Highly active Cry1Ac mutant and its application

Abstract

The present invention belongs to the field of biotechnology, and discloses a highly active Cry1Ac mutant and its application. The present invention uses site-directed mutagenesis technology to carry out site-directed mutagenesis on key amino acids, and constructs a series of Cry1Ac mutants. The phenylalanine at position 371 on the amino acid sequence of the Cry1Ac protein is mutated into glutamine, the asparagine at position 372 is mutated into glutamic acid, the isoleucine at position 373 is mutated into glutamine, or the isoleucine at position 375 is mutated into glutamine. The mutant protein obtained is 4.5, 2.7, 6.3 times and 3.4 times more toxic to the diamondback moth than the wild type. The present invention provides theoretical guidance for the transformation of Cry toxins and provides new ideas for insect resistance management.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a high-activity Cry1Ac mutant and an application thereof. Background Art

[0002] The diamondback moth (Plutellaxylostella) is a highly destructive Lepidoptera pest and is considered one of the most important agricultural pests in the world. The diamondback moth mainly harms cruciferous plants, but its diet is very wide and it can feed on more than 40 different host plants, including many important economic crops such as cabbage, kale, cauliflower, etc. These plants are major vegetable crops in many parts of the world, and the infestation of the diamondback moth poses a serious threat to the growth and harvest of these crops. Due to the long-term and large-scale use of chemical pesticides, the diamondback moth is highly resistant to more than 50 commonly used chemical synthetic pesticides. [1] This resistance has greatly increased the difficulty of controlling diamondback moth and caused significant economic losses worldwide. In my country alone, the economic losses caused by diamondback moth to vegetable production amount to billions of yuan each year. [2] .

[0003] Bacillus thuringiensis (Bt) is a Gram-positive, spore-forming soil bacterium that is widely used in biological control and genetically modified crops. During the spore formation process, Bt bacteria produce crystalline toxins (Cry proteins), which have a highly effective insecticidal effect on a variety of insects. [3,4] Cry crystals are composed of three domains, namely Domain I, Domain II and Domain III [5] Domain I is related to the formation of holes in the midgut epithelial cell membrane after insects feed on proteins, Domain II is related to the recognition and binding of specific receptors in the insect midgut, and Domain III may be related to maintaining the integrity of protein molecules and specific binding to receptors. [6-8] After being ingested by insects, Bt Cry toxins are first dissolved in the alkaline intestine and then activated by midgut proteases to become active proteins with insecticidal effects. Specific receptors on the brush border membrane vesicles of insect midguts sequentially bind and accumulate activated Cry toxins on the membrane. These receptors include ABC transporters [9,10] , Cadherin (CAD)

[11] , alkaline phosphatase (ALP)

[12] and aminopeptidase (APN)

[13] After binding to these specific receptors, Cry toxins form holes, destroy intestinal cells, and ultimately cause the death of pests.

[14] Bt toxins are key insecticidal proteins used in genetically modified crops, which offer enormous agronomic, economic and environmental advantages. However, insect resistance to Bt toxins threatens their continued effectiveness.

[15] In 1985, the world's first report of the laboratory population of Indian meal borer (Plodia interpunctella) resistant to Bt was published.

[16] The diamondback moth is the first insect reported to be resistant to Bt formulations in the field and has become a model insect for studying Bt resistance.

[0004] Bt toxins produce toxic effects on target pests through a series of complex multi-step processes, and any changes in these processes may lead to pest resistance. It can be seen that changes in specific toxin receptor interactions can lead to high levels of resistance to Bt toxins in insects. Through positional cloning experiments in resistant insects, ABC transporters were found for the first time as targets of Bt toxins.

[10] Cry1Ac is the most widely studied of the Bt toxin family, and has a highly effective insecticidal effect on a variety of lepidopteran pests (such as diamondback moth, cotton bollworm, corn borer, tobacco hornworm, etc.). Physiological and biochemical studies in recent years have found that insect ABC transporters can act as functional receptors for the Bt toxin Cry1Ac. Among them, the ABC transporter ABCC2 is considered to be the most important receptor for Cry1Ac, and different mutations in the ABCC2 gene can lead to a high level of resistance to Cry1Ac. [10,17,18] ABCC2 may be involved in promoting the insertion of Cry1Ac oligomers into the membrane.

[19] To address the resistance problem, obtaining Cry1Ac protein with high insecticidal activity through point mutation is an effective strategy to deal with resistance. [20,21] Since Cry1Ac domain II plays a role in receptor binding, domain II has become a key focus for modification.

[0005] References:

[0006] 1. Furlong, MJ, DJ Wright, and LMDosdall, Diamondback moth ecology and management: problems, progress, and prospects. Annu Rev Entomol, 2013.58: p.517-41.

[0007] 2.Li,Z.,et al.,Biology,Ecology,and Management of the Diamondback Mothin China.Annu Rev Entomol,2016.61:p.277-96.

[0008] 3.Schnepf,E.,et al.,Bacillus thuringiensis and its pesticidal crystalproteins.Microbiol Mol Biol Rev,1998.62(3):p.775-806.

[0009] 4.Crickmore,N.,et al.,Revision of the nomenclature for the Bacillusthuringiensis pesticidal crystal proteins.Microbiol Mol Biol Rev,1998.62(3):p.807-13.

[0010] 5.Evdokimov,A.G.,et al.,Structure of the full-length insecticidalprotein Cry1Ac reveals intriguing details of toxin packaging into invivoformed crystals.Protein Sci,2014.23(11):p.1491-7.

[0011] 6.Gómez,I.,et al.,Role of receptor interaction in the mode of actionof insecticidal Cry and Cyt toxins produced by Bacillusthuringiensis.Peptides,2007.28(1):p.169-73.

[0012] 7.Zhang,Q.,G.Hua,and M.J.Adang,Effects and mechanisms of Bacillusthuringiensis crystal toxins for mosquitolarvae.Insect Sci,2017.24(5):p.714-729.

[0013] 8.Galitsky,N.,et al.,Structure of the insecticidal bacterial delta-endotoxin Cry3Bb1 of Bacillus thuringiensis.Acta Crystallogr D BiolCrystallogr,2001.57(Pt8):p.1101-9.

[0014] 9.Wu,C.,et al.,Insect ATP-Binding Cassette(ABC)Transporters:Roles inXenobiotic Detoxification and Bt Insecticidal Activity.Int J Mol Sci,2019.20(11).

[0015] 10.Gahan,L.J.,et al.,An ABC transporter mutation is correlated withinsect resistance toBacillus thuringiensis Cry1Ac toxin.PLoS Genet,2010.6(12):p.e1001248.

[0016] 11.Gahan,L.J.,F.Gould,and D.G.Heckel,Identification of a geneassociated with Bt resistance in Heliothis virescens.Science,2001.293(5531):p.857-60.

[0017] 12.Jurat-Fuentes,J.L.and M.J.Adang,Characterization of a Cry1Ac-receptor alkaline phosphatase in susceptible and resistant Heliothisvirescens larvae.Eur J Biochem,2004.271(15):p.3127-35.

[0018] 13.Knight,P.J.,N.Crickmore,and D.J.Ellar,The receptor for Bacillusthuringiensis CrylA(c)delta-endotoxin in the brush border membrane of thelepidopteran Manduca sexta is aminopeptidase N.Mol Microbiol,1994.11(3):p.429-36.

[0019] 14.Pardo-López,L.,M.Soberón,and A.Bravo,Bacillus thuringiensisinsecticidal three-domain Cry toxins:mode of action,insect resistance andconsequences for cropprotection.FEMS Microbiol Rev,2013.37(1):p.3-22.

[0020] 15.Tabashnik,B.E.,et al.,Insect resistance to Bt crops:evidenceversus theory.Nat Biotechnol,2008.26(2):p.199-202.

[0021] 16.McGaughey,W.H.,Insect Resistance to the Biological InsecticideBacillus thuringiensis.Science,1985.229(4709):p.193-5.

[0022] 17. Xiao, Y., et al., Mis-splicing of the ABCC2 gene linked with Bt toxin resistance in Helicoverpa armigera. Sci Rep, 2014. 4: p. 6184.

[0023] 18. Tanaka, S., et al., The ATP-binding cassette transporter subfamily C member 2 in Bombyx mori larvae is a functional receptor for Cry toxins from Bacillus thuringiensis. Febs j, 2013. 280(8): p. 1782-94.

[0024] 19. Ocelotl, J., et al., ABCC2 is associated with Bacillus thuringiensis Cry1Ac toxin oligomerization and membrane insertion in diamondback moth. Sci Rep, 2017. 7(1): p. 2386.

[0025] 20. Pardo-López, L., et al., Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis. Peptides, 2009. 30(3): p. 589-95.

[0026] 21. Dean, D. H., et al., Probing the mechanism of action of Bacillus thuringiensis insecticidal proteins by site-directed mutagenesis--a mini review. Gene, 1996. 179(1): p. 111-7. Summary of the Invention

[0027] Therefore, it is necessary to provide a method for constructing a Cry1Ac mutant with high activity by using site-directed mutagenesis technology to improve the insecticidal effect. Furthermore, the application of the Cry1Ac protein mutant in pest control.

[0028] The invention provides a highly active Cry1Ac mutant, which is obtained by having one or more mutations of F371Q, N372E, I373Q and I375Q on the basis of the amino acid sequence of wild-type Cry1Ac, wherein the amino acid sequence of the wild-type Cry1Ac has a reference NCBI accession number of AAU87037.1.

[0029] Preferably, it is obtained by mutation of F371Q, N372E, I373Q or I375Q based on the amino acid sequence of wild-type Cry1Ac.

[0030] The present invention provides a nucleic acid encoding the highly active Cry1Ac mutant, which is optionally obtained by mutation based on the nucleotide sequence shown in NCBI accession number AY730621.1.

[0031] The present invention further provides an expression vector containing the nucleic acid.

[0032] The present invention also provides a recombinant host bacteria containing the nucleic acid or the expression vector.

[0033] The present invention further provides the use of the highly active Cry1Ac mutant in the preparation of drugs for controlling insects.

[0034] Preferably, the insect is a Lepidoptera pest, specifically, the insect is Plutella xylostella.

[0035] The present invention also provides the use of the highly active Cry1Ac mutant in controlling insects. Preferably, the insect is a lepidopteran pest, specifically, Plutella xylostella.

[0036] The beneficial effects of the present invention are as follows: the present invention uses site-directed mutagenesis technology to carry out site-directed mutagenesis on key amino acids, and constructs a series of Cry1Ac mutants. The phenylalanine (Phe) at position 371 on the amino acid sequence of the Cry1Ac protein is mutated into glutamine (Gln), and the obtained mutant protein F371Q has a 4.5-fold increase in toxicity to the diamondback moth compared with the wild type; the asparagine (Asn) at position 372 is mutated into glutamic acid (Glu), and the obtained mutant protein N372E has a 2.7-fold increase in toxicity to the diamondback moth compared with the wild type; the isoleucine (Ile) at position 373 is mutated into glutamine (Gln), and the obtained mutant protein I373Q has a 6.3-fold increase in toxicity to the diamondback moth compared with the wild type; and the isoleucine (Ile) at position 375 is mutated into glutamine (Gln), and the obtained mutant protein I375Q has a 3.4-fold increase in toxicity to the diamondback moth compared with the wild type. The present invention provides theoretical guidance for the modification of Cry toxins and new ideas for insect resistance management. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 :Cry1Ac mutant design (orange is Cry1Ac, blue is ABCC2).

[0038] Figure 2 : SDS-PAGE gel images of wild-type Cry1Ac and mutant protoxins.

[0039] Figure 3 : Anion exchange chromatography and SDS-PAGE gel images of wild-type Cry1Ac and mutants. A to E are anion exchange chromatography images of wild-type Cry1Ac, mutants F371Q, N372E, I373Q and I375Q, respectively, and F is an SDS-PAGE gel image.

[0040] Figure 4 : Gel filtration chromatograms and SDS-PAGE gel images of wild-type Cry1Ac and mutants. A to E are gel filtration chromatograms of wild-type Cry1Ac, mutants F371Q, N372E, I373Q and I375Q, respectively, and F is an SDS-PAGE gel image.

[0041] Figure 5 :The results of bioassay of wild-type Cry1Ac and mutants in treating Plutella xylostella larvae. A to E are the results of wild-type Cry1Ac, mutants F371Q, N372E, I373Q and I375Q respectively. DETAILED DESCRIPTION

[0042] In order to explain the technical content, achieved objectives and effects of the technical solution in detail, the following is a detailed description in conjunction with specific embodiments and accompanying drawings.

[0043] Example 1: Structure prediction and determination of mutation sites

[0044] The interaction interface between the ABC transporter ABCC2 and domain II of Cry1Ac (NCBI accession number AAU87037.1, the corresponding nucleotide sequence NCBI accession number AY730621.1) was analyzed by structural prediction. Figure 1 ), it was found that N372 on Cry1Ac was close to R235 on ABCC2, and the mutant N372E was designed to form a salt bridge with R235 to enhance the interaction ( Figure 1 In the predicted structure, the main chain of F371 in Cry1Ac interacts with R122 in ABCC2. In order to enhance the interaction between the side chain of 371 and R122, 371 was mutated to Q, i.e., F371Q ( Figure 1 Similarly, to enhance the interaction between Cry1Ac I373 and I375 and R1221 and T355 in ABCC2, mutants I373Q and I375Q ( Figure 1 The wild-type Cry1Ac and mutant plasmids were synthesized by gene synthesis (Nanjing GenScript), and then the proteins were expressed and purified. Finally, bioassays were performed in Plutella xylostella larvae to verify whether the insecticidal activity of these mutants was improved.

[0045] Example 2: Expression and purification of wild-type Cry1Ac and mutants

[0046] 1) Synthesis of target genes: The cDNA sequences of wild-type Cry1Ac (NCBI accession number of the nucleotide sequence is AY730621.1) and mutant protein genes were fully synthesized;

[0047] 2) Construction of expression vector: The expression vector is pHT315-8E21b (provided by Wang Zeyu, Institute of Plant Protection, Chinese Academy of Agricultural Sciences), with restriction sites of BamHI and SaII. After restriction digestion, the target gene is connected to the vector under the action of ligase. After successful connection, it is transformed into the cloning strain Escherichia coli DH5α by heat shock method, and then plasmid is extracted. Positive clones are identified by sequencing. After correct identification, the positive clones are cultured to extract the target plasmid;

[0048] 3) Recombinant expression of protein: The wild-type Cry1Ac and mutant plasmids were transferred into the crystal-free mutant strain HD73- using an electroporation method (900V, 25μF). Transformants were screened using LB plates containing erythromycin, and PCR was used to detect whether the transformants contained the Cry1Ac mutant gene. The wild-type Cry1Ac and mutant strains were inoculated into 10 mL of LB liquid culture medium, cultured overnight at 30°C and 220rpm, and then transferred to 1000 mL of LB culture medium at a 1% inoculum and cultured for 36 hours at 30°C and 220rpm.

[0049] 4) Extraction of Cry1Ac protein: (1) Centrifuge at 4°C, 8000 g for 10 min to collect the mixture of spores and crystals in the culture medium;

[0050] (2) Wash the cells with pre-cooled 1 M NaCl to remove spores and foreign proteins in the culture medium, centrifuge at 8000 g for 10 min at 4°C, and discard the supernatant;

[0051] (3) Wash twice with precooled sterile water, centrifuge at 9000g for 15 min at 4°C, and discard the supernatant;

[0052] (4) Add Na2CO3 lysis solution (50 mM Na2CO3, pH = 10.5, add 0.5% DTT immediately before use) to the cytosolic mixture precipitate, stir evenly, and place on ice and shake for 1 h (70 rpm);

[0053] (5) Centrifuge at 9000 g for 20 min at 4°C, remove the supernatant, add 1 / 7 volume of 4 M NaAc and let it settle on ice for 2 h;

[0054] (6) Centrifuge at 9000 g for 20 min at 4°C, discard the supernatant, resuspend the precipitate in sterile water, centrifuge at 9000 g for 20 min at 4°C, and resuspend once more in sterile water;

[0055] (7) Centrifuge at 9000 g for 20 min at 4°C, discard the supernatant, and dissolve the precipitate in 50 mM Na2CO3-NaHCO3 buffer;

[0056] (8) 8-12% SDS-PAGE protein electrophoresis was used to detect protein extraction.

[0057] 5) Purification of Cry1Ac activated toxin

[0058] The extracted Cry1Ac protoxin was activated by incubating with trypsin at a mass ratio of 1:1 at 37° for 2 hours, and then the protoxin was purified by an anion exchange column (Hi Trap TM 5mL Q HP) purification, the purification instrument is AKTA avant 150 (GE Company, USA), the purification steps are as follows:

[0059] (1) The activated protein was centrifuged at 12,000 g for 20 minutes at 4°C, the supernatant was collected, filtered through a 0.25 μm filter, and purified.

[0060] (2) Pump A, pump B and the system were cleaned one by one with 50 mM Na2CO3-NaHCO3 (pH 9.6) buffer (buffer A). The upper limit of the column pressure was set to 0.3 MPa, and the anion exchange column was flushed with 50 mM Na2CO3-NaHCO3 buffer (buffer B) containing 1 M NaCl, and the flow rate was set to 2 mL / min.

[0061] (3) The protein to be purified was injected and loaded, the flow rate was set to 2 mL / min, and buffer A was continuously flowed to allow the protein to hang on the column under this condition.

[0062] (4) Wash the column with at least 5 column volumes of buffer A and collect the flow-through peak, which consists of excess protein or impurity proteins that are not attached to the column.

[0063] (5) Gradient elution was performed with 0-50% buffer B within 30 min, and each protein peak was collected. The flow rate was set to 1.5 mL / min until all proteins were eluted.

[0064] (6) The collected proteins were subjected to SDS-PAGE detection.

[0065] (7) Use HiLoad 26 / 600 Superdex 75pg column for further purification to remove other small molecular proteins. On the other hand, it can also remove the NaCl contained in the protein purified by the anion exchange column. Use 50mM Na2CO3-NaHCO3 (pH9.6) buffer (buffer A) to clean pumpA and the system in turn, then inject the protein, set the flow rate to 0.5mL / min, collect the protein peak, perform SDS-PAGE detection, and after confirming that there is no problem, store the purified protein at -80℃.

[0066] The results are as follows Figure 2 As shown, it is the SDS-PAGE gel image result of the wild-type Cry1Ac and mutant protoxin protein obtained after purification. The protein size is 120kDa. It can be seen from the figure that the expression and purification results of the mutant protein and the wild-type Cry1Ac are similar, indicating that this mutation is feasible and will not affect the protein expression and purification.

[0067] The original toxin protein was then activated by trypsin and purified using an anion exchange column. Figure 3As shown, the protein peak marked by the red asterisk was collected and the size was observed to be 60 kDa when running SDS-PAGE gel, which is the desired target protein. Compared with the wild type, the mutation did not affect the trypsin cleavage effect.

[0068] Finally, gel chromatography column was used for further purification to remove other small molecular proteins and NaCl. Figure 4 As shown, the protein peak marked with a red asterisk was collected and SDS-PAGE was run to identify the protein purity. It was found that the wild-type Cry1Ac and the mutant protein had single bands, achieving the purification purpose.

[0069] Example 3: Diamondback moth bioassay experiment

[0070] (1) Dilute the activated wild-type Cry1Ac and mutants into 6 different concentrations in a 50 mL centrifuge tube;

[0071] (2) Select fresh cruciferous vegetable leaves (of uniform size) and soak them in a centrifuge tube containing the sample to be tested for 5 min. Take out the leaves, place them on filter paper and dry them at room temperature (turn them over once in the middle), and then divide them into culture dishes, which are lined with filter paper sprayed with sterile water;

[0072] (3) Place the second-instar larvae of the diamondback moth in a culture dish, with 30 larvae per dish, and repeat each treatment three times. Place the dish in a 25°C light incubator with a photoperiod of 14L:10D. Observe the leaves every day to see if they are rotten or dry, and if there is condensation. Count the number of dead and live larvae for 48 hours, and calculate the corrected mortality rate and LC 50 .

[0073] The bioassay was performed using Plutella xylostella larvae. The larval mortality rates obtained using different concentrations of wild-type Cry1Ac and mutants were as follows: Figure 5 The LC values ​​of wild-type Cry1Ac and mutants F371Q, N372E, I373Q and I375Q were calculated. 50 The values ​​were 0.0022 mg / L, 0.0004 mg / L, 0.0006 mg / L, 0.0003 mg / L and 0.0005 mg / L, respectively, that is, the insecticidal activity of mutants F371Q, N372E, I373Q and I375Q were 4.5 times, 2.7 times, 6.3 times and 3.4 times higher than that of the wild type, respectively.

Claims

1. A highly active Cry1Ac mutant, characterized in that: It is obtained by only having the mutation of I373Q based on the amino acid sequence of the wild-type Cry1Ac. The amino acid sequence of the wild-type Cry1Ac has a reference NCBI accession number of AAU87037.

1.

2. A nucleic acid encoding the highly active Cry1Ac mutant according to claim 1.

3. The nucleic acid according to claim 2, characterized in that It is obtained by mutation based on the nucleotide sequence shown in NCBI accession number AY730621.

1.

4. An expression vector comprising the nucleic acid according to claim 2 or 3.

5. A recombinant host bacteria containing the nucleic acid according to claim 2 or 3 or the expression vector according to claim 4.

6. Use of the highly active Cry1Ac mutant according to claim 1 or 2 in the preparation of drugs for controlling insects; the insect is Plutella xylostella.

7. Use of the highly active Cry1Ac mutant according to claim 1 in controlling insects; the insect is Plutella xylostella.

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