A Cry2Ab protein mutant and its application

Abstract

The present invention provides a Cry2Ab protein mutant and its application, belonging to the field of genetic engineering and biological control technology. The mutant is Cry2Ab-L183I, and its amino acid sequence is shown in SEQ ID NO.2. The mutant protein Cry2Ab-L183I obtained by the present invention has a 107.0% higher toxicity to diamondback moth than the wild type, and can effectively replace traditional Bt insecticides for biological control of pests.

Description

[0001] This application is a divisional application. The application number of the original application is 202210430242.9 and the application date is April 22, 2022. On the day, the application was named "A Cry2Ab protein mutant and its application." Technical Field

[0002] The invention belongs to the technical field of genetic engineering and biological control, and specifically relates to a Cry2Ab protein mutant and application thereof in killing lepidopteran agricultural pests. Background Art

[0003] The diamondback moth (Plutella xylostella) is a worldwide lepidopteran pest, widely distributed in various provinces of my country, and its diet is very wide, and it can feed on more than 40 host plants. At present, the prevention and control of the diamondback moth mainly adopts the means of spraying chemical pesticides, and the diamondback moth has developed a high level of resistance to them. Current research shows that the diamondback moth has developed resistance to more than 50 commonly used chemical synthetic pesticides, becoming the number one pest in my country's vegetable cultivation, causing huge economic losses to my country. For many years, people have been committed to the study of the mechanism of the diamondback moth's resistance to pesticides, and the prevention and control of the diamondback moth has become an important topic that cannot be ignored worldwide.

[0004] Bacillus thuringiensis (Bt) is the most widely used microbial insecticide, used to control agricultural pests around the world, and the diamondback moth is the only pest reported to be resistant to Bt toxins in the field. One of the characteristics of Bt is that parasporal crystals are produced during spore formation, the main component of which is δ-endotoxin with insecticidal effect. The insecticidal protein encoded by the Cry gene, Cry protein, is one of the δ-endotoxins with the most thorough insecticidal mechanism research and the most widely used.

[0005] At present, the main strategies for managing Bt resistance are: high dose / refuge, multi-gene strategy, development of new toxins, and modification of Bt Cry toxins. The limitation of high dose / refuge and multi-gene strategies is that they can only control pest resistance at a low level, and long-term use may produce more serious resistance; the research and development of new toxins is currently unable to meet the management of resistant insects due to low screening efficiency and long research and development time. The modification of Cry toxins is achieved by replacing, adding or deleting key amino acid residues to modify the toxins. The modification of Cry toxins can quickly change the properties of proteins, such as increasing the stability of Cry toxins, improving the binding of Cry toxins to insect midgut receptors, and enhancing the oligomerization ability or membrane perforation ability of Cry toxins in insect midgut cell membranes, thereby improving the insecticidal activity of Cry toxins and achieving the purpose of overcoming insect resistance.

[0006] Since the modification of Cry toxins is highly targeted and efficient, it has become an important means of managing Bt resistance. The N-terminus of Cry protein plays an important role in the insecticidal activity, and the amino acid region related to the insecticidal specificity of Cry protein to Plutella xylostella is 145 SW 198 However, most of the mutants reported are mutations with reduced activity, and there are few successful cases of improving activity by single-point mutations. Summary of the invention

[0007] Therefore, it is necessary to provide a method for constructing a Cry2Ab mutant with high activity by using site-directed mutagenesis technology to improve the insecticidal effect.

[0008] To achieve the above purpose, the inventors provide the following technical solutions:

[0009] A Cry2Ab protein mutant, wherein the mutant is Cry2Ab-L183I, and its amino acid sequence is shown in SEQ ID NO.2.

[0010] The gene sequence encoding the Cry2Ab-L183I mutant is shown in SEQ ID NO.1.

[0011] Furthermore, the Cry2Ab protein mutant is used in the preparation of insecticides.

[0012] Furthermore, the Cry2Ab protein mutant is used in killing lepidopteran pests.

[0013] The beneficial effects of the present invention are:

[0014] The present invention uses site-directed mutagenesis technology to perform site-directed mutagenesis on key amino acids and constructs a series of Cry2Ab mutants. The leucine (Leu) at position 183 is mutated to isoleucine (Ile), and the mutant protein L183I obtained has a 107.0% higher toxicity to 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. It effectively solves the problem of finding Bacillus thuringiensis insecticidal protein genes that produce high toxicity to diamondback moth, and delays the resistance of pests to engineered bacteria and transgenic plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the Cry2Ab mutant sequence described in the specific implementation manner, and the result of alignment with the Wild-type original sequence.

[0016] Figure 2The results of protein expression of Cry2Ab and mutant genes in E. coli (BL21) described in the specific implementation method are shown in Figure 1. 1: Cry2Ab Wild-type original protein; 2: Cry2Ab-N151H mutant protein; 3: Cry2Ab-L183I mutant protein; KDA: protein marker, molecular weight 11-180KD. DETAILED DESCRIPTION

[0017] 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.

[0018] Example 1

[0019] 1 Materials and reagents

[0020] 1.1 Strain source

[0021] The Bacillus thuringiensis strain BtLSZ9408 is a highly toxic strain to lepidopteran pests isolated and screened by the Biotechnology Center of Fujian Academy of Agricultural Sciences. It belongs to the Kustaki subspecies (B.thuringiensis var.kurstaki) and has a serotype of H1; competent Escherichia coli BL21 was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. and Yueyang Pharmaceutical Development (Guangzhou) Co., Ltd.

[0022] 1.2 Culture medium

[0023] LB liquid medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, dilute to 1000 mL with deionized water, and store at 4°C after sterilization (LB solid medium plus 15 g of agar; Kanamycin-LB medium plus kanamycin to a final concentration of 50 μg / ml).

[0024] 1.3 Buffer

[0025] Buffer 1: 20mM CaCl2 + 80mM MgCl2

[0026] Buffer 2: 100 mM CaCl2 + 10% glycerol

[0027] Ni-50 buffer (PH = 8.0): NaH2PO4 50mM, NaCl 300mM, imidazole 50mM

[0028] Ni-100 buffer (PH = 8.0): NaH2PO4 50mM, NaCl 300mM, imidazole 100mM

[0029] Ni-500 buffer (PH = 8.0): NaH2PO4 50mM, NaCl 300mM, imidazole 500mM

[0030] 2 Experimental methods

[0031] 2.1 Construction of Cry2Ab mutants

[0032] 2.1.1 Preparation of competent E. coli BL21

[0033] 1. Take the E. coli BL21 bacterial solution and streak it on the antibiotic-free LB plate, and culture it in an incubator overnight (37°C);

[0034] 2. Pick a single colony of E. coli BL21 every other day, inoculate it into 10 mL of antibiotic-free LB liquid medium, and culture it at 37°C and 220 rpm for 7-8 hours;

[0035] 3. Inoculate E. coli BL21 into 100 mL of fresh LB liquid medium at a ratio of 1:100, and culture at 37°C with shaking at 220 rpm for 2-3 hours until the OD600nm of the bacteria is between 0.4 and 0.5;

[0036] 4. Add the E. coli BL21 bacterial solution into a sterilized 50 mL centrifuge tube and centrifuge at 4°C and 5000 rpm for 10 min.

[0037] 5. Discard the supernatant and add 5 mL of pre-cooled buffer 1 to resuspend; then add 25 mL of pre-cooled buffer 1, place on ice for 20 min, and centrifuge at 4°C and 5000 rpm for 10 min;

[0038] 6. Discard the supernatant, add 3 mL of pre-cooled buffer 2, gently suspend the precipitate, and place on ice for 5 minutes.

[0039] 7. The prepared competent cells are packaged in 100 μL / tube and stored at -80°C. There are two main points to pay attention to when making competent cells: ensure sterility and low temperature throughout the process.

[0040] 2.1.2 Cry2Ab plasmid extraction

[0041] Select the recombinant plasmid pET30a-cry2Ab as the vector for prokaryotic expression. Pick the E. coli containing the pET30a-cry2Ab plasmid and inoculate it into 3-5 mL of LB liquid medium containing Kana, and culture it overnight at 37°C and 220rpm. Use a rapid plasmid extraction kit to extract plasmid DNA. The collected plasmid solution is stored at -20°C.

[0042] 2.1.3 Mutant Primer Whole Plasmid PCR

[0043] In order to construct mutants at key sites of Bacillus thuringiensis, the Cry2Ab183 site was selected, and site-directed mutants with mutations in key amino acid residues were designed for site-directed mutagenesis. Different forward and reverse primers were designed, and the primer sequences are shown in Table 1.

[0044] Table 1 Primer sequences of target mutants

[0045]

[0046] PCR system for amplifying the upstream DNA fragment of Cry2Ab mutant (10 μL system) for full plasmid PCR reaction conditions:

[0047]

[0048] PCR program settings: 95°C pre-denaturation for 5 min; 98°C denaturation for 20 s, 60°C annealing for 20 s, 72°C extension for 90 s, 25 cycles; 72°C extension for 10 min; 4°C hold.

[0049] 2.1.4Q.CutDpn1 digestion system

[0050] The PCR product was digested with restriction endonuclease Q.CutDpn1. The digestion reaction system was as follows (11 μL system):

[0051]

[0052] The above system was reacted in a water bath at 30°C for 90 min to complete the digestion reaction.

[0053] 2.1.5 Ethanol dehydrogenation

[0054] The digested product was added with 4 times the volume of anhydrous ethanol, followed by -20°C low-temperature treatment for 20 min, and then centrifuged at 5000 rpm for 1 min. The supernatant was aspirated, the remaining ethanol was dried with a hair dryer, and 10 μL of sterile water was repeatedly blown and sucked along the tube wall with a pipette to obtain the purified plasmid.

[0055] 2.1.6 Transformation of mutant recombinant plasmid into competent BL21

[0056] The ethanol dehydrogenation product was transformed into competent Escherichia coli BL21 by heat shock method. The specific method is as follows:

[0057] 1. Take 10 μL of ethanol dehydrogenation product and add 100 μL of competent E. coli BL21, mix well by pipetting slowly, and place in an ice box for 30 min.

[0058] 2. Place the competent E. coli BL21 in a 42°C water bath for 90 seconds, quickly take it out and place it in an ice box for 5 minutes.

[0059] 3. Add 900 μL of fresh LB culture medium to each tube of competent E. coli BL21 and culture at 37°C and 220 rpm for 60 min.

[0060] 4. Centrifuge the cultured bacterial solution at 5000 rpm for 1 min; retain about 100 μL of the supernatant, fully suspend the bacterial pellet, spread it on a Kana-resistant solid LB plate (final Kana concentration of 50 μg / mL), and culture it in a 37°C incubator for 16 h.

[0061] 2.1.7 Identification of positive clones

[0062] After culturing in a 37°C incubator for 16 h, positive clones were picked and sent to the company for DNA sequencing to obtain mutant sequences and compare them with the original sequences.

[0063] 2.2 Protein IPTG-induced expression and purification

[0064] 2.2.1 IPTG-induced protein expression of Cry2Ab key amino acid residue mutants

[0065] First, the mutant strain of the key amino acid residue of Cry2Ab was expanded and cultured. One night in advance, a small amount of mutant strain was picked up with a small-range pipette tip and added to 10 mL of liquid LB medium, and Kanamycin was added to make the final concentration 50 μg / mL, and then shaken on a shaker overnight, with the temperature set to 36.5°C and the speed set to 170 rpm.

[0066] The next day, take a fresh 100mL bottle of liquid LB medium, inoculate the overnight cultured bacteria into the large bottle for expansion, add Kanamycin to a final concentration of 50μg / mL, and continue to culture on a shaker at 37°C and 180rpm for 4-5h until the OD600nm reaches about 0.5. Then add IPTG for protein induction expression. The culture temperature is 25°C, the shaker speed is 150rpm, and the induction time is 24h. Under this condition, the protein induction expression efficiency is the highest.

[0067] 2.2.2 Protein purification of Cry2Ab key amino acid residue mutants

[0068] (1) Ultrasonic cell disruptor for bacterial lysis

[0069] 1. The BL21 bacterial solution of the Cry2Ab key amino acid residue mutant induced by IPTG was dispensed into 50 mL centrifuge tubes, the centrifuge temperature was set to 4 ° C, the speed was set to 7000 rpm, and after centrifugation for 15 minutes, the supernatant was removed;

[0070] 2. Resuspend the precipitate, add the appropriate amount of Ni-50 in proportion, and mix it by repeatedly blowing and sucking with a pipette. Then use an ultrasonic cell disruptor to disrupt the cell. This process requires low temperature because high temperature will destroy the structure of the protein. Before putting it into the ultrasonic cell disruptor, you can put the centrifuge tube in a beaker filled with ice-water mixture to ensure low temperature.

[0071] 3. The ultrasonic time is set to 3s of ultrasound, 2s of pause, and the whole process is set to 20min.

[0072] 4. Take out the centrifuge tube and centrifuge it in a pre-cooled 4℃ centrifuge at 6000rpm. After the end, keep the supernatant and discard the precipitate. The soluble target protein is present in the supernatant.

[0073] (2) Purification of Cry2Ab key amino acid residue mutant protein

[0074] Balance and activate the IDA-Ni column: Pour 40 mL of pH 8.0 Ni-50 buffer into the IDA-Ni column. Connect an infusion tube with a controller under the column to facilitate flow rate control. After all the Ni-50 buffer flows through the IDA-Ni column, add the supernatant containing the mutant protein of the key amino acid residue of Cry2Ab obtained after centrifugation in the previous step.

[0075] Adsorption of target protein: Control the speed of each supernatant passing through the IDA-Ni column at about 5mL / min. After the end, add about 30mL of Ni-100 buffer and control the flow rate at about 10mL / min. The purpose of this operation is to remove impurities. At different stages when the Ni-100 buffer flows through the IDA-Ni column, use Coomassie Brilliant Blue G-250 solution to detect the protein content of the effluent. At the beginning, after the effluent is drawn and added to the Coomassie Brilliant Blue G-250 solution, it will be found that it immediately turns blue; but when the Ni-100 buffer flows out of the IDA-Ni column immediately, the effluent will not appear blue after being added to the Coomassie Brilliant Blue G-250 solution.

[0076] Elution of target protein: Use Ni-500 to elute the target protein of Cry2Ab key amino acid residue mutant. Add Ni-500 buffer, 1 mL each time, and collect the effluent from each tube with a 1.5 mL centrifuge tube. Collect 6-10 tubes to obtain a higher concentration of target protein. Use Coomassie Brilliant Blue G-250 solution to detect the protein. The effluent from tubes 1-3 will hardly turn the Coomassie Brilliant Blue G-250 solution blue, and the blue color of tubes 4-6 is darker. The more effluent is collected later, the less likely it is to turn the Coomassie Brilliant Blue G-250 solution blue. Glycerol can be added to the collected Cry2Ab key amino acid residue mutant target protein to prevent its precipitation.

[0077] 2.2.3 SDS-PAGE protein electrophoresis

[0078] While inducing the expression of mutant proteins, the BL21 bacterial solution containing the wild-type Cry2Ab plasmid was used as a control group. After the induction, the induction status was detected using SDS-PAGE electrophoresis.

[0079] 2.2.4 Desalting of Cry2Ab protein

[0080] The Cry2Ab protein was replaced into Na2CO3 buffer (50 mM, pH 9.5) using a desalting column PD-10.

[0081] 2.2.5 Protein concentration determination

[0082] The protein concentration was determined by the BCA method. A standard curve was drawn with OD562nm as the ordinate and BSA concentration as the abscissa to calculate the protein concentration in the sample.

[0083] 3 Experimental results

[0084] 3.1 Construction of prokaryotic expression vectors for Cry2Ab series saturation mutants

[0085] The mutant recombinant plasmid obtained by PCR cloning was transformed into BL21 competent cells by heat shock method. After the plate was coated, positive mutant colonies were randomly picked on the plate for sequencing. The gene sequence of Cry2Ab-L183I is shown in SEQ ID NO.1, and its corresponding amino acid sequence is shown in SEQ ID NO.2.

[0086] The sequencing results were compared and analyzed. Figure 1 The experimental results showed that the leucine (Leu) at position 183 in the amino acid sequence of Cry2Ab protein mutated into isoleucine (Ile), indicating that the Cry2Ab-L183I mutant had been successfully constructed.

[0087] 3.2 Induced expression and purification of Cry2Ab series mutants

[0088] IPTG induced BL21 cells containing mutant recombinant plasmids to express the corresponding mutant protein. After SDS-PAGE electrophoresis, Cry2Ab-L183I mutant expressed the target protein with a size of 65kDa ( Figure 2 ) and is consistent in size with the original toxin.

[0089] Example 2

[0090] 1 Experiments on Plutella xylostella

[0091] The diamondback moth and its artificial diet used in this experiment were purchased from Keyun Biotechnology Co., Ltd.

[0092] 1.1 Configuration of artificial culture medium for Plutella xylostella

[0093] Take the artificial diet of diamondback moth, add double distilled water and boil it, place it in a 100℃ water bath and stir it quickly to fully dissolve the artificial diet powder; spread the artificial diet in a 10cm diameter round-bottom insect box and dry it naturally. The prepared artificial culture medium is sealed and stored at 4℃ for one week.

[0094] 1.2 Artificial Rearing of Plutella xylostella

[0095] The 10cm insect box is used for ordinary feeding and group feeding of insects. After cutting the egg card into small square pieces of 1×2cm, place it upright or sideways on the feed to prevent the egg card from getting damp and affecting the hatching rate of the diamondback moth. The eggs are incubated at 27±2℃. When the diamondback moth is hatched, it is transferred to a 10cm insect box with flat feed for feeding. After the larvae are cultured to the second instar, the insecticide experiment can be carried out. Whether hatching diamondback moth eggs or raising diamondback moth larvae, a piece of paper towel should be laid flat on the top before closing the lid of the 10cm insect box to prevent the feed from drying out. The artificial feed should be replaced every 3-4 days; the insect box for raising diamondback moths is placed in an artificial climate incubator with the temperature set to 27±2℃; the humidity set to 70%; and the photoperiod set to 14:10h (light / dark).

[0096] 1.3 Bioassay experiment on Plutella xylostella

[0097] The insecticidal toxicity of wild-type Cry2Ab and its mutants to Plutella xylostella was determined by the plate feeding method. 2 mL of artificial culture medium was added to an insect box with a diameter of 35 mm and dried naturally. The Cry2Ab protein was replaced into Na2CO3 buffer (50 mM, pH 9.5) using a desalting column PD-10. Five or six concentration gradients were set for each Cry2Ab protein sample. 300 μL of protein was spread on the artificial culture medium for each concentration gradient, and 300 μL of Na2CO3 buffer was added as a negative control, and dried naturally. 20 healthy late second-instar or early third-instar Plutella xylostella larvae were selected into each culture dish, and the mortality rate of Plutella xylostella was counted after 48 hours (the moth did not move or react slowly when touched by a brush as the standard of death of Plutella xylostella); 3 replicates were made for each protein concentration; the LC of Plutella xylostella larvae was calculated using the PROBIT program in SPSS 17.0 software. 50 The corresponding Cry2Ab concentration.

[0098] 2 Experimental results

[0099] After the target protein was successfully induced and purified, the second-instar diamondback moth was used as the research object to detect the insecticidal activity of the Cry2Ab-L183I mutant (with Wild-type Cry2Ab as a positive control). The results are shown in Table 2.

[0100] LC of wild type against Plutella xylostella 50 0.267 μg / cm 2 , Cry2Ab-L183I against LC of Plutella xylostella 50 0.129 μg / cm 2 The experimental results showed that the LC of Cry2Ab-L183I 50 It was significantly lower than the wild type, indicating that the toxicity was increased after the leucine (Leu) at position 183 was mutated to isoleucine (Ile). The toxicity of the mutant protein L183I to Plutella xylostella was increased by 107.0% compared with the wild type.

[0101] Table 2 Analysis of insecticidal activity of Cry2Ab-N151H and Cry2Ab-L183I mutants against Plutella xylostella

[0102]

[0103] In summary, by using the site-directed mutagenesis method to mutate the leucine (Leu) at position 183 on the amino acid sequence of the Cry2Ab protein into isoleucine (Ile), a Bacillus thuringiensis expressed protein Cry2Ab mutant with high toxicity to the diamondback moth was obtained. Corresponding genetically engineered bacteria are constructed according to the mutant, or insecticides are made using the mutant protein and applied to actual production for biological control, which can delay the resistance of pests to engineered bacteria and transgenic plants. The mutant can produce toxic effects on Lepidoptera insects other than the diamondback moth, such as the beet armyworm, the cutworm, etc.

[0104] It should be noted that, although the above embodiments have been described in this article, the patent protection scope of the present invention is not limited thereby. Therefore, based on the innovative concept of the present invention, changes and modifications made to the embodiments described herein, or equivalent structures or equivalent process changes made using the contents of the present invention specification and drawings, directly or indirectly applying the above technical solutions to other related technical fields, are all included in the patent protection scope of the present invention.

Claims

1. A Cry2Ab protein mutant, characterized in that: The mutant is Cry2Ab-L183I, and its amino acid sequence is shown in SEQ ID NO.

2.

2. The coding gene of the Cry2Ab protein mutant according to claim 1, characterized in that: The gene sequence encoding the Cry2Ab-L183I mutant is shown in SEQ ID NO.

1.

3. Use of the Cry2Ab protein mutant as claimed in claim 1 in the preparation of a Plutella xylostella insecticide.

4. Use of the Cry2Ab protein mutant as claimed in claim 1 in killing lepidopteran pests.

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