A mutant insecticidal protein and its application in controlling the gypsy moth

Abstract

The present invention relates to a mutant insecticidal protein and its application in controlling the gypsy moth. The present invention finds that after the Cry9Aa655 protein is mutated into the 316LRG318AAA protein, its insecticidal activity against the gypsy moth is significantly improved. In addition, the synergistic effect of the insecticidal activity against the gypsy moth produced by the combination of the 316LRG318AAA mutant protein and Cry1Ah is significantly higher than that of the combination of the Cry9Aa655 protein and Cry1Ah.

Description

Technical Field

[0001] The invention relates to the field of insecticides, and in particular to a mutant insecticidal protein and application thereof in preventing and controlling the gypsy moth. Background Art

[0002] American white moth ( Hyphantria cunea ) is a polyphagous, voracious herbivorous pest with more than 600 host species, including forests, fruit trees and a variety of crops.

[0003] Common control measures for the American white moth include chemical control, physical control and biological control. Chemical agents have the advantage of rapid effect, but the 3R (resistance, recurrence and residue) problems caused by long-term use cannot be ignored. Physical control mainly adopts manual removal or mechanical equipment, but it is not suitable for promotion and large-scale control. Biological control mainly uses Bacillus thuringiensis ( Bacillus thuringiensis , Bt), Beauveria bassiana ( Beauveria bassiana ), Hyphantria cunea nuclear polyhedrosis virus (HcNPV), etc., among which Bt preparations have become a promising choice due to their specificity, sustainability and safety.

[0004] So far, it has been discovered cry1Ab , cry1Ah wait cry1 Such genes have insecticidal activity against the larvae of the gypsy moth. However, in order to prevent resistance caused by a single insect-resistant gene, it is very important to find new genes or genomes that have insecticidal activity against the gypsy moth. Summary of the invention

[0005] One aspect of the present invention provides a mutant insecticidal protein, the amino acid sequence of which is shown in SEQ ID No. 4.

[0006] The second aspect of the present invention provides a composition comprising the insecticidal protein as described in the first aspect of the present invention and a pesticide acceptable carrier.

[0007] In a specific embodiment, the composition further comprises Cry1Ah protein.

[0008] In a specific embodiment, the GenBank accession number of the Cry1Ah protein is AF281866.1.

[0009] In a specific embodiment, the mass ratio of the insecticidal protein according to one of the present invention to the Cry1Ah protein is 1:1.

[0010] The third aspect of the present invention provides a nucleic acid encoding the insecticidal protein as described in one of the present inventions.

[0011] In a specific embodiment, the base sequence of the nucleic acid is shown as SEQ ID No. 3.

[0012] The fourth aspect of the present invention provides a microorganism into which the nucleic acid as described in the third aspect of the present invention is transferred, and the nucleic acid can be expressed in the microorganism to synthesize the insecticidal protein as described in the first aspect of the present invention.

[0013] In a specific embodiment, the microorganism is Escherichia coli or Bacillus thuringiensis.

[0014] The fifth invention provides the use of at least one of the insecticidal protein according to the first invention, the composition according to any one of the second invention, the nucleic acid according to the third invention and the microorganism according to the fourth invention in controlling the American white moth.

[0015] Beneficial effects of the present invention: The present invention found that after the Cry9Aa655 protein was mutated into the 316LRG318AAA protein, its insecticidal activity against the gypsy moth was significantly improved. In addition, the synergistic effect of the insecticidal activity against the gypsy moth produced by the combination of the 316LRG318AAA mutant protein and Cry1Ah was significantly higher than that of the combination of the Cry9Aa655 protein and Cry1Ah. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Shows the cry9Aa655 Gene mutation information.

[0017] Figure 2 The biological activity assay of Cry9Aa655 and its mutant proteins against the gypsy moth is shown. DETAILED DESCRIPTION

[0018] 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.

[0019] Unless otherwise specified, the reagents in the embodiments of the present invention can be purchased through commercial channels.

[0020] Example 1: Expression of Cry9Aa655 protein

[0021] cry9Aa655 The nucleotide sequence of the gene is shown in SEQ ID No. 1, and the amino acid sequence of the Cry9Aa655 protein is shown in SEQ ID No. 2.

[0022] Will cry9Aa655The gene was connected to the pEB vector to obtain the pEB-cry9Aa655 recombinant plasmid, and then the positive recombinant plasmid was transformed into Escherichia coli Rosetta (DE3) to screen out the positive transformants Rosetta (DE3) / pEB-cry9Aa655.

[0023] Pick a fresh colony of BL21(DE3) / pEB-cry9Aa655 and inoculate it into 5 mL of LB liquid medium for activation for 8 h. Then inoculate it into a conical flask containing 300 mL of sterilized LB medium (with 1 / 1000 ampicillin) at a 1% inoculum and culture at 37 degrees Celsius and 220 rpm until OD 600nm When the concentration of IPTG was 0.6, IPTG was added to a final concentration of 0.5 mmol / L, the induction temperature was set at 16 degrees Celsius, the induction culture was carried out for 14 hours, and the induced bacteria were collected by centrifugation at 4 degrees Celsius, 8000 rcf, and 15 minutes. The bacteria were then suspended evenly with 20 mmol / L Tris-HCl (pH 8.0). Next, the bacteria were ultrasonically disrupted at 40% power for 10 minutes (3 seconds of supersonication and 5 seconds of stopping), and then centrifuged at 4 degrees Celsius, 8000 rcf for 20 minutes, and the supernatant and precipitate were collected respectively, among which the precipitate was suspended with 20 mmol / L Tris-HCl (pH 8.0). The supernatant and the suspended precipitate were detected by SDS-PAGE and quantified by BSA. The results showed that the expression amount of Cry9Aa655 protein in the supernatant (soluble component) was more than double that in the precipitate (insoluble component). Therefore, the insecticidal activity analysis was carried out with the soluble component.

[0024] Example 2: Expression of Cry9Aa655 mutant protein

[0025] according to Figure 1 Information on cry9Aa655The gene was mutated and each mutant gene was connected to the pET-28b vector to obtain pET-307PIG309AAA recombinant plasmid, pET-316LRG318AAA recombinant plasmid, pET-364TDR366AAA recombinant plasmid, pET-393HTT395AAA recombinant plasmid, pET-416NDT418AAA recombinant plasmid, pET-422VNR424AAA, pET-R313A recombinant plasmid, pET-S314A recombinant plasmid, pET-R368A recombinant plasmid, pET-V369A recombinant plasmid, pET-R403A recombinant plasmid, pET-S498A recombinant plasmid, pET-R569A recombinant plasmid, pET-Q571A recombinant plasmid, and then the positive recombinant plasmids were transformed into Escherichia coli BL21 (DE3) to screen the positive transformants BL21 (DE3) / p ET-307PIG309AAA, BL21(DE3) / pET-316LRG318AAA, BL21(DE3) / pET-364TDR366AAA, BL21(DE 3) / pET-393HTT395AAA, BL21(DE3) / pET-416NDT418AAA, BL21(DE3) / pET-422VNR424AAA, BL21 (DE3) / pET-R313A, BL21(DE3) / pET-S314A, BL21(DE3) / pET-R368A, BL21(DE3) / pET-V369A, BL 21(DE3) / pET-R403A, BL21(DE3) / pET-S498A, BL21(DE3) / pET-R569A, BL21(DE3) / pET-Q571A.

[0026] Among them, the nucleotide sequence of the 316LRG318AAA gene is shown in SEQ ID No. 3, and the amino acid sequence of the 316LRG318AAA protein is shown in SEQ ID No. 4.

[0027] The extraction of each mutant protein and the detection of protein distribution by protein electrophoresis were the same as in Example 1.

[0028] The results showed that the expression levels of each mutant protein of Cry9Aa655 in the supernatant (soluble fraction) were higher than those in the precipitate (insoluble fraction). Therefore, the insecticidal activity was analyzed using the soluble fraction.

[0029] Example 3: Protein purification

[0030] The protein samples in the supernatant of the induced expression of E. coli in Example 1 and Example 2 were purified by nickel affinity chromatography, and the column material was Ni SEPHAROSE 6 Fast Flow. The solutions used in the entire purification process were filtered and sterilized before use.

[0031] Take 2 mL of column material and place it in an empty purification column to allow it to settle naturally. Wash the 20% ethanol in the column material with 5 column volumes of ultrapure water, and balance the column material with 10 column volumes of binding buffer; add the supernatant sample containing the target protein, collect the flow-through, and repeat the loading 3 times. Elute the impurities with 10 column volumes of binding buffer, collect the flow-through; elute the target protein with 5 mL of elution buffer, and collect the flow-through; after use, rinse the column material with 5 column volumes of ultrapure water; rinse the column material with 5 column volumes of 20% ethanol, and soak the column material in 20% ethanol and store it at 4°C.

[0032] The purified protein was desalted using an AKTA-Avant automated purification system, and the desalted protein was finally obtained in a 20 mmol / L pH 8.0 Tris-HCl aqueous solution.

[0033] The protein was purified by polyacrylamide gel electrophoresis and then quantified by BCA kit for the biological activity assay against the gypsy moth.

[0034] Example 4: Expression of Cry1Ah protein

[0035] The GenBank accession number of Cry1Ah protein is AF281866.1; cry1Ah The GenBank accession number of the gene is AF281866.1, of which positions 530 to 4078 are the coding region. cry1Ah The gene was ligated to the pSXY422 vector (erythromycin resistance) to obtain pSXY422-1Ah, which was then transformed into Bacillus thuringiensis HD73 — The engineered bacterium Biot1Ah was obtained, see CN2004100099189 for details.

[0036] The Biot1Ah strain was inoculated into 1 / 2LB liquid medium and grown at 30 degrees Celsius and 220 r / min until 50% of the crystals were released; the pellet was collected and washed with 1.0 mol / L NaCl and then with distilled water after centrifugation at 8500 rcf for 10 min at 4 degrees Celsius; the pellet was resuspended in lysis buffer and incubated on ice (shaking at 70 r / min) for 4 h; the supernatant was collected after centrifugation at 8500 rcf for 15 min at 4 degrees Celsius; 3.0 mol / L sodium acetate-acetate buffer was added and incubated at 4 degrees Celsius for at least 1 h; after centrifugation at 8000 rcf for 15 min at 4 degrees Celsius, the pellet was collected and washed twice with pre-cooled distilled water; the pellet was dissolved in 50 mmol / L Na 2 CO 3 (pH 9.5) to obtain a Cry1Ah protein solution, and the Cry1Ah protein was activated by trypsin at 37 degrees Celsius in a water bath for 2 hours to obtain an activated Cry1Ah protein. The mass ratio of Cry1Ah protein to trypsin was 10:1.

[0037] The activated Cry1Ah protein was purified and desalted using an AKTA-Avant automated purification system, and finally the activated Cry1Ah desalted protein dissolved in a 20 mmol / L pH 8.0 Tris-HCl aqueous solution was obtained.

[0038] Example 5: Determination of insecticidal activity of Cry9Aa655 and its mutant proteins against Hyphantria cunea

[0039] The gypsy moth was provided by the Chinese Academy of Forestry.

[0040] Feed formula for the American white moth: 30g agar, 110g soybean powder, 210g wheat bran or wheat germ powder, 40g yeast powder, 4g sorbic acid, 55g casein, 4g ascorbic acid, 3mL multivitamin, 3mL formaldehyde, 6mL acetic acid, 1mL tecodol, and 1800mL distilled water.

[0041] Insecticidal activity determination steps: weigh 8 g of artificial feed into a sterilized culture dish, add the protein sample to be tested, mix thoroughly, and place at room temperature until all excess water in the feed evaporates; use a brush to inoculate healthy, active, and unfed newly hatched larvae of the American white moth into a 6 cm diameter culture dish containing feed, and inoculate 20 insects per dish. After inoculation, cover the mouth of the culture dish with toilet paper, then cover the dish lid, tie it tightly with a rubber band, and place it upright in a biochemical incubator at 25 degrees Celsius, a photoperiod of 16L:8D, and a relative humidity of 50%. Each treatment has 20 insects and 3 replicates. Use 20 mmol / L Tris-HCl (pH 8.0) as a blank control. Observe every day to check the light, humidity, temperature, and whether the feed is moldy and whether there is condensation of water vapor; after 4 days, investigate the number of dead insects, and then calculate the mortality rate and corrected mortality rate. The results are shown in Figure 2 .

[0042] Analysis of the insecticidal activity of these mutants against the larvae of the gypsy moth revealed that the insecticidal activity of the R313A, R569A, 307PIG309AAA, 364TDR366AAA, and 416NDT418AAA mutants was reduced, while the insecticidal activity of the mutant 316LRG318AAA was increased, and there was no significant change in the activity of the remaining mutants.

[0043] Example 6: LC of 316LRG318AAA mutant protein and its combination with Cry1Ah protein 50 Determination

[0044] The Cry9Aa655 protein and the Cry1Ah protein were mixed in a mass ratio of 1:1 to obtain the Cry9Aa655+Cry1Ah protein.

[0045] The 316LRG318AAA protein and the Cry1Ah protein were mixed at a mass ratio of 1:1 to obtain the 316LRG318AAA + Cry1Ah protein.

[0046] Cry9Aa655, 316LRG318AAA protein, Cry1Ah, Cry9Aa655+Cry1Ah protein and 316LRG318AAA+Cry1Ah protein were diluted into five different concentration gradients for bio-LC of the American moth 50 Activity assay.

[0047] The insecticidal activity determination operation was the same as in Example 5. After 4 days, the number of dead insects was investigated, and then the mortality rate and corrected mortality rate were calculated. The results of the bioassay were analyzed using SPSS. The results are as follows: LC of Cry9Aa655 50 The LC value of 316LRG318AA was 5.87 μg / g, with a 95% confidence interval of 4.37 to 7.78 μg / g. 50The LC value of Cry1Ah was 3.01 μg / g, with a 95% confidence interval of 1.31 to 4.27 μg / g. 50 The expected LC of Cry9Aa655+Cry1Ah is 4.36 μg / g, with a 95% confidence interval of 3.42 to 5.42 μg / g. 50 5.00 μg / g, the measured LC 50 The LC value of 316LRG318AAA+Cry1Ah was 1.02 μg / g, with a 95% confidence interval of -0.47 to 2.02 μg / g. 50 Expected LC 50 The measured LC 50 The value was 0.43 μg / g, with a 95% confidence interval of -1.87 to 1.43 μg / g.

[0048] According to the results, the insecticidal activity of 316LRG318AAA was 1.95 times higher than that of Cry9Aa655. Cry9Aa655 and its mutant protein were mixed with Cry1Ah protein in a 1:1 ratio for biological activity assay, and it was found that Cry9A and its mutant protein had synergistic effects with Cry1Ah protein, with synergistic factors of 4.90 and 8.24, respectively. Among them, the mutant 316LRG318AA mixed with Cry1Ah protein had a LC-1 inhibitory effect on the lycaenid moth. 50 Significantly lower than the LC of the mixture of Cry9Aa655 and Cry1Ah proteins against the nymphalidae 50。

Claims

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

4.

2. A composition comprising the insecticidal protein according to claim 1 and a pesticidal acceptable carrier.

3. The composition according to claim 2, characterized in that The composition also includes Cry1Ah protein.

4. The composition according to claim 3, characterized in that The GenBank accession number of the Cry1Ah protein is AF281866.

1.

5. The composition according to claim 3, characterized in that The mass ratio of the insecticidal protein according to claim 1 to the Cry1Ah protein is 1:

1.

6. A nucleic acid encoding the insecticidal protein of claim 1.

7. The nucleic acid according to claim 6, characterized in that The base sequence of the nucleic acid is shown in SEQ ID No.

3.

8. A microorganism, into which the nucleic acid according to claim 6 or 7 is transferred, and the nucleic acid can be expressed in the microorganism to synthesize the insecticidal protein according to claim 1.

9. The microorganism according to claim 8, characterized in that The microorganism is Escherichia coli or Bacillus thuringiensis.

10. Use of at least one of the insecticidal protein according to claim 1, the composition according to any one of claims 2 to 5, the nucleic acid according to claim 6 or 7, and the microorganism according to claim 8 or 9 in controlling the gypsy moth.

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