CN117229377B - Insecticidal protein and application thereof in gill-prevention and control of scarab beetles - Google Patents
Insecticidal protein and application thereof in gill-prevention and control of scarab beetles Download PDFInfo
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 35
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 31
- 230000000749 insecticidal effect Effects 0.000 title claims abstract description 21
- 241000254062 Scarabaeidae Species 0.000 title description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 4
- 241000270708 Testudinidae Species 0.000 claims abstract description 3
- 241000346653 Holotrichia parallela Species 0.000 claims description 15
- 102000039446 nucleic acids Human genes 0.000 claims description 8
- 108020004707 nucleic acids Proteins 0.000 claims description 8
- 150000007523 nucleic acids Chemical class 0.000 claims description 8
- 244000005700 microbiome Species 0.000 claims description 7
- 241000193388 Bacillus thuringiensis Species 0.000 claims description 6
- 229940097012 bacillus thuringiensis Drugs 0.000 claims description 6
- 239000010931 gold Substances 0.000 claims description 4
- 229910052737 gold Inorganic materials 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 241001465965 Holotrichia Species 0.000 claims description 2
- 230000002265 prevention Effects 0.000 abstract description 3
- 239000002689 soil Substances 0.000 description 14
- 239000000047 product Substances 0.000 description 12
- 239000007788 liquid Substances 0.000 description 9
- 244000000626 Daucus carota Species 0.000 description 7
- 235000002767 Daucus carota Nutrition 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 239000002244 precipitate Substances 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 239000006285 cell suspension Substances 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 102000008300 Mutant Proteins Human genes 0.000 description 4
- 108010021466 Mutant Proteins Proteins 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 3
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- 238000012360 testing method Methods 0.000 description 3
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000270666 Testudines Species 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
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- 230000002588 toxic effect Effects 0.000 description 2
- 244000144730 Amygdalus persica Species 0.000 description 1
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- 235000006040 Prunus persica var persica Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 241001106462 Ulmus Species 0.000 description 1
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- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to insecticidal protein and application thereof in gill-golden tortoise prevention and treatment. The amino acid sequence of the insecticidal protein is shown as SEQ ID No. 8.
Description
Technical Field
The invention relates to the field of biological control, in particular to application of protein in controlling gill-gold tortoise.
Background
Holotrichia parallela (Holotrichia parallela) is widely distributed in China, and larvae grubs are root tubers and tubers of harmful crops, especially grain crops such as potatoes and cash crops such as peanuts, and adults harm leaves of trees such as poplar, elm and peach, so that huge losses are brought to agriculture, forestry and urban greening. The larvae live in soil in a concealed manner, which causes certain difficulty in traditional chemical control.
Bacillus thuringiensis (Bacillus thuringiensis, bt) and its chaperonin development and utilization provide an effective means for preventing and controlling Holotrichia parallela larvae, and Bt toxic proteins effective on Holotrichia parallela, such as Cry8E, cry8F, cry8H, cry G, are discovered successively. It is still necessary to find or artificially mutate to obtain a toxic protein with better insecticidal activity.
Disclosure of Invention
The invention provides an insecticidal protein, and the amino acid sequence of the insecticidal protein is shown as SEQ ID No. 8.
The second invention provides a composition containing the insecticidal protein according to the first invention.
The third invention provides a nucleic acid encoding the insecticidal protein according to one of the invention.
In a specific embodiment, the nucleotide sequence of the nucleic acid is shown in SEQ ID No. 7.
The fourth invention provides a microorganism carrying a nucleic acid according to the third invention and capable of expressing an insecticidal protein according to one of the invention.
In a specific embodiment, the microorganism is E.coli and/or Bacillus thuringiensis.
In a specific embodiment, the Bacillus thuringiensis is a HD8E-R163H strain.
The fifth invention provides the use of one of the insecticidal proteins according to one of the invention, the composition according to the second invention, the nucleic acid according to the third invention and the microorganism according to any one of the fourth invention for controlling Holotrichia.
In a specific embodiment, the gill-gold turtle is Holotrichia parallela (Holotrichia parallela).
In a specific embodiment, the gill-gold turtle is a larva of Holotrichia parallela (Holotrichia parallela).
The invention has the beneficial effects that: the Cry8Ea1-R163H protein of the invention has obviously improved insecticidal activity on Holotrichia parallela, is 9.5 times of the wild type protein Cry8Ea1, obviously reduces the protein usage amount in the actual prevention and control operation, and is favorable for biological prevention and control on Holotrichia parallela with lower cost.
Drawings
FIG. 1 shows the SDS-PAGE detection result of the protein.
Detailed Description
The above-described aspects of the invention are described in further detail below in the form of preferred embodiments, which are not to be construed as limiting the invention.
Reagents for use in the examples of the invention are commercially available unless otherwise specified.
The HD8E strain is obtained by transforming Bt amorphous mutant strain HD 73-into pSTK-Cry8Ea1 plasmid, contains Cry8Ea1 gene (shown as SEQ ID No. 1), and the amino acid sequence of Cry8Ea1 protein coded by the Cry8Ea1 gene is shown as SEQ ID No. 2.
Example 1
Cloning and expression of muteins
Cloning of mutant proteins
Primers 8E-H-F (shown as SEQ ID No. 3), 8E-R (shown as SEQ ID No. 4), 8E-F (shown as SEQ ID No. 5) and 8E-H-R (shown as SEQ ID No. 6) were designed. Primers were synthesized by the division of biological engineering (Shanghai).
And (3) taking 8E-H-F and 8E-R as primers, taking the HD8E strain as a template, carrying out PCR amplification to obtain a 3009bp PCR product, and then carrying out gel recovery on the PCR product to obtain a gel-recovered PCR product 1. Double digestion and gel recovery are carried out on the pSTK empty vector by using BamHI and SalI restriction enzymes, and a gel recovered enzyme digestion product 1 is obtained. The gel-recovered PCR product 1 and the gel-recovered cleavage product 1 were ligated to obtain a positive recombinant plasmid pSTK-Cry8Ea1-3009. And (3) taking 8E-F and 8E-H-R as primers, taking the HD8E strain as a template, carrying out PCR amplification to obtain a 486 bp PCR product, and then carrying out gel recovery on the PCR product to obtain a gel-recovered PCR product 2. The pSTK-Cry8Ea1-3009 was subjected to single cleavage with BamHI and gel recovery to give gel recovered cleavage product 2. The PCR product 2 recovered by the gel is connected with the enzyme digestion product 2 recovered by the gel to obtain a positive recombinant plasmid pSTK-Cry8Ea1-R163H, so that AGA at 487 th to 489 th positions of Cry8Ea1 genes is mutated into CAT, R at 163 th positions of Cry8Ea1 proteins is mutated into H, and mutant genes (shown as SEQ ID No. 7) and mutant proteins (shown as SEQ ID No. 8) are shown as Cry8Ea 1-R163H.
The positive recombinant plasmid pSTK-Cry8Ea1-R163H is transferred into a Bt crystal-free mutant strain HD73-, positive transformants are selected and named HD8E-R163H.
Expression of muteins
(1) Picking single colonies: picking HD8E-R163H single colony, and shake culturing in LB liquid medium 5 mL containing kanamycin with a final concentration of 50 μg/mL at 30 ℃ and 220 rpm for 12 hours to obtain activated bacterial liquid;
(2) Inoculating the activated bacteria liquid into a 1L triangular flask filled with 300 mL of 1/2 LB liquid medium (containing 50 mug/mL kanamycin) according to 1% volume, and performing shaking culture at 220 rpm at 30 ℃ until about 80% of bacteria are cracked in microscopic examination to obtain a fermentation broth;
(3) Centrifuging the fermentation broth at a low temperature of 6,500×g for 15 min, removing the first supernatant, and collecting the first precipitate;
(4) Washing the first precipitate with pre-chilled 1M NaCl for 1 time, centrifuging at 8,000Xg for 15 minutes at low temperature, and removing the second supernatant to obtain a second precipitate;
(5) Washing the second precipitate with precooled sterilized water for 1-2 times, mixing thoroughly during the washing process, centrifuging after washing, and removing the third supernatant to obtain a third precipitate;
(6) With 50 mM NaCO pH 9.5 3 Suspending the third precipitate in water solution, standing at 4deg.C for 12 hr to dissolve crystallin in NaCO 3 An aqueous solution to obtain a protein-dissolving suspension;
(7) Centrifuging the protein dissolution suspension at a low temperature of 8,000Xg for 10 min, and collecting supernatant to be tested;
(8) Extracting HD8E protein by the same operation as in (1) to (7), and finally harvesting Cry8Ea1 protein supernatant;
(9) The supernatants of the test and Cry8Ea1 proteins were subjected to SDS-PAGE and the results are shown in FIG. 1.
As can be seen from the results of FIG. 1, cry8Ea1-R163H was successfully expressed in HD 73-.
Example 2
Insecticidal Activity assay
Single colonies of the HD8E-R163H strain were picked up, added to 1 mL LB liquid medium containing kanamycin (50. Mu.g/ml), cultured at 28℃and 200 rpm for 12H activation to obtain an activated bacterial liquid, and the activated bacterial liquid was added to 100 mL LB liquid medium containing kanamycin (50. Mu.g/ml) at 1% by volume, and cultured at 28℃with shaking at 200 rpm. 48 After h, detecting the cracking condition of the companion crystals, and harvesting the culture solution to prepare a cell crystal suspension when 50% of the companion crystals are cracked. The method comprises the following steps: the culture broth was centrifuged at 12000 rpm, the supernatant was discarded, and the pellet was resuspended in sterile water to obtain a suspension of HD8E-R163H cells.
A suspension of HD8E cells was produced in the same manner as described above.
Soil was autoclaved at 121 ℃ for 30 min for insecticidal activity testing.
CFU of two kinds of cell suspension are measured and calculated, the volume of the cell suspension with the highest concentration is determined, and the cell suspension of HD8E-R163H and HD8E is diluted by 5 times in gradient, so that the final concentration of the cell suspension with each concentration is 2.1875X10 after the cell suspension is mixed with 60 g soil 8 CFU/g soil, 0.4375×10 8 CFU/g soil, 0.0875X10 8 CFU/g soil, 0.0175×10 8 CFU/g soil and 0.0035×10 8 CFU/g soil. The method comprises the steps of preparing carrot filaments, washing the carrot filaments with clear water, and airing until the surface of the carrot filaments has no water. Immersing shredded carrot in the cytokinesis suspension at each concentration for 20 min, taking out shredded carrot and uniformly placing the shredded carrot in a 6-hole biological test plate, wherein about 4 to 5 shredded carrot are placed in each hole, and stirring the residual bacterial liquid in 60 g soil. Packaging the mixed soil into 6-hole raw measurement plates with corresponding concentration, inoculating 1 head of larva of Holotrichia parallela of 3 days old into each hole, and placingFor a temperature of 26 ℃, the light was fed in an incubator with L: d=16:8. Each repetition had 24 heads, with 3 repetitions. Sterile water was used as a negative control. After 7 days the number of dead and live insects was investigated, the average mortality was calculated, the mortality was corrected, and then the concentration in death was calculated using poleplus software (LC 50 ）。
Results LC of HD8E-R163H 50 1.43×10 6 CFU/g soil with confidence interval of 5.1X10 5 To 2.94×10 6 CFU/g soil; LC of HD8E 50 1.358×10 7 CFU/g soil with confidence interval of 3.31X10 6 Up to 6.294X 10 7 CFU/g soil. LC according to both 50 It was found that, after R163H mutation was performed on the Cry8Ea1 protein, the insecticidal activity of the mutant proteins Cry8Ea1-R163H was 9.5 times that of the wild type protein Cry8Ea1, indicating that the insecticidal activity of the mutant proteins Cry8Ea1-R163H was significantly improved over that of the wild type protein Cry8Ea 1.
Claims (9)
1. An insecticidal protein has an amino acid sequence shown in SEQ ID No. 8.
2. A composition comprising the insecticidal protein of claim 1.
3. A nucleic acid encoding the insecticidal protein of claim 1.
4. A nucleic acid according to claim 3, wherein the base sequence of the nucleic acid is as shown in SEQ ID No. 7.
5. A microorganism carrying the nucleic acid of claim 3 or 4 and expressing the insecticidal protein of claim 1, said microorganism being escherichia coli and/or bacillus thuringiensis.
6. The microorganism of claim 5, wherein the bacillus thuringiensis is a strain HD8E-R163H.
7. Use of one of the insecticidal proteins according to claim 1, the compositions according to claim 2, the microorganisms according to claim 5 or 6 for controlling gill-gold tortoises (Holotrichia).
8. The use according to claim 7, characterized in that the gill-mossback is Holotrichia parallela (Holotrichia parallela).
9. The use according to claim 7, characterized in that the gill-mossback is a larva of holotrichia parallela (Holotrichia parallela).
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US11021716B2 (en) * | 2016-11-01 | 2021-06-01 | Pioneer Hi-Bred International, Inc. | Insecticidal proteins and methods for their use |
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