CN107602678B - Acetamiprid-coupled Bt insecticidal toxin and preparation method and application thereof - Google Patents
Acetamiprid-coupled Bt insecticidal toxin and preparation method and application thereof Download PDFInfo
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Abstract
本发明提供一种啶虫脒偶联的Bt杀虫毒素，其是在Bt杀虫蛋白Cry2Ab，和羧基啶虫脒在偶联剂EDC、NHS的共同作用下键合形成的一个新的偶联型杀虫毒素，该Bt杀虫毒素具有较强的毒力，其对小菜蛾具有更好的杀虫效果，即对防治农业害虫具有更好的应用前景，能够作为针对小菜蛾的农业杀虫剂；且同时揭露了该Bt杀虫毒素的制备方法，该制备方法过程简单便捷、可操作性强。
The present invention provides a Bt insecticidal toxin coupled with acetamiprid, which is a new coupling formed by bonding the Bt insecticidal protein Cry2Ab and carboxyl acetamiprid under the joint action of coupling agents EDC and NHS. type insecticidal toxin, the Bt insecticidal toxin has strong virulence, and it has better insecticidal effect on diamondback moth, that is, it has better application prospects for the control of agricultural pests, and can be used as an agricultural insecticide against diamondback moth At the same time, a preparation method of the Bt insecticidal toxin is disclosed, and the preparation method is simple, convenient, and highly operable.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the field of agricultural pest control, and particularly relates to an acetamiprid-coupled Bt insecticidal toxin, and a preparation method and application thereof.
[ background of the invention ]
In recent years, agricultural science is rapidly developed, and the prevention and the treatment of pesticides are promoted to a certain extent. The pesticide can effectively prevent and control pests such as agricultural diseases, insects, grasses, rats and the like, and ensures an important link of agricultural production increase and harvest. But the generation of the drug resistance of diseases is a difficult problem which cannot be avoided by chemical pesticides or biological pesticides; the pest resistance to pesticide is to overcome the action of pesticide site and make it ineffective and passivated. In practical applications, pesticide alternation application, pesticide mixing application, increasing the contact area of pesticide to pests, breeding of sensitive strains of pests, increasing ecological diversity to increase pest diversity, and the like, substantially maintain sufficient sensitivity of pesticide action sites, and seek means for overcoming resistance. The biological coupling is to couple two toxins to form a new toxin by utilizing a biological coupling technology, and has important significance for maintaining the sensitivity of pesticide sites and the like.
Bacillus thuringiensis (Bt for short) is a microbial pesticide with the largest production quantity and the most extensive application in the world at present, and the crystal protein of the Bacillus thuringiensis has insecticidal toxicity to more than 570 lepidoptera pests and dozens of pests such as diptera, hymenoptera and coleoptera. It is generally thought that after feeding on crystallin, the insect can carry out enzymolysis to active toxin protein by virtue of its own midgut protease, and the toxin protein is then combined with receptors on Brush Border Membranes (BBMVs) of midgut epithelial cells and further inserted into the membranes to form holes or ion channels, so that ion leakage is caused, epithelial cells on intestinal walls are damaged, intestinal solutes permeate into blood cavities, septicemia is caused, and the insect is killed; however, the resistance of insects to drugs sharply reduces the control effect of the existing Bt toxin, so that the improvement of the Bt toxin and the provision of the Bt toxin with stronger toxicity are eagerly desired by practitioners.
[ summary of the invention ]
The invention aims to solve the technical problem of providing an acetamiprid-coupled Bt insecticidal toxin and a preparation method and application thereof.
The invention solves the technical problems through the following technical scheme:
an acetamiprid-coupled Bt insecticidal toxin, wherein the chemical structural formula of the Bt insecticidal toxin is as follows:
also discloses the acetamipridThe preparation method of the coupled Bt insecticidal toxin comprises the following specific operations: respectively weighing carboxyl acetamiprid, EDC and NHS according to the molar ratio of 1:1.5:1.5, dissolving in a DMSO solvent to activate the carboxyl of the carboxyl acetamiprid to obtain activated carboxyl acetamiprid for later use; preparing Na containing 5mmol/L Bt Cry2Ab insecticidal protein from Bt Cry2Ab insecticidal protein2CO3/ NaHCO3A buffer solution, namely a Bt Cry2Ab insecticidal protein solution, for later use; respectively measuring the prepared activated carboxyl acetamiprid and the Bt Cry2Ab insecticidal protein solution, and stirring for 1 hour to perform coupling reaction to obtain the Bt insecticidal toxin.
Further, the preparation steps of the Bt Cry2Ab insecticidal protein are as follows:
(1) transforming the cry2Ab gene into an escherichia coli engineering bacterium by a hot shock method to obtain the escherichia coli engineering bacterium containing the cry2Ab gene, then inoculating the escherichia coli engineering bacterium onto an LB solid culture medium, and placing the LB solid culture medium at the constant temperature of 30 ℃ for 24h to carry out strain activation;
(2) inoculating the activated engineering bacteria strain of Escherichia coli into LB liquid culture medium, placing in shake flask, fermenting and culturing at 37 deg.C with rotation speed set to 180r/min, and waiting for bacterial liquid OD600After reaching 0.5 ℃, adjusting the temperature to 25 ℃, and continuing culturing for 24 hours at 180 r/min; then placing the fermentation broth obtained by fermentation culture at 4 deg.C and under 10000r/min for centrifuging for 10min, taking out precipitate after centrifugation, and resuspending in Na2CO3Performing ultrasonic lysis on lysate at 4 deg.C for 30min, centrifuging the lysed solution at 4 deg.C at 10000r/min for 30min, and collecting supernatant;
(3) passing the supernatant obtained in the step (2) through an IDA-Ni affinity chromatography column, removing impure proteins by adopting a Ni50 buffer solution, and eluting target proteins by adopting a Ni500 buffer solution; desalting the eluted target protein by a desalting column PD-10 to obtain Bt Cry2Ab insecticidal protein, and storing the Bt Cry2Ab insecticidal protein at-20 ℃ for later use.
Further, the formulation of the Ni50 buffer solution is NaCl 300mM, NaH2PO450mM, imidazole 50 mM; the formulation of the Ni500 buffer solution is NaCl 300mM and NaH2PO450mM, imidazole 500 mM.
Further, the preparation process of the carboxyl acetamiprid comprises the following steps: dissolving mercaptopropionic acid, potassium hydroxide and acetamiprid in an organic solvent DMSO according to a mass ratio of 1:1:2, and reacting for 2 hours at a constant temperature of 100 ℃; after the reaction is finished, cooling the reaction system to room temperature, pouring the reaction system into a clean beaker filled with secondary distilled water, and adding 12 mol.L-1HC1 adjusted the pH of the reaction solution to 3; then extracting the reaction liquid for multiple times by using dichloromethane, and collecting an organic phase; and then carrying out reduced pressure concentration on the organic phase to obtain a crude product, and carrying out silica gel column chromatography separation on the crude product to obtain the carboxyl acetamiprid.
The invention also discloses application of the Bt insecticidal toxin as an agricultural insecticide.
The invention has the beneficial effects that: the Bt insecticidal toxin coupled with the acetamiprid is provided, has stronger toxicity, has better insecticidal effect on diamond back moths, can be used as an agricultural insecticide, and has better application prospect on preventing and controlling agricultural pests; and simultaneously discloses a preparation method of the Bt insecticidal toxin, and the preparation method has simple and convenient process and strong operability.
[ description of the drawings ]
The invention will be further described with reference to the following examples with reference to the accompanying drawings.
FIG. 1 is a SDS-PAGE profile of example 4 of the present invention.
[ detailed description ] embodiments
For better understanding of the present invention, the following examples and application examples are further set forth to illustrate the present invention, but are not intended to limit the scope of the present invention.
Example 1
Preparation of carboxyl acetamiprid
Acetamiprid (2.26g) was dissolved in 15mL of DMSO in a three-necked flask, KOH (1.13g) was added, and 10mL of beta-mercaptopropionic acid (1.13g) dissolved in DMSO was added dropwise through a constant pressure funnel under stirring, followed by gradually raising the temperature to 100 ℃ and reacting for 2 hours; after the reaction is finished, cooling the reaction system to room temperature, pouring the reaction system into a clean beaker filled with secondary distilled water, and adding 12 mol.L-1HC1 adjusting the pH of the reaction solution3; and then extracting the reaction liquid for multiple times by using dichloromethane, collecting an organic phase, concentrating the organic phase under reduced pressure to obtain a crude product, and separating the crude product by silica gel column chromatography to obtain the carboxyl acetamiprid.
The structure of the acetamiprid was identified by MS and NMR techniques:
13C NMR(100MHz,DMSO)：δ173.05,157.91,157.43,149.18,136.70, 128.16,122.19,118.16,50.47,38.01,34.59,25.10,19.52；
HR-ESI-MS:C13H15N4O2S([M-H]-):291.0975。
thus, the chemical structure of the carboxyl acetamiprid is determined, and the reaction chemical formula involved in the example is as follows:
example 2
Preparation of insecticidal protein of Bacillus thuringiensis, namely Bt Cry2Ab insecticidal protein
(1) Transforming the cry2Ab gene into an escherichia coli engineering bacterium by a hot shock method to obtain the escherichia coli engineering bacterium containing the cry2Ab gene, then inoculating the escherichia coli engineering bacterium containing the cry2Ab gene onto an LB solid culture medium, and placing the LB solid culture medium at the constant temperature of 30 ℃ for 24h to carry out strain activation;
(2) inoculating the activated engineering bacteria strain of Escherichia coli into LB liquid culture medium, placing in shake flask, fermenting and culturing at 37 deg.C with rotation speed set to 180r/min, and waiting for bacterial liquid OD600After reaching 0.5 ℃, adjusting the temperature to 25 ℃, and continuing culturing for 24 hours at 180 r/min; then placing the fermentation broth obtained by fermentation culture at 4 deg.C and under 10000r/min for centrifuging for 10min, taking out precipitate after centrifugation, and resuspending in Na2CO3Performing ultrasonic lysis on lysate at 4 deg.C for 30min, centrifuging the lysed solution at 4 deg.C at 10000r/min for 30min, and collecting supernatant;
(3) passing the supernatant obtained in step (2) through IDA-Ni affinity chromatography column, and first using Ni50 buffer solution (NaCl 300mM, NaH)2PO450mM, imidazole 50mM) to remove the heteroprotein; rear endNi500 buffer (NaCl 300mM, NaH) was used2PO450mM, imidazole 500mM) eluting the target protein; desalting the eluted target protein by a desalting column PD-10 to obtain Bt Cry2Ab insecticidal protein, and storing the Bt Cry2Ab insecticidal protein at-20 ℃ for later use.
Example 3
Preparation of Bt insecticidal toxin
Dissolving the carboxyl acetamiprid (0.5mmol) prepared in the example 1 in 1mL of DMSO, adding EDC (0.75mmol) and NHS (0.75mmol), stirring at room temperature for 2h, and activating the carboxyl of the carboxyl acetamiprid to obtain activated carboxyl acetamiprid for later use; taking the Bt Cry2Ab insecticidal protein prepared in example 2 to prepare Na containing 5mmol/L Bt Cry2Ab insecticidal protein2CO3/NaHCO3A buffer solution, namely a Bt Cry2Ab insecticidal protein solution, for later use; and then adding 100 mu L of activated carboxyl acetamiprid into 1mL of Bt Cry2Ab insecticidal protein solution at 4 ℃, and stirring for 1 hour to perform coupling reaction to obtain the Bt insecticidal toxin.
Example 4
SDS-PAGE detection of coupling effects
The coupling effect of the coupling reaction process in example 3 was detected by SDS-PAGE, and the detection results are shown in FIG. 1 (in FIG. 1, M: marker; 10: sampling after 10min of coupling; 20: sampling after 20min of coupling; 30: sampling after 30min of coupling; 40: sampling after 40min of coupling; 50: sampling after 50min of coupling; C: Bt Cry2Ab insecticidal protein before coupling); as can be seen from FIG. 1, after 50min from the beginning of coupling, the Bt Cry2Ab protein and the activated carboxyl acetamiprid are obviously coupled, and the molecular weight is increased by about 1Kd before and after coupling, namely the coupling is successful.
Application example 1
Insecticidal Activity assay of Bt insecticidal toxins
(1) Preparation of diamondback moth
Collecting indoor diamondback moth pupa, eclosion, collecting adult moth eggs, collecting diamondback moth eggs once every 24 hours, and feeding the collected diamondback moth eggs in the same batch in a climatic chamber under the same condition for LC50The conditions of the artificial climate box are measured as follows: the temperature is 25 ℃ and the relative humidity is 70%And photoperiod 16: 8 (L: D).
(2) Verification method and results
The mortality of Bt Cry2Ab insecticidal protein, acetamiprid and Bt insecticidal toxin prepared in example 3 to diamondback moth second-instar larvae is determined by adopting a 24-hole plate feeding method, and an experimental group and two control groups (one control group and the second control group respectively) are arranged at the same time. The specific experimental method is as follows: diluting Bt insecticidal toxin to obtain Bt insecticidal toxin solutions with different concentrations; then sucking 1mL of feed under aseptic environment, subpackaging in 24-pore plate, naturally drying, sucking 100 μ L of prepared Bt insecticidal toxin solution with different concentrations, uniformly coating on the surface of feed, and uniformly coating with 100 μ L of Na2CO3/ NaHCO3Using buffer solution as blank control, marking each group, and airing in a natural state; inoculating diamondback moth of second age to 24-well plate (5-7 in each well), repeating each concentration gradient for 4 wells, and determining death rate of diamondback moth after 48 hours (death rate of diamondback moth is determined by touching diamondback moth with writing brush for immobility) to obtain LC50. In one of the control groups, the only difference from the experimental group was that the Bt Cry2Ab insecticidal protein was used as the effector. In the second control group, the only difference from the experimental group is that acetamiprid is used as the effector. The test results are shown in table 1 below.
TABLE 1 insecticidal Effect of Bt insecticidal toxins
Analysis via table 1 and using a regression model of the SPSS software gave: the LC50 of the Bt Cry2Ab insecticidal protein in one of the control groups to the diamondback moth is 23.825 mug/mL, the LC50 of the Bt insecticidal toxin in the experimental group to the diamondback moth is 9.881 mug/mL, and the insecticidal toxicity is improved by 2.41 times; LC50 for diamondback moth of the control group was 276.53 μ g/mL. The result shows that the Bt insecticidal toxin can improve the insecticidal toxicity to the plutella xylostella.
In conclusion, the Bt insecticidal toxin formed by coupling has stronger toxicity, and the Bt insecticidal toxin has better insecticidal effect on diamond back moths, in other words, the Bt insecticidal toxin can be used as an agricultural insecticide for killing the diamond back moths, and has better application prospect for preventing and controlling agricultural pests.
Claims (2)
1. A preparation method of acetamiprid-coupled Bt insecticidal toxin is characterized by comprising the following steps: the structural formula of the Bt insecticidal toxin is as follows:
the preparation method comprises the following specific operations: respectively weighing carboxyl acetamiprid, EDC and NHS according to the molar ratio of 1:1.5:1.5, dissolving in a DMSO solvent to activate the carboxyl of the carboxyl acetamiprid to obtain activated carboxyl acetamiprid for later use; preparing Na containing 5mmol/L Bt Cry2Ab insecticidal protein from Bt Cry2Ab insecticidal protein2CO3/ NaHCO3A buffer solution, namely a Bt Cry2Ab insecticidal protein solution, for later use; respectively measuring the prepared activated carboxyl acetamiprid and the Bt Cry2Ab insecticidal protein solution, and stirring for 1 hour to perform coupling reaction to obtain Bt insecticidal toxin;
the preparation process of the carboxyl acetamiprid comprises the following steps: dissolving mercaptopropionic acid, potassium hydroxide and acetamiprid in an organic solvent DMSO according to a mass ratio of 1:1:2, and reacting for 2 hours at a constant temperature of 100 ℃; after the reaction is finished, cooling the reaction system to room temperature, pouring the reaction system into a clean beaker filled with secondary distilled water, and adding 12 mol.L-1HC1 adjusted the pH of the reaction solution to 3; then extracting the reaction liquid for multiple times by using dichloromethane, and collecting an organic phase; and then carrying out reduced pressure concentration on the organic phase to obtain a crude product, and carrying out silica gel column chromatography separation on the crude product to obtain the carboxyl acetamiprid.
2. The method for preparing the acetamiprid-coupled Bt insecticidal toxin according to claim 1, characterized in that: the preparation steps of the Bt Cry2Ab insecticidal protein are as follows:
(1) will be provided withcry2AbThe gene is transformed into Escherichia coli engineering bacteria by heat shock method to obtain the productcry2AbOf genesInoculating the engineering bacteria of the escherichia coli to an LB solid culture medium, and culturing the LB solid culture medium at the constant temperature of 30 ℃ for 24 hours to activate the strains;
(2) inoculating the activated engineering bacteria strain of Escherichia coli into LB liquid culture medium, placing in shake flask, fermenting and culturing at 37 deg.C with rotation speed set to 180r/min, and waiting for bacterial liquid OD600After reaching 0.5 ℃, adjusting the temperature to 25 ℃, and continuing culturing for 24 hours at 180 r/min; then placing the fermentation broth obtained by fermentation culture at 4 deg.C and under 10000r/min for centrifuging for 10min, taking out precipitate after centrifugation, and resuspending in Na2CO3 Performing ultrasonic lysis on lysate at 4 deg.C for 30min, centrifuging the lysed solution at 4 deg.C at 10000r/min for 30min, and collecting supernatant;
(3) passing the supernatant obtained in the step (2) through an IDA-Ni affinity chromatography column, removing impure proteins by adopting a Ni50 buffer solution, and eluting target proteins by adopting a Ni500 buffer solution; desalting the eluted target protein by a desalting column PD-10 to obtain Bt Cry2Ab insecticidal protein, and storing the Bt Cry2Ab insecticidal protein at-20 ℃ for later use; the formulation of the Ni50 buffer solution is NaCl 300mM and NaH2PO450mM, imidazole 50 mM; the formulation of the Ni500 buffer solution is NaCl 300mM and NaH2PO450mM, imidazole 500 mM.
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| WO2010096874A1 (en) * | 2009-02-25 | 2010-09-02 | The Crown in the right of the State of New South Wales | Use of benzodioxole derivatives and bt toxins in the control of pets |
| CN104968672A (en) * | 2012-07-02 | 2015-10-07 | 先锋国际良种公司 | Novel insecticidal proteins and methods for their use |
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| CN102675411A (en) * | 2012-05-08 | 2012-09-19 | 福建省农业科学院农业生物资源研究所 | Method for preparing biotoxin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010096874A1 (en) * | 2009-02-25 | 2010-09-02 | The Crown in the right of the State of New South Wales | Use of benzodioxole derivatives and bt toxins in the control of pets |
| CN104968672A (en) * | 2012-07-02 | 2015-10-07 | 先锋国际良种公司 | Novel insecticidal proteins and methods for their use |
Non-Patent Citations (2)
| Title |
|---|
| PxAPN5 serves as a functional receptor of Cry2Ab in Plutella xylostella (L.) and its binding domain analysis;Zhi-Zhen Pan 等;《International Journal of Biological Macromolecules》;20170716;第105卷;第516-521页 * |
| 苏云金芽孢杆菌一新菌株的鉴定及其杀虫活性;孟鑫睿 等;《植物保护学报》;20150630;第42卷(第3期);第410-417页 * |
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