the specification of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0028] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0029] The reagents used in the embodiments of the present invention are all purchased through commercial channels.

[0030] Preparation method of liquid LB medium: Dissolve 10 g of tryptone, 10 g of NaCl, and 10 g of yeast extract in 1 L of distilled water, adjust the pH to 7.0, and autoclave at 121 °C for 20 min.

[0031] Other reagents include: Kanamycin, concentration 50 mg / mL; Isopropyl-β-D-thiogalactoside (IPTG), concentration 50 mg / mL.

[0032] Example 1

[0033] 1. Gene cloning and protein induction expression

[0034] The coding gene of the insecticidal protein Rknp1 from Bacillus thuringiensis was cloned into the pET30a(+) vector and transformed into Escherichia coli BL21(DE3) to obtain the transformed strain pET30a(+)-Rknp1. The transformed strain BL21(DE3)-pET30a(+) obtained by transforming Escherichia coli BL21(DE3) with the pET30a(+) empty vector was used as the negative control for protein expression analysis; the transformed strain pET30a(+)-Rknp1 ( Figure 1 in lane 2) obtained by transforming Escherichia coli BL21(DE3) with the pET30a(+)-Rknp1 vector was used as the control for protein expression analysis.

[0035] The amino acid sequence of the insecticidal protein Rknp1 is shown in SEQ ID NO.1, with a length of 382 aa. The nucleotide sequence of the Rknp1 coding gene is shown in SEQ ID NO.2, with a length of 1149 bp.

[0036] SEQ ID NO.1:

[0037] LPYIGIIFLKLTIFKGVEKYMEIRNDTYYKIVNMNSGLVADVKAKSTSNSALLEQLAWQNETFQQWLVFQLDGELYAFTNRNSGLIMDVKSKSTSNSADLEQLDWYGETFQQWMISETDTEGYYKITNQNSGLVMDVKGKSTSNSALLEQLDWYNESFQKWAFEPLDSITLPSVETQPLPNVPQYTSADENTLPDQTDPVITNYTLMPCIMVTDNNWSASNKITNTPYYMLIKTQYWKKIDSHTFAPNTKYTSTTEYGMNQTDQESMTETTSISVTADAGFSFGKMSSSISTTVTNELQVSKSTTTELMTDVTTTEEISNPYTYEIKWTKYALVTQYDLKRADGTSVTASWTVIDKNEEQESYYPLDSPPEVMASKSEEGVK。

[0038] SEQ ID NO.2:

[0039]

[0040] Experimental procedures:

[0041] (1) Activation of bacterial liquid

[0042] Pick a single colony of E. coli BL21(DE3) and inoculate it into 10 mL of LB liquid medium (added with kanamycin at a final concentration of 100 μg / mL). Culture it at 37 °C and 220 rpm for 12 h to obtain the activated bacterial liquid.

[0043] (2) Induced expression

[0044] Inoculate the activated bacterial liquid into 1 L of LB liquid medium (also added with kanamycin at a final concentration of 100 μg / mL) at an inoculation amount of 1%. Culture it at 37 °C and 220 rpm for about 4 h. When the OD 600 reaches 0.8, add IPTG at a final concentration of 1 mM. Continue to culture it at 25 °C and 180 rpm for 16 h to obtain the induced fermentation broth.

[0045] (3) Centrifugation and collection

[0046] Place the fermentation broth at 4 °C and centrifuge it at 8000 rpm for 10 min. Discard the supernatant.

[0047] (4) Resuspension and disruption

[0048] Resuspend the precipitate with 50 mL of NaCl-Tris-HCl buffer (pH 8.0, 50 mM Tris-HCl, 500 mM NaCl, 10% glycerol), and transfer it to a 100 mL beaker. Disrupt the cells by ultrasonic wave (power 300 W, ultrasonic working for 5 s, interval 5 s, total time 10 min) to obtain the disrupted bacterial liquid.

[0049] (5) Centrifugal separation

[0050] Place the disrupted bacterial liquid at 4 °C and centrifuge it at 13000 g for 10 min. The centrifugation product is divided into a soluble fraction (supernatant) and an insoluble fraction (precipitate). The precipitate is further resuspended with NaCl-Tris-HCl buffer.

[0051] (6) SDS-PAGE protein detection

[0052] Perform SDS-PAGE electrophoresis analysis on the supernatant and the resuspended precipitate. The results are as Figure 1 shown. The insecticidal protein Rknp1 mainly exists in the soluble fraction (supernatant).

[0053] 2. Protein purification

[0054] 2.1 Preparation of nickel affinity chromatography column

[0055] 1) Wash the empty column with 20% ethanol, shake well the nickel filler Ni-NTAAgarose (QIAGEN), and take 1 mL to load into the affinity chromatography column (12 mL).

[0056] 2) Wash the chromatography column with 5 column volumes of ultrapure water.

[0057] 3) Equilibrate the column filler with 5 column volumes of binding buffer

[0058] The preparation method of the binding buffer is as follows: Dissolve 0.7 g of imidazole (final concentration 20 mM), 29.2 g of NaCl (final concentration 0.5 M), and 20 mL of 1 M Tris-HCl (pH = 8.0) in ultrapure water and make up the volume to 1 L.

[0059] 2.2 Passing the protein supernatant through the column

[0060] 1) Pretreat the bacterial solution after ultrasonic disruption: Centrifuge at 4 °C and 13000 g for 20 min, and transfer the supernatant to a new 50 mL centrifuge tube.

[0061] 2) Pass the supernatant through the column 3 times in sequence to allow the protein to bind fully to the nickel filler.

[0062] 3) Wash the column with 5 column volumes of gradient binding buffer (20 mM and 50 mM imidazole buffer, the preparation method is the same as that of the binding buffer) to remove the miscellaneous proteins.

[0063] 4) Elute the target protein with 3 column volumes of elution buffer (containing 17.0 g of imidazole, final concentration 250 mM; 29.2 g of NaCl, final concentration 0.5 M; 20 mL of 1 M Tris-HCl, pH = 8.0; made up to 1 L with ultrapure water), and repeat the elution 2 times. Collect the eluted protein solution containing imidazole and analyze the protein in the eluate by SDS-PAGE.

[0064] 5) Further elute the non-specifically bound miscellaneous proteins with 5 column volumes of elution buffer (containing 34.0 g of imidazole, final concentration 500 mM; 29.2 g of NaCl, final concentration 0.5 M; 20 ml of 1 M Tris-HCl, pH = 8.0; made up to 1 L with ultrapure water) to ensure that the nickel column is free from contamination by miscellaneous proteins and can be reused.

[0065] 6) Wash the chromatography column with 5 column volumes of ultrapure water, then wash it 3 times with 20% ethanol, and place the chromatography column in 20% ethanol for long-term storage at 4 °C.

[0066] 2.3 Desalting and purification of the protein

[0067] Desalt and purify the protein solution containing imidazole by dialysis treatment. The treatment steps of the dialysis bag are as follows:

[0068] 1) Cut the dialysis bag into small segments of 10 - 20 cm.

[0069] 2) Boil the dialysis bag in a large volume of 2% (w / v) sodium bicarbonate and 1 mM EDTA (pH = 8.0) solution for 10 min.

[0070] 3) Thoroughly wash the dialysis bag with distilled water.

[0071] 4) Boil the dialysis bag in 1 mM EDTA (pH = 8.0) solution again for 10 min.

[0072] 5) After cooling, store the dialysis bag at 4 °C, ensuring it is always immersed in 30% ethanol solution. Wear clean gloves when using the dialysis bag.

[0073] 6) Before use, fill the dialysis bag with water, squeeze out the water, and then wash it clean.

[0074] Load 30 mL of protein solution into the treated dialysis bag (about 15 cm long), place it in a beaker containing 3 L of NaCl-Tris-HCl buffer (pH = 8.0), and dialyze at 4 °C for 24 h, changing the buffer every 12 h. After dialysis is completed, collect the desalted protein solution and analyze the Rknp1 protein in the desalted solution by SDS-PAGE. The results are as Figure 2 shown.

[0075] 3. Protein quantification

[0076] Use a BCA protein concentration assay kit (Beyotime) to measure the protein concentration in the desalted protein solution by the microplate reader method. The specific steps are as follows:

[0077] (1) Prepare the standard solution

[0078] Dilute the BSA protein standard solution (25 mg / mL) with PBS buffer. Take an appropriate amount of the BSA standard solution and dilute it to a final concentration of 0.5 mg / mL. Aliquot and store it at -20 °C for long-term preservation.

[0079] (2) Configure the standard curve

[0080] Add the diluted BSA standards to different wells of a 96-well plate at 0, 1, 2, 4, 8, 12, 16, 20 μL respectively; add the standard diluent to each well to make the total volume up to 20 μL, keeping the total volume consistent.

[0081] (3) Add the sample

[0082] Add an appropriate volume of the sample to the sample wells of a 96-well plate; if the sample volume is less than 20 μL, make it up to 20 μL with the standard diluent.

[0083] (4) Add BCA working solution

[0084] Add 200 μL of BCA working solution to each well; incubate at 37 °C for 20 - 30 min to ensure sufficient reaction.

[0085] (5) Measure absorbance

[0086] Use a microplate reader to measure the absorbance (A 562 ) at a wavelength of 562 nm for each well.

[0087] (6) Calculate protein concentration

[0088] Based on the linear regression equation obtained from the BSA standard curve, as well as the measured absorbance and volume of the sample, calculate the protein concentration in the sample.

[0089] 4. Bioactivity assay

[0090] In the present invention, Meloidogyne enterolobii and Culex quinquefasciatus are used as target pests to verify the insecticidal effect of the protein.

[0091] 4.1 Bioactivity assay of root-knot nematodes

[0092] (1) Propagation and collection of root-knot nematodes

[0093] Inoculate second-stage juveniles (J2) onto the roots of water spinach planted in sterile soil for nematode propagation and preservation. Select the roots of severely diseased water spinach, wash the roots, and pick nematode egg masses with forceps. Immerse the egg masses in 0.05% NaClO solution for 5 min for disinfection, then rinse with sterile water to remove residual NaClO, and repeat this operation 3 times. Place the disinfected nematode egg masses in a 6-well plate, and add an appropriate amount of sterile water to each well. Incubate the 6-well plate in an incubator at 28 °C for 3 days to obtain a large number of highly active nematodes.

[0094] (2) Activity detection by 96-well plate method

[0095] Add 30 second-stage juveniles (J2) to each well, and add Rknp1 protein solution with a final concentration of 300 μg / mL; set 3 replicates for each treatment. Set up a blank control group (add sterile water) and a negative control group (add an equal volume of 50 mM Tris-HCl buffer). Incubate the 96-well plate in an incubator at 28 °C for 72 h, and observe and count the nematode death situation using a stereomicroscope every 24 h. Calculate the corrected mortality rate according to the following formula.

[0096] Mortality rate (%) = 100 × number of dead nematodes / total number of nematodes;

[0097] Corrected mortality rate (%) = 100×(mortality rate of treatment group - mortality rate of blank control group) / (1 - mortality rate of blank control group).

[0098] 4.2 Bioactivity determination of *Culex pipiens quinquefasciatus*

[0099] Activity detection by 6-well plate method: Add 10 second-instar mosquito larvae to each well, and add Rknp1 protein solution with a final concentration of 300 μg / mL; set 3 replicates for each treatment. Set up a blank control group (add sterile water) and a negative control group (add an equal volume of 50 mM Tris-HCl buffer). Place the 6-well plate in an incubator at 28 °C for 72 h, and record the death of mosquito larvae. Calculate the corrected mortality rate according to the following formula.

[0100] Mortality rate (%) = 100×number of dead mosquito larvae / total number of mosquito larvae;

[0101] Corrected mortality rate (%) = 100×(mortality rate of treatment group - mortality rate of blank control group) / (1 - mortality rate of blank control group).

[0102] 4.3 Experimental results

[0103] The experiment lasted for 3 days. By recording the number of dead and surviving individuals, the mortality rate, survival rate and corrected mortality rate were calculated. The specific results are shown in Table 1.

[0104] Table 1 Bioactivity determination of Rknp1 expressed protein against target pests

[0105]

[0106] Note: a indicates no significant difference in mortality rate between the negative control group and the blank control group in the same column (P>0.05).

[0107] Then, use IBM SPSS Statistics 27 software for virulence regression analysis and calculate LC 50 value. The results are shown in Table 2.

[0108] Table 2 LC 50 Calculation

[0109]

[0110] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. An insecticidal protein, characterized in that, The amino acid sequence of the insecticidal protein is shown in SEQ ID NO.

1.

2. The coding gene of the insecticidal protein as described in claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

3. A recombinant vector comprising the encoding gene according to claim 2.

4. An engineered bacterium comprising the recombinant vector according to claim 3.

5. Use of the insecticidal protein according to claim 1, or the encoding gene according to claim 2, or the recombinant vector according to claim 3, or the engineered bacterium according to claim 4 in the preparation of a product for controlling pests.

6. The application according to claim 5, characterized in that The pests are Meloidogyne incognita, Meloidogyne enterolobii or Culex quinquefasciatus.

7. A product for controlling pests, characterized in that, The product has the insecticidal protein according to claim 1 as the sole active ingredient.

8. The product according to claim 7, wherein In the product, the concentration of the insecticidal protein is not less than 200 μg / mL.

9. Use of the insecticidal protein according to claim 1, or the product according to claim 7 or 8 in controlling pests.

10. The application according to claim 9, wherein The pests are Meloidogyne incognita, Meloidogyne enterolobii or Culex quinquefasciatus.

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