CN116948045B - Artificial intelligence assisted insecticidal protein and application thereof - Google Patents
Artificial intelligence assisted insecticidal protein and application thereof Download PDFInfo
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- CN116948045B CN116948045B CN202311218202.9A CN202311218202A CN116948045B CN 116948045 B CN116948045 B CN 116948045B CN 202311218202 A CN202311218202 A CN 202311218202A CN 116948045 B CN116948045 B CN 116948045B
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 46
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 46
- 230000000749 insecticidal effect Effects 0.000 title abstract description 25
- 238000013473 artificial intelligence Methods 0.000 title abstract description 11
- 210000004027 cell Anatomy 0.000 claims description 13
- 102000039446 nucleic acids Human genes 0.000 claims description 9
- 108020004707 nucleic acids Proteins 0.000 claims description 9
- 150000007523 nucleic acids Chemical class 0.000 claims description 9
- 241000256251 Spodoptera frugiperda Species 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 3
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 abstract description 6
- 239000000575 pesticide Substances 0.000 abstract description 3
- 241000238631 Hexapoda Species 0.000 description 8
- 230000000694 effects Effects 0.000 description 5
- 238000000034 method Methods 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 230000009261 transgenic effect Effects 0.000 description 3
- 241000256248 Spodoptera Species 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 230000000853 biopesticidal effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 241000208838 Asteraceae Species 0.000 description 1
- 241000193388 Bacillus thuringiensis Species 0.000 description 1
- 241000219193 Brassicaceae Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 241000255777 Lepidoptera Species 0.000 description 1
- 241000256259 Noctuidae Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 238000013528 artificial neural network Methods 0.000 description 1
- 229940097012 bacillus thuringiensis Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 244000038559 crop plants Species 0.000 description 1
- 238000013135 deep learning Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 238000000455 protein structure prediction Methods 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
- A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
- A01N47/44—Guanidine; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P7/00—Arthropodicides
- A01P7/04—Insecticides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Pest Control & Pesticides (AREA)
- Organic Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Dentistry (AREA)
- Agronomy & Crop Science (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Insects & Arthropods (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The application relates to an artificial intelligence-assisted insecticidal protein and application thereof, and belongs to the field of protein engineering. The application obtains a batch of novel insecticidal proteins based on active site prediction and amino acid sequence generation of an artificial intelligence algorithm. The insecticidal proteins have application value in the fields of preparing pesticides or cultivating genetically engineered plants.
Description
Technical Field
The application relates to an artificial intelligence-assisted insecticidal protein and application thereof, and belongs to the field of protein engineering.
Background
Insecticidal proteins derived from bacillus thuringiensis are currently the proteins of interest commonly used in the development of transgenic crops and biopesticides. However, with the large-scale application of these commercial pesticides and insect-resistant crops, pests generally develop resistance to existing insecticidal proteins, and there is an urgent need to find or create new insecticidal proteins.
Spodoptera frugiperda (subject name: spodoptera frugiperda) belongs to the genus spodoptera of the family spodoptera, and larvae thereof can gnaw a large amount of gramineous crops such as rice, sugarcane and corn, and various crops such as asteraceae and cruciferae, so that serious economic loss is caused. The major transgenic crop plants such as the united states were grown mainly using Cry1Fa, vip3Aa proteins to cultivate new varieties of corn and soybean for control of spodoptera frugiperda, however, due to the strong migration and rapid propagation characteristics of this insect, more and more resistant lines to Cry1Fa, vip3Aa have been found (motoreto J C, michel a P, silva Filho M C, silva n. Adaptive potential of fall armyworm (Lepidoptera: noctuidae) limits Bt trait durability in Brazil J, intelgr, pest manag, 2017, 8, 17). Thus, future spodoptera frugiperda resistant crop varieties are required to obtain novel insecticidal proteins that are completely different from Cry1Fa and Vip3 Aa.
The function of a protein in a cell is determined by its three-dimensional structure. However, it is time-consuming and laborious to determine the protein structure by experiment. With the development of artificial intelligence technology, protein structure prediction has made breakthrough progress. This provides new opportunities for massive research and creation of protein structures. The alpha field artificial intelligent network developed by google deep has given the three-dimensional structure of hundreds of thousands of proteins, including each protein which can be manufactured by human body, and the results are expected to bring more surprise to the fields of medicine and drug design. Although more accurate protein folding, ligand combination and other details have much space to be optimized, the method can realize the prediction of protein structure, experimental test, optimization improvement and finally generate new engineering functional protein by combining the mode of functional verification and screening of experimental layers through artificial intelligence algorithms of deep learning and neural networks.
CN202311033251.5 patent discloses a batch of insecticidal proteins generated with the assistance of artificial intelligence, wherein WBY-1 has higher insecticidal activity. In order to further improve the insecticidal effect of the protein, the application obtains a batch of insecticidal proteins with better effects based on active site prediction and amino acid sequence generation of an artificial intelligence algorithm.
Disclosure of Invention
In order to solve the problems, the application adopts the following technical scheme:
the application provides an artificial intelligence-assisted insecticidal protein, which is characterized by being formed by fusing 468-1169 positions of SEQ ID NO. 4 and SEQ ID NO. 1.
The application also provides a nucleic acid molecule which is characterized in that the nucleic acid molecule codes for the protein.
The application also provides a vector, which is characterized by comprising the nucleic acid molecule.
The application also provides a recombinant cell, which is characterized in that the recombinant cell contains the nucleic acid molecule or the vector.
In some embodiments, the recombinant cell is a prokaryotic cell.
In some embodiments, the recombinant cell is an E.coli cell.
The application also provides application of the protein, the nucleic acid molecule, the vector and the recombinant cell in insect resistance or preparation of insect resistance preparations or cultivation of insect resistance plants.
In some embodiments, the insect-resistant agent is an agent that has insecticidal activity against Spodoptera frugiperda.
The application has the beneficial effects that: the application obtains a insecticidal protein with better effect based on the active region prediction and amino acid sequence generation of the artificial intelligence algorithm, and can be used for developing biological pesticides or cultivating novel insect-resistant plants.
Detailed Description
The following definitions and methods are provided to better define the present application and to guide those of ordinary skill in the art in the practice of the present application. Unless otherwise indicated, terms are to be construed according to conventional usage by those of ordinary skill in the relevant art. All patent documents, academic papers, industry standards, and other publications cited herein are incorporated by reference in their entirety.
The following examples are illustrative of the application and are not intended to limit the scope of the application. Modifications and substitutions to methods, procedures, or conditions of the present application without departing from the spirit and nature of the application are intended to be within the scope of the present application. Examples follow conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular cloning: a laboratory manual, 2001), or conditions recommended by the manufacturer's instructions, unless otherwise indicated. Unless otherwise indicated, all chemical reagents used in the examples were conventional commercial reagents, and the technical means used in the examples were conventional means well known to those skilled in the art.
Example 1 active region prediction and sequence Generation of proteins
The application uses WBY-1 protein (the sequence is shown as SEQ ID NO. 1) in the CN202311033251.5 patent as a template for active site analysis and sequence generation. Since the D1 and D2 domains are improving insecticidal effectThe protein has great effect on activity. Thus utilize alpha Fold2、The RoseTTAFold on-line tool predicts the active regions of the D1 and D2 domains (positions 1-467) of protein WBY-1, and then uses the database and the PROTEINGAN algorithm prediction tool to build a model to generate and design the amino acid sequence of the active region. The protein structure was generated using a Chimera 1.17 visual comparison.
6 proteins are selected from WBY-1 active region modified proteins through scoring ranking, the proteins are respectively named WBY-1.01-WBY-1.06, the amino acid sequence of the N end is shown as SEQ ID NO. 2-SEQ ID NO. 7, and the C end is the same as 468-1169 of SEQ ID NO. 1.
Example 2 testing of the insecticidal Effect of the newly produced proteins
These protein entities were synthesized using a protein expression experimental system and tested for their insecticidal effect on spodoptera frugiperda.
Protein synthesis and insecticidal efficacy test methods refer to the disclosure in example 2 of the CN202311033251.5 patent.
The test results are shown in Table 1. WBY-1.03 has higher insecticidal activity than the original WBY-1 protein, and can be used as a novel insecticidal protein with higher activity for development of biopesticides and insect-resistant transgenic plants.
Table 1 insecticidal Activity of proteins
| Protein name | Amino acid sequence | Insecticidal Activity (LC 50) 1 ） |
| WBY-1 | SEQ ID NO. 1 | 50±12 |
| WBY-1.01 | SEQ ID NO. 2+SEQ ID NO. 1 position 468-1169 | 34±15 |
| WBY-1.02 | SEQ ID NO. 3+SEQ ID NO. 1 position 468-1169 | 40±8 |
| WBY-1.03 | SEQ ID NO. 4+SEQ ID NO. 1 position 468-1169 | 25±4* |
| WBY-1.04 | SEQ ID NO. 5+SEQ ID NO. 1 position 468-1169 | 48±6 |
| WBY-1.05 | SEQ ID NO. 6+SEQ ID NO. 1 position 468-1169 | 55±28 |
| WBY-1.06 | SEQ ID NO. 7+SEQ ID NO. 1 position 468-1169 | 30±23 |
1: units ng/g; * Indicating a significant difference (α=0.05) compared to WBY-1.
While the application has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the application and are intended to be within the scope of the application as claimed.
Claims (7)
1. A protein is characterized in that the protein is formed by fusing 468-1169 positions of SEQ ID NO. 4 and SEQ ID NO. 1 from the N end to the C end in sequence.
2. A nucleic acid molecule encoding the protein of claim 1.
3. A vector comprising the nucleic acid molecule of claim 2.
4. A recombinant cell comprising the nucleic acid molecule of claim 2 or the vector of claim 3.
5. The recombinant cell of claim 4, wherein the recombinant cell is a prokaryotic cell.
6. The recombinant cell of claim 5, wherein the recombinant cell is an e.
7. Use of a protein according to claim 1, or a nucleic acid molecule according to claim 2, or a vector according to claim 3, or a recombinant cell according to any one of claims 4 to 6, for combating spodoptera frugiperda, or for preparing an anti-spodoptera frugiperda preparation, or for growing an anti-spodoptera frugiperda plant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311218202.9A CN116948045B (en) | 2023-09-21 | 2023-09-21 | Artificial intelligence assisted insecticidal protein and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311218202.9A CN116948045B (en) | 2023-09-21 | 2023-09-21 | Artificial intelligence assisted insecticidal protein and application thereof |
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| CN116948045A CN116948045A (en) | 2023-10-27 |
| CN116948045B true CN116948045B (en) | 2023-11-24 |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3787350D1 (en) * | 1986-11-20 | 1993-10-14 | Monsanto Co | Insect resistant tomato plants. |
| CN104488945A (en) * | 2014-12-22 | 2015-04-08 | 北京大北农科技集团股份有限公司 | Application of insecticidal protein |
| CN116396368A (en) * | 2023-04-20 | 2023-07-07 | 湖北科技学院 | Insect-resistant protein and preparation method and application thereof |
| CN116514936A (en) * | 2023-06-29 | 2023-08-01 | 莱肯生物科技(海南)有限公司 | Insect-resistant protein and preparation method and application thereof |
| CN116768990A (en) * | 2023-08-16 | 2023-09-19 | 莱肯生物科技(海南)有限公司 | Artificial intelligence auxiliary generated insecticidal protein |
-
2023
- 2023-09-21 CN CN202311218202.9A patent/CN116948045B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3787350D1 (en) * | 1986-11-20 | 1993-10-14 | Monsanto Co | Insect resistant tomato plants. |
| CN104488945A (en) * | 2014-12-22 | 2015-04-08 | 北京大北农科技集团股份有限公司 | Application of insecticidal protein |
| CN116396368A (en) * | 2023-04-20 | 2023-07-07 | 湖北科技学院 | Insect-resistant protein and preparation method and application thereof |
| CN116514936A (en) * | 2023-06-29 | 2023-08-01 | 莱肯生物科技(海南)有限公司 | Insect-resistant protein and preparation method and application thereof |
| CN116768990A (en) * | 2023-08-16 | 2023-09-19 | 莱肯生物科技(海南)有限公司 | Artificial intelligence auxiliary generated insecticidal protein |
Non-Patent Citations (2)
| Title |
|---|
| Bacillus thuringiensis Cry1Ab domain III β-22 mutants with enhanced toxicity to spodoptera frugiperda(J.E.Smith);Gomez, I, et al.;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;第86卷(第22期);第e01580-20页 * |
| 我国草地贪夜蛾的生物学、生态学和防治研究概况与展望;梁沛 等;《昆虫学报》;第63卷(第5期);第624-638页 * |
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