Short peptide simulating cry1ac protein anti-insect function epitope and application thereof

Abstract

The present application relates to the technical field of pest control, in particular to a short peptide simulating a Cry1Ac protein anti-insect function epitope and application thereof. The present application takes a cotton bollworm middle intestine cadherin fragment (HaCad-TBR) as a target, and carries out screening on a phage display random 6-peptide library constructed by the laboratory, to obtain a 6-peptide LTETGK capable of combining with the cotton bollworm middle intestine cadherin, i.e. the 6-peptide has the effect of simulating a key anti-insect function epitope of the Cry1Ac protein, and a recombinant insecticidal protein C8CL2 having good insecticidal activity on the cotton bollworm can be obtained by replacing Loop2 of Domain II in the Cry1Ac. The present application expands the diversity of anti-insect materials of the Cry1Ac protein, and provides a potential alternative new material for preventing and controlling target pests, and can be used for the prevention and control of Lepidoptera pests, and provides technical support for the development of Lepidoptera pest control products.

Description

Technical Field

[0001] This invention relates to the field of pest control technology, and in particular to a short peptide that mimics the insect-resistant functional epitope of the Cry1Ac protein and its application. Background Technology

[0002] Cry1Ac protein is a protein produced by Bacillus thuringiensis (Bacillus thuringiensis). Bacillus thuringiensis Insecticidal crystal proteins (ICPs) produced by transgenic insecticides (Bt) belong to the Cry family and exhibit specific toxicity to various insects (such as Lepidoptera and Coleoptera), and are widely used in the research and development of biopesticides and insect-resistant crops. However, with the long-term and widespread application of crops transgenic with Cry1Ac protein, issues such as accelerated pest resistance and potential ecological safety risks have been a hot topic of scientific research. Exploring novel safe bio-derived insect-resistant materials, especially expanding the Cry1Ac protein family and even exploring bioactive materials that can replace the insect-resistant function of Cry1Ac protein, has become a research hotspot in the field of green biopesticides. Summary of the Invention

[0003] This invention provides a short peptide that mimics the insect-resistant functional epitope of the Cry1Ac protein and its application. The short peptide provided by this invention mimics the key insect-resistant functional epitope of the Cry1Ac protein, expanding the diversity of insect-resistant materials containing the Cry1Ac protein. It provides a potential new alternative material for controlling target pests and can be used for the control of lepidopteran pests, providing technical support for the development of lepidopteran pest control products.

[0004] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a short peptide that mimics the insect-resistant functional epitope of the Cry1Ac protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0005] Preferably, the nucleotide sequence of the gene encoding the short peptide is shown in SEQ ID NO.2.

[0006] This invention provides a recombinant Cry1Ac protein, in which the Loop2 region of Domain II in the Cry1Ac protein is replaced with the short peptide described in the above technical solution.

[0007] Preferably, the amino acid sequence of the recombinant is shown in SEQ ID NO.5.

[0008] Preferably, the nucleotide sequence of the coding gene of the recombinant is shown in SEQ ID NO.6.

[0009] This invention provides the application of the short peptides or recombinants described in the above-mentioned technical solutions in the control of lepidopteran pests.

[0010] Preferably, the lepidopteran pests include the cotton bollworm.

[0011] This invention provides the application of the short peptide or recombinant protein described in the above-mentioned technical solution in the preparation of insecticides for lepidopteran pests.

[0012] Preferably, the lepidopteran pests include the cotton bollworm.

[0013] This invention provides an insecticide whose active ingredient includes the recombinant strain described in the above-mentioned technical solution.

[0014] Beneficial effects: This invention provides a short peptide that mimics the insecticidal epitope of the Cry1Ac protein, with the amino acid sequence shown in SEQ ID NO. 1. Targeting the midgut cadherin fragment (HaCad-TBR) of the cotton bollworm, this invention screened a phage display random 6-peptide library constructed in our laboratory, obtaining the 6-peptide LTETGK, which can bind to the midgut cadherin of the cotton bollworm. This 6-peptide mimics the key insecticidal epitope of the Cry1Ac protein. Replacing Loop2 of Domain II in Cry1Ac with this peptide yields the recombinant insecticidal protein C8CL2, which exhibits good insecticidal activity against cotton bollworm. This invention expands the diversity of insecticidal materials containing the Cry1Ac protein and provides a potential alternative material for controlling pesticide resistance in target pests. It can be used for the control of lepidopteran pests, providing technical support for the development of lepidopteran pest control products. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0016] Figure 1 This refers to the ELISA data on the binding of polyclonal phages during the screening process in Example 1; Figure 2 This includes binding ELISA data and competitive ELISA identification data of some monoclonal phages from Example 1; Figure 3 This refers to the data from the indoor bollworm activity assay of the recombinant insect-resistant protein in Example 2. Different letters indicate that there are significant differences between different groups. Detailed Implementation

[0017] This invention provides a short peptide that mimics the insect-resistant functional epitope of the Cry1Ac protein, the amino acid sequence of which is shown in SEQ ID NO. 1. As one embodiment, the nucleotide sequence of the gene encoding the short peptide is shown in SEQ ID NO. 2.

[0018] This invention targets the midgut cadherin fragment (HaCad-TBR) of the cotton bollworm, screening a phage-displayed random 6-peptide library constructed in our laboratory. The resulting 6-peptide, LTETGK, binds to the midgut cadherin of the cotton bollworm. This 6-peptide mimics a key insecticidal epitope of the Cry1Ac protein. Replacing Loop2 of Domain II in Cry1Ac with this 6-peptide yields the recombinant insecticidal protein C8CL2, which exhibits good insecticidal activity against cotton bollworm. This invention expands the diversity of insecticidal materials containing Cry1Ac protein and provides a potential alternative material for controlling pesticide resistance in target pests. It can be used for the control of lepidopteran pests, providing technical support for the development of lepidopteran pest control products. The 6-peptide provided by this invention can also prevent target pests from developing resistance to Cry1Ac protein and its mimics. Furthermore, this 6-peptide is expected to be used directly as a synergist to enhance the insecticidal activity and insecticidal spectrum of Cry1Ac protein and its mimics.

[0019] Based on the above advantages, the present invention provides a recombinant Cry1Ac protein, in which the Loop2 region of Domain II in the Cry1Ac protein is replaced with the short peptide described in the above technical solution.

[0020] As one embodiment, the amino acid sequence of the recombinant is shown in SEQ ID NO.5.

[0021] As one embodiment, the nucleotide sequence of the coding gene of the recombinant is shown in SEQ ID NO.6.

[0022] Based on the above advantages, this invention provides the application of the short peptide or recombinant protein described in the above technical solution in the control of lepidopteran pests. As one embodiment, the lepidopteran pest includes the cotton bollworm.

[0023] Based on the above advantages, this invention provides the application of the short peptide or recombinant protein described in the above technical solution in the preparation of insecticides for lepidopteran pests. As one embodiment, the lepidopteran pest includes the cotton bollworm.

[0024] Based on the above advantages, the present invention provides an insecticide whose active ingredient includes the recombinant strain described in the above technical solution. As one embodiment, the insecticide further includes pharmaceutically acceptable excipients.

[0025] To further illustrate the present invention, the following detailed description, in conjunction with embodiments and accompanying drawings, describes a short peptide that mimics the insect-resistant functional epitope of the Cry1Ac protein and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1 Using HaCad-TBR as the target, a phage display random 6-peptide library constructed in the laboratory was screened. The phage display random 6-peptide library and its construction method are disclosed in Chinese Patent CN113912684A. The screening process is as follows: a. The 6-well plate was coated overnight at 4°C with 1 mL of 200 μg / mL HaCad-TBR. The next day, it was washed three times with 5 mL PBS (0.2 g KCl, 8 g NaCl, 3.6 g Na2HPO4 and 0.24 g KH2PO4 dissolved in 1 L sterile water, pH 7.4). The expression method of HaCad-TBR can be found in the literature [Gao, M., Dong, S., Hu, X., Zhang, X., Liu, Y., Zhong, J., Lu, L., Wang, Y., Chen, L., & Liu, X. (2019). Roles of Midgut Cadherin from Two Moths in Different Moths]. Bacillus thuringiensis Action Mechanisms:Correlation among Toxin Binding, Cellular Toxicity, and Synergism. Journal of agricultural and food chemistry , 67 (48), 13237–13246. https: / / doi.org / 10.1021 / acs.jafc.9b04563; b. Add 4 mL of blocking buffer 5% MPBS (PBS + 5% [w / v] skim milk powder, purchased from Solarbio [Shanghai] Co., Ltd.), and incubate at 37℃ for blocking. After 2 h, wash 3 times with 5 mL PBS. c. Take 200 μL of a titer of 2 × 10⁻⁶. 11 A CFU / mL phage display random 6-peptide library was mixed with 800 μL of 5% MPBS and added to the well. The mixture was then slowly shaken at 150 rpm for 1 h at 25 °C and allowed to stand at 25 °C for 1 h. d. The well was washed 7 times with 4 mL PBST and 3 times with PBS. Then, 1 mL of 500 μg / mL Cry1Ac protein (purchased from Meiyan [Beijing] Agricultural Technology Co., Ltd.) was added and the mixture was slowly shaken at 100 rpm for 1 h at 25℃. The eluent at this time is the first round of screening product.

[0027] e. The products from each round of screening were amplified and used in the next round of screening. The methods for the second and third rounds of screening were similar to the first round. In the second round, the HaCad-TBR coating concentration was 100 μg / mL, and the number of PBST washes was increased to 12. In the third round, the HaCad-TBR coating concentration was 50 μg / mL, and the number of PBST washes was increased to 20. The polyclonal phage binding ELISA data during the three rounds of screening are shown below. Figure 1 .

[0028] Example 2 The enriched products from the third round of screening in Example 1 were identified by monoclonal ELISA. The identification process is as follows: Add 100 μL of 10 μg / mL HaCad-TBR to each well of a 96-well plate and incubate overnight at 4°C. After washing three times with 300 μL PBST, add 250 μL of 5% MPBS to each well and incubate at 37°C for 1.5 h. After washing three times with 300 μL PBST, add 100 μL of monoclonal rescue supernatant to each well and incubate at 37°C for 1 h. After washing three times with 300 μL PBST, add 100 μL (1:5000 dilution) of anti-M13-HRP (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.) to each well and incubate at 37°C for 40 min. After washing five times with 300 μL PBST, add TMB single-component chromogenic solution to each well and incubate at 37°C for 10 min. Stop the chromogenic process by adding 50 μL of 2 M H2SO4 to each well and measure the OD. 450 Each monoclonal antibody was used as a negative control by coating wells with 5% MPBS. OD values ​​from the HaCad-TBR-coated group were selected. 450 With the corresponding negative OD 450 Monoclonal clones with a ratio greater than 2.5 are considered positive clones and proceeded to the next step of competitive ELISA identification.

[0029] Positive clones were then subjected to competitive ELISA detection. The procedure was similar to that of monoclonal ELISA identification, but 100 μL of monoclonal rescue supernatant was added to each well, along with HaCad-TBR containing 100 μg / mL of Cry1Ac toxin, which was also coated with the same solution. 100 μL of monoclonal rescue supernatant was added to each well as a control.

[0030] like Figure 2As shown, competitive ELISA identification revealed that Cry1Ac toxin can competitively bind to HaCad-TBR with a 6-peptide named C8. Figure 2 Meanwhile, a 6-peptide named B5, which did not exhibit competitive activity, was provided as a control; the amino acid sequence of C8 is shown in SEQ ID NO.1, and the corresponding nucleotide sequence is shown in SEQ ID NO.2; the amino acid sequence of B5 is shown in SEQ ID NO.3, and the corresponding nucleotide sequence is shown in SEQ ID NO.4, as detailed below: SEQ ID NO.1: LTETGK; SEQ ID NO.2: 5'-CTGACGGAGACTGGTAAG-3'; SEQ ID NO.3: QMKPND; SEQ ID NO. 4: 5'-CAGATGAAGCCGAATGAT-3'.

[0031] Example 3: Insecticidal Experiment with Recombinant Protein Replacing C8 with Loop 2 of Domain II in Cry1Ac in Example 2 yielded the C8CL2 recombinant protein, and replacing B5 with Loop 2 of Domain II in Cry1Ac yielded the B5CL2 recombinant protein. Following the method disclosed in Example 2 of Chinese Patent CN113912684A, purified C8CL2 and B5CL2 recombinant proteins were prepared by General Biosystems (Anhui) Co., Ltd. The amino acid sequence of the C8CL2 recombinant protein is shown in SEQ ID NO.5, and the coding sequence is shown in SEQ ID NO.6; the amino acid sequence of the B5CL2 recombinant protein is shown in SEQ ID NO.7, and the coding sequence is shown in SEQ ID NO.8, as detailed below: SEQ ID NO.5: MDNNPNINECIPYNCLSNPEVEVLGGERIETGYTPIDISLSLTQFLLSEFVPGAGFVLGLVDIIWGIFGPSQWDAFLVQIEQLINQRIEEFARNQAISRLEGLSNLYQIYAESFREWEADPTNPALREEMRIQFNDMNSALTTAIPLFAVQNYQVPLLSVYVQAANLHLSVLRDVSVFGQRWGFDAATINSRYNDLTRLIGNYTDYAVRWYNTGLERVWGPDSRDWVRYNQFRRELTLTVLDIVALFPNYDSRRYPIRTVSQLTREIYTNPVLENFDGSFRGSAQGIERSIRSPHLMDILNSITIYTDAHRGYYYWSGHQIMASPVGFSGPEFTFPLYGTMGNAAPQQRIVAQLGQGVYRTLSSTLYRRPLTETGKNQQLSVLDGTEFAYGTSSNLPSAVYRKSGTVDSLDEIPPQNNNVPPRQGFSHRLSHVSMFRSGFSNSSVSIIRAPMFSWIHRSAEFNNIIASDSITQIPAVKGNFLFNGSVISGPGFTGGDLVRLNSSGNNIQNRGYIEVPIHFPSTSTRYRVRVRYASVTPIHLNVNWGNSSIFSNTVPATATSLDNLQSSDFGYFESANAFTSSLGNIVGVRNFSGTAGVIIDRFEFIPVTLE; SEQ ID NO.6: SEQ ID NO.7: MDNNPNINECIPYNCLSNPEVEVLGGERIETGYTPIDISLSLTQFLLSEFVPGAGFVLGLVDIIWGIFGPSQWDAFLVQIEQLINQRIEEFARNQAISRLEGLSNLYQIYAESFREWEADPTNPALREEMRIQFNDMNSALTTAIPLFAVQNYQVPLLSVYVQAANLHLSVLRDVSVFGQRWGFDAATINSRYNDLTRLIGNYTDYAVRWYNTGLERVWGPDSRDWVRYNQFRRELTLTVLDIVALFPNYDSRRYPIRTVSQLTREIYTNPVLENFDGSFRGSAQGIERSIRSPHLMDILNSITIYTDAHRGYYYWSGHQIMASPVGFSGPEFTFPLYGTMGNAAPQQRIVAQLGQGVYRTLSSTLYRRPQMKPNDNQQLSVLDGTEFAYGTSSNLPSAVYRKSGTVDSLDEIPPQNNNVPPRQGFSHRLSHVSMFRSGFSNSSVSIIRAPMFSWIHRSAEFNNIIASDSITQIPAVKGNFLFNGSVISGPGFTGGDLVRLNSSGNNIQNRGYIEVPIHFPSTSTRYRVRVRYASVTPIHLNVNWGNSSIFSNTVPATATSLDNLQSSDFGYFESANAFTSSLGNIVGVRNFSGTAGVIIDRFEFIPVTLE; SEQ ID NO.8:

[0032] Artificial feed was spread on 24-well plates. Purified C8CL2 and B5CL2 recombinant proteins were diluted to 20 μg / mL with PBS, and 200 μL of recombinant protein was spread on the surface of the feed in each well. After drying, one second-instar cotton bollworm was inoculated into each well. Cry1Ac protein served as a positive control, and B5CL2 recombinant protein served as a negative control and a blank control. The plates were incubated at 28℃±1℃, 80±5% relative humidity, and a photoperiod (L:D) of 16 h:8 h. Mortality was observed and recorded after 5 days. Each treatment was inoculated with 72 larvae, and the experiment was repeated three times. The mortality rate was calculated after 5 days. The results are shown below. Figure 3 As shown.

[0033] The results showed that at a concentration of 20 μg / mL, the lethality rates of Cry1Ac, C8CL2, B5CL2 and PBS against cotton bollworms at 5 days were 91.67±1.96%, 69.44±1.96%, 2.77±1.96% and 0±1.31%, respectively.

[0034] The above examples demonstrate that the recombinant protein obtained by replacing Loop2 with the 6-peptide LTETGK can be used for the control of cotton bollworm, providing technical support for the development of control products.

[0035] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A short peptide mimicking the insect-resistant functional epitope of the Cry1Ac protein, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. The short peptide according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the short peptide is shown in SEQ ID NO.

2.

3. A recombinant form of Cry1Ac protein, characterized in that, Replace the Loop2 region of Domain II in the Cry1Ac protein with the short peptide described in claim 1 or 2.

4. The recombinant according to claim 3, characterized in that, The amino acid sequence of the recombinant is shown in SEQ ID NO.

5.

5. The recombinant according to claim 3 or 4, characterized in that, The nucleotide sequence of the coding gene of the recombinant is shown in SEQ ID NO.

6.

6. The use of the short peptide according to claim 1 or 2 or the recombinant according to any one of claims 3 to 5 in the control of lepidopteran pests.

7. The application according to claim 6, characterized in that, The lepidopteran pests include the cotton bollworm.

8. The use of the short peptide according to claim 1 or 2 or the recombinant according to any one of claims 3 to 5 in the preparation of insecticides for lepidopteran pests.

9. The application according to claim 8, characterized in that, The lepidopteran pests include the cotton bollworm.

10. An insecticide, characterized in that, The active ingredient includes the recombinant as described in any one of claims 3 to 5.

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