An insecticidal protein and its application in preventing and controlling Aedes albopictus

Abstract

The present invention relates to an insecticidal protein and its application in preventing and controlling Aedes albopictus. The amino acid sequence of the insecticidal protein is shown as SEQ ID No.2.

Description

Technical Field

[0001] The invention relates to the field of biological control, and in particular to application of a protein in controlling Aedes albopictus. Background Art

[0002] Aedes albopictus originated in Southeast Asia and has now spread to tropical, subtropical and temperate regions around the world. Aedes albopictus is widely distributed in China, from Hainan Island in the south to southern Liaoning in the north and Baoji in Shaanxi in the west. Aedes albopictus is an important vector of mosquito-borne infectious diseases such as dengue fever and is also the most invasive mosquito species in the world.

[0003] At present, the prevention and control of Aedes albopictus mainly adopts strategies such as comprehensive environmental management, chemical control, and biological control. However, the widespread and improper use of chemical pesticides has led to an increasingly serious problem of pesticide resistance and is prone to environmental pollution.

[0004] Biological control mainly relies on insect pathogens, such as Bacillus thuringiensis (Bt). However, with the continuous application of Bt mosquito killers, mosquitoes gradually develop resistance to them.

[0005] Therefore, it is necessary to find new proteins with insecticidal activity against Aedes albopictus. Summary of the invention

[0006] One of the present inventions provides a protein, whose amino acid sequence is shown as SEQ ID No.2.

[0007] The second aspect of the present invention provides a composition containing the protein as described in the first aspect of the present invention.

[0008] The third aspect of the present invention provides a nucleic acid encoding the protein as described in one of the present inventions.

[0009] In a specific embodiment, the base sequence of the nucleic acid is shown as SEQ ID No.1.

[0010] A fourth aspect of the present invention provides a microorganism, which carries the nucleic acid as described in claim 3 or 4 and can express the protein as described in claim 1.

[0011] In a specific embodiment, the microorganism is Escherichia coli and / or Bacillus thuringiensis.

[0012] The fifth aspect of the present invention provides the use of one of the protein according to the first aspect of the present invention, the composition according to the second aspect of the present invention, the nucleic acid according to the third aspect of the present invention, and the microorganism according to the fourth aspect of the present invention in controlling Aedes albopictus. Beneficial effects of the present invention:

[0013] The protein Anc0 of the present invention, whose amino acid is shown in SEQ ID No. 2, has insecticidal activity against Aedes albopictus, which lays a foundation for the development of a new generation of biological mosquito killers and enriches the gene resource library of mosquito-killing engineering microorganisms in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The results of SDS-PAGE detection of Anc0 are shown. DETAILED DESCRIPTION

[0015] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation to the present invention.

[0016] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.

[0017] Example 1

[0018] Expression of Anc0 protein

[0019] 1. Gene synthesis and protein expression

[0020] Based on the nucleotide sequence shown in SEQ ID No. 1, gene synthesis was performed by Sangon Biotech (Shanghai) Co., Ltd. Wherein, the nucleic acid sequence shown in SEQ ID No. 1 encodes the protein shown in SEQ ID No. 2 (ie, Anc0 protein).

[0021] The synthesized insecticidal protein gene was connected to the PUC18-p1Ac-GFP vector to obtain the p1Ac-GFP-Anc0 recombinant plasmid, which was transformed into Escherichia coli TOP10. The transformant was named TOP10 / p1Ac-GFP-Anc0, in which the promoter used was the Cry1Ac1 promoter.

[0022] The transformant TOP10 / PUC18-p1Ac-GFP obtained by transforming Escherichia coli TOP10 with empty vector PUC18-p1Ac-GFP was used as a negative control for protein expression analysis.

[0023] 2. Protein Extraction

[0024] (1) Picking a single colony: Pick a single colony of TOP10 / p1Ac-GFP-Anc0 and place it in 5 mL of LB liquid culture medium containing ampicillin, and culture it at 37°C and 220 rpm for 12 hours to obtain an activated bacterial solution;

[0025] (2) inoculating 1% of the activated bacterial solution into a 1 L Erlenmeyer flask containing 800 mL of LB liquid medium (containing ampicillin), and shaking culture at 37° C. and 220 rpm for 72 h to obtain a fermentation broth;

[0026] (3) centrifuging the fermentation broth at 8,000 × g at 4°C for 10 min, discarding the supernatant, and resuspending the precipitate in pre-cooled 20 mmol / L Tris-HCl (pH = 8.0) buffer to obtain a first bacterial suspension;

[0027] (4) The first bacterial suspension was transferred to a 50 mL centrifuge tube, centrifuged at 8,000 × g for 10 min at 4°C, the supernatant was discarded, and the precipitate was suspended in pre-cooled 20 mmol / L Tris-HCl (pH = 8.0) to prepare the second bacterial suspension;

[0028] (5) Ultrasonication is used to disrupt the cell walls of the second bacterial suspension (power 70%, 5 min, ultrasonication for 3 s, and rest for 5 s) to obtain a disrupted bacterial solution;

[0029] (6) Centrifuge the disrupted bacterial solution at 8,000 × g for 10 min at 4°C and collect the precipitate;

[0030] (7) Wash the precipitate 2 to 3 times with pre-cooled sterile water;

[0031] (8) Add 5 ml of 20 mmol / L Tris-HCl (pH = 8.0) buffer to resuspend the precipitate to obtain a protein suspension to be tested;

[0032] (9) Extract TOP10 / PUC18-p1Ac-GFP protein using the same procedures as above (1) to (8) to obtain a negative control protein suspension;

[0033] (10) The protein suspension to be tested and the negative control protein suspension were subjected to SDS-PAGE detection. The results are shown in Figure 1 .

[0034] according to Figure 1 The results show that Anc0 was successfully expressed in E. coli, and the expression product was in the precipitate after cell disruption. Therefore, the protein suspension to be tested is the protein suspension containing Anc0.

[0035] Prior to the activity assay, the protein suspension containing Anc0 was quantified with BSA.

[0036] Example 2

[0037] The Cry2A proteins discovered so far mainly include Cry2Aa, Cry2Ab, Cry2Ac, Cry2Ad, Cry2Ae, Cry2Af, Cry2Ag, Cry2Ah, Cry2Ai, Cry2Ak and Cry2Al.

[0038] The nucleic acid accession number of Cry2Aa is MK184464.1, and the amino acid accession number is AAA22335.1.

[0039] The nucleic acid accession number of Cry2Ab is MK184479.1, and the amino acid accession number is AAA22342.1.

[0040] The nucleic acid accession number of Cry2Ac is X57252.1, and the amino acid accession number is CAA40536.1.

[0041] The nucleic acid accession number of Cry2Ad is CP014853.1, and the amino acid accession number is AAF09583.1.

[0042] The nucleic acid accession number of Cry2Ae is EA286849.1, and the amino acid accession number is AAQ52362.1.

[0043] The nucleic acid accession number of Cry2Af is EF439818.1, and the amino acid accession number is ABO30519.1.

[0044] The nucleic acid accession number of Cry2Ag is FJ032364.1, and the amino acid accession number is ACH91610.1.

[0045] The nucleic acid accession number of Cry2Ah is FJ550343.1, and the amino acid accession number is ACL80665.1.

[0046] The nucleic acid accession number of Cry2Ai is FJ788388.1, and the amino acid accession number is ACV97158.1.

[0047] The nucleic acid accession number of Cry2Ak is FJ493542.1, and the amino acid accession number is AGU13825.1.

[0048] The nucleic acid accession number of Cry2A1 is KJ149819.1, and the amino acid accession number is AHH35153.1.

[0049] The nucleic acids encoding Cry2Aa, Cry2Ab, Cry2Ac, Cry2Ad, Cry2Ae, Cry2Af, Cry2Ag, Cry2Ah, Cry2Ai, Cry2Ak and Cry2A1 proteins were connected to the PUC18-p1Ac-GFP vector in the same manner as in Example 1 to obtain the p1Ac-GFP-Cry2Aa recombinant plasmid, p1Ac-GFP-Cry2Ab recombinant plasmid, p1Ac-GFP-Cry2Ac recombinant plasmid, p1Ac-GFP-Cry2Ad recombinant plasmid, p1Ac-GFP-Cry2Ae recombinant plasmid, p1Ac-GFP-Cry2Af recombinant plasmid, p1Ac-GFP-Cry2Ah recombinant plasmid, p1Ac-GFP-Cry2Ai recombinant plasmid and p1Ac-GFP-Cry2A1 recombinant plasmid. P-Cry2Ak recombinant plasmid and p1Ac-GFP-Cry2Al recombinant plasmid, and each recombinant plasmid was transformed into Escherichia coli TOP10, respectively, to obtain the recombinant strains TOP10 / p1Ac-GFP-Cry2Aa, TOP10 / p1Ac-GFP-Cry2Ab, TOP10 / p1Ac-GFP-Cry2Ac, TOP10 / p1Ac-GFP-Cry2Ad, TOP10 / p1Ac-GFP-Cry2Ae, TOP10 / p1Ac-GFP-Cry2Af, TOP10 / p1Ac-GFP-Cry2Ag, TOP10 / p1Ac-GFP-Cry2Ah, TOP10 / p1Ac-GFP-Cry2Ai, TOP10 / p1Ac-GFP-Cry2Ak and TOP10 / p1Ac-GFP-Cry2Al respectively.

[0050] The recombinant strain was expressed, proteins were extracted and the extracted proteins were tested for the presence of the above proteins in the same manner as in Example 1. The results showed that the above proteins were successfully expressed in E. coli and the expression products were in the precipitate after cell disruption.

[0051] Prior to the activity assay, the protein suspension containing the above proteins was quantified with BSA.

[0052] Example 3

[0053] The amino acid accession numbers of Cry2Aa, Cry2Ab, Cry2Ac, Cry2Ad, Cry2Ae, Cry2Af, Cry2Ag, Cry2Ah, Cry2Ai, Cry2Ak and Cry2A1 proteins are shown in Example 2.

[0054] BioEdit software was used to align the sequences of Anc0, Cry2Aa, Cry2Ab, Cry2Ac, Cry2Ad, Cry2Ae, Cry2Af, Cry2Ag, Cry2Ah, Cry2Ai, Cry2Ak and Cry2Al, and the results are shown in Table 1.

[0055] Table 1

[0056]

[0057] From the results in Table 1, it can be seen that the amino acid sequence identities of the 11 Cry2A proteins and the Anc0 protein range from 81.30% to 96.50%, among which the amino acid sequence identity of Cry2Aa and the Anc0 protein is the highest.

[0058] Example 4

[0059] Determination of mosquito-killing activity against Aedes albopictus

[0060] The water source used for the biological test of Aedes albopictus is dechlorinated tap water.

[0061] The Anc0 protein and 11 Cry2A protein suspensions were diluted to 1 μg / ml with dechlorinated tap water, and mixed thoroughly to obtain the protein samples to be tested. The volume of 20 mmol / L Tris-HCl (pH=8.0) contained in the protein samples to be tested was added to dechlorinated tap water as a negative control.

[0062] 20 mL of the protein sample or negative control was added to 13 disposable sterile plastic cups, and 30 healthy, active 2nd-instar Aedes albopictus larvae were gently picked with a brush and put into each plastic cup. Each treatment was repeated 3 times, and then placed in an artificial climate box and cultured at 26°C, a photoperiod of 14L:10D, and a relative humidity of 60%. The samples were observed every day to check the light, humidity, and temperature. After 72 hours, the number of dead and live insects was investigated, and the corrected mortality rate was calculated. The results are shown in Table 2.

[0063] The adjusted mortality rate was calculated as follows:

[0064]

[0065] Table 2

[0066] protein Adjusted mortality rate (%) Anc0 96.67 Cry2A 0 Cry2Ab 0 Cry2Ac 0 Cry2Ad 0 Cry2Ae 0 Cry2A 0 Cry2Ag 0 Cry2A 0 Cry2Ai 0 Cry2Ak 0 Cry2Al 0

[0067] According to the results in Table 2 , protein Anc0 has high activity against Aedes albopictus, while 11 Cry2A proteins, including Cry2Aa, Cry2Ab, Cry2Ac, Cry2Ad, Cry2Ae, Cry2Af, Cry2Ag, Cry2Ah, Cry2Ai, Cry2Ak and Cry2Al, have no insecticidal activity at equivalent protein concentrations.

[0068] The insecticidal activity of Anc0 against Aedes albopictus was further tested at gradient concentrations of 10 ng / ml, 30 ng / ml, 50 ng / ml, 100 ng / ml and 200 ng / ml, and the operation was the same as above, and the corrected mortality rate at each gradient concentration was finally calculated. The corrected mortality rate at the five gradient concentrations was further analyzed using SPSS27 software to determine the median lethal concentration (LC) of Anc0 protein. 50 ), the results showed that Anc0 protein has an inhibitory effect on the LC of Aedes albopictus 50 It is 68.76 (49.19-88.27) ng / ml.

Claims

1. A protein, the amino acid sequence of which is shown in SEQ ID No.

2.

2. A composition comprising the protein according to claim 1.

3. A nucleic acid encoding the protein according to claim 1.

4. The nucleic acid according to claim 3, characterized in that The base sequence of the nucleic acid is shown in SEQ ID No.

1.

5. A microorganism carrying the nucleic acid according to claim 3 or 4 and capable of expressing the protein according to claim 1.

6. The microorganism according to claim 5, characterized in that The microorganism is Escherichia coli and / or Bacillus thuringiensis.

7. Use of one of the protein according to claim 1, the composition according to claim 2, the nucleic acid according to claim 3 or 4, and the microorganism according to claim 5 or 6 in controlling Aedes albopictus.

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