CN117003841B - Method for controlling soybean leaf roller pests - Google Patents
Method for controlling soybean leaf roller pests Download PDFInfo
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- CN117003841B CN117003841B CN202311274162.XA CN202311274162A CN117003841B CN 117003841 B CN117003841 B CN 117003841B CN 202311274162 A CN202311274162 A CN 202311274162A CN 117003841 B CN117003841 B CN 117003841B
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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
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
- A01H1/022—Genic fertility modification, e.g. apomixis
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
- A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
- A01H1/127—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance for insect resistance
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/10—Seeds
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
- A01H6/54—Leguminosae or Fabaceae, e.g. soybean, alfalfa or peanut
- A01H6/542—Glycine max [soybean]
-
- 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
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- 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
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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Abstract
The application relates to a method for controlling soybean leaf roller pests, belonging to the field of pest control. The test shows that the WBY-7.06 protein has better insecticidal activity on the soybean leaf rollers, and provides a new means for preventing and controlling soybean leaf rollers.
Description
Technical Field
The application relates to a method for controlling soybean leaf roller pests, belonging to the field of pest control.
Background
Lepidopteran insects (Lepidoptera) belong to a genus below the subclass ptera. The known species in the world reach 20 ten thousand species, the known species in China are about 8000 species, and most of the larvae are harmful to various plants. The pests such as cotton bollworms, cabbage butterflies, plutella xylostellas, pod borers, corn borers and the like are farmland pests with very wide pests, cause great barriers to normal production of agriculture, and bring about very high control cost.
Soybean leaf roller (academic name:Lamprosema indicata) Is a lepidoptera moth-killing insect which is mainly harmful to leguminous crops such as soybean, cowpea, kidney bean, hyacinth bean, mung bean, red bean and the like, and is one of main pests of leguminous crops. Distributed in Zhejiang, jiangsu, jiangxi, fujian, taiwan, guangdong, hubei, sichuan, henan, hebei, inner Mongolia etc. provinces and cities. Is an important pest for soybean. The larvae wind the leaves into a tube, especiallyThe soybean is serious in flowering and pod bearing period.
Besides the traditional physical and chemical control methods, the development of bioengineering bacteria or transgenic plants by using insecticidal proteins can achieve the effect of controlling pests, and has lower control cost and more friendly environment. It is therefore important to find the insecticidal protein corresponding to each pest.
The WBY-7.06 protein in the CN202311218205.2 patent is a novel insecticidal protein which is generated by utilizing the assistance of an artificial intelligence technology, and has higher insecticidal activity on spodoptera frugiperda. However, it is not known whether the protein is active against other pests. The insecticidal spectrum of the protein can be tested to provide a new control means for specific pests.
Disclosure of Invention
In order to solve the problems, the application adopts the following technical scheme:
the application provides a method for controlling soybean leaf roller pests, which is characterized by comprising the step of contacting the soybean leaf roller pests with at least WBY-7.06 protein.
In some embodiments, the WBY-7.06 protein is present in a host cell that produces at least the WBY-7.06 protein, and the soybean leaf roller pest is contacted with at least the WBY-7.06 protein by feeding the host cell.
In some embodiments, the WBY-7.06 protein is present in a bacterium or transgenic plant that produces at least the WBY-7.06 protein, and the soybean leaf roller pest is contacted with at least the WBY-7.06 protein by feeding on the bacterium or tissue of the transgenic plant, the soybean leaf roller pest growth being inhibited and/or causing death after the contact, to effect control of a soybean leaf roller pest plant.
In some embodiments, the transgenic plant is a leguminous plant.
In some embodiments, the tissue of the transgenic plant is root, leaf, stalk, fruit, tassel, female ear, anther, or filament.
In some embodiments, the WBY-7.06 protein has the amino acid sequence set forth in SEQ ID NO. 1.
In some embodiments, the WBY-7.06 protein has a nucleotide sequence set forth in SEQ ID NO. 2 in bacteria.
In some embodiments, the transgenic plant further comprises at least one second nucleotide different from the nucleotide encoding the WBY-7.06 protein.
In some embodiments, the second nucleotide encodes a Cry insecticidal protein, a Vip insecticidal protein, a protease inhibitor, a lectin, an alpha-amylase, or a peroxidase.
In some embodiments, the second nucleotide is a dsRNA that inhibits a gene of interest in the insect pest of interest.
The application also provides an application of the WBY-7.06 protein in controlling soybean leaf roller pests.
The present application also provides a method of producing a plant for controlling soybean leaf roller pests, comprising introducing into the genome of said plant a polynucleotide sequence encoding a WBY-7.06 protein.
The present application also provides a method of producing a plant seed for controlling soybean leaf roller pests, comprising crossing a first plant obtained by the above method with a second plant, thereby producing a seed comprising a polynucleotide sequence encoding a WBY-7.06 protein.
The application also provides a method for culturing plants for controlling soybean leaf roller pests, which is characterized by comprising the following steps: planting at least one plant seed comprising in its genome a polynucleotide sequence encoding a WBY-7.06 protein; growing the plant seeds into plants; growing the plant under conditions in which the soybean leaf roller pest and/or soybean leaf roller pest naturally occurs at risk is artificially inoculated, and harvesting plants having reduced plant damage and/or increased plant yield as compared to other plants not having the polynucleotide sequence encoding the WBY-7.06 protein.
The application has the beneficial effects that: according to the application, through testing the insecticidal activity of WBY-7.06 protein on insects such as carpopodium borer, oriental armyworm, asian corn borer, cotton bollworm, cutworm, prodenia litura and soybean leaf roller, the insecticidal spectrum of the protein is clarified, and the WBY-7.06 protein has better insecticidal activity on soybean leaf roller, so that a new means is provided for preventing and controlling soybean leaf roller.
Drawings
FIG. 1 results of soybean leaf roller protein assay.
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 preparation of proteins Synthesis
These protein entities were synthesized using a protein expression experimental system and tested for their insecticidal effect against several lepidopteran pests.
The nucleic acid sequence encoding the sequence was first designed on the basis of the amino acid sequence (http:// www.friendbio.com/codon. Html. In the following in-line tool), with codons set to E.coli (K12 strain) preference and avoiding XhoI and HindIII cleavage sites. The amino acid sequence of WBY-7.06 protein is shown as SEQ ID NO. 1, and the nucleic acid sequence of the obtained coded protein is shown as SEQ ID NO. 2.
The nucleic acid molecule with the sequence is artificially synthesized and cloned between the sites of restriction enzymes XhoI and HindIII in the expression vector pET28a to obtain the protein expression vector. The vector is transferred into an escherichia coli BL21 cell line and protein expression is carried out. The method comprises the following specific steps:
inoculating single colony to 0.5 mL LB liquid culture medium, culturing at 37deg.C for 4 h until the culture medium is turbid, adding IPTG (isopopyl-beta-D-thiohinging) into 100 uL bacterial liquid to a final concentration of 0.8 mM, simultaneously taking 100 uL bacterial liquid as negative control, continuously culturing for 4 h, adding 25 μl of loading buffer into 100 μl bacterial liquid for sample preparation electrophoresis, and comparing according to negative control and result induced by adding IPTG, and judging whether expression exists. The remaining 20. Mu.L of the expression was inoculated into 2 mL of LB liquid medium and cultured at 37℃for 12-16 h as a seed solution, the seed solution was inoculated into 250 mL of LB liquid medium again to OD600 = 0.5-0.6, then IPTG (isopopyl-beta-D-thiogaside) was added to a concentration of 0.8 mM, and the culture was continued under the same conditions for 4 hours. The culture medium was centrifuged at 5000 g for 10 minutes to pellet E.coli cells, and the supernatant was discarded to collect the pellet. The precipitate was sonicated with 30 mL of 20mM Tris-50mM NaCl buffer. After centrifugation, the supernatant was examined for the presence of recombinant proteins and quantified.
Example 2 insecticidal Effect test
The recombinant proteins obtained in the above examples were further subjected to a test for insecticidal activity. The method comprises the following steps:
the biological assay is carried out by adopting a surface smearing method, firstly, about 1 mL of non-coagulated artificial feed (about 1 g) is added into a 24-hole plate, the feed is paved on the bottom of the hole plate by slight shaking, after the feed is coagulated, protein solutions (20 mu L/hole) with different concentrations are added, after the addition, the liquid medicine is evenly paved on the surface of the feed by slight shaking, and the feed is naturally dried in the air in a fume hood for 1 h. Experiment set up 6 gradient concentrations (0.001, 0.01, 0.1, 0.5, 1, 2. Mu.g/cm) 2 ) And a blank (buffer), each treatment joint 24 is artificially raised target insect initially hatched larva (hatching time is 2-12 h), 3 times of repetition are set, the treatment joints are placed in a pest-raising room with temperature of 26+/-2 ℃ and photoperiod of 14:10 (L: D) h, relative humidity of 50-70% are cultured, and death rate is investigated after 7 days. To be touched by a writing brushThe larvae were regarded as dead at the tail and dead without 2 years of development.
Mortality and corrected mortality were calculated according to the following formulas, and LC50 values were calculated using graphpad.
(equation 1)
(equation 2)
The test result shows that the LC50 value of WBY-7.06 on soybean leaf rollers, prodenia litura and oriental armyworms is less than 1 mug/g (see table 1), the insecticidal activity is better, and the requirements on pest control can be met. Therefore, WBY-7.06 can be used to control soybean leaf roller pests (FIG. 1).
TABLE 1 insect bioassay results
| Insect | Insecticidal Activity (LC 50) 1 ） |
| Peach borerDichocrocis punctiferalis | 1.65 |
| Oriental armywormMythimna separata | 0.46 |
| Asian corn borerOstrinia furnacalis | 1.27 |
| Bollworm (Bowls)Helicoverpa armigera | 1.57 |
| Radix seu herba Gei aleppiciAgrotis ipsilon | 1.43 |
| Prodenia litura (L.) DCSpodoptera litura | 0.11 |
| Soybean leaf rollerLamprosema indicate | 0.99 |
1: unit μg/g
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 (6)
1. A method of controlling soybean leaf roller pests comprising contacting the soybean leaf roller pests with at least WBY-7.06 protein; the amino acid sequence of the WBY-7.06 protein is shown as SEQ ID NO. 1.
2. The method of claim 1, wherein said WBY-7.06 protein is present in a host cell that produces at least said WBY-7.06 protein, and said soybean leaf roller pest is contacted with at least said WBY-7.06 protein by feeding said host cell.
3. The method of claim 2, wherein said WBY-7.06 protein is present in at least said WBY-7.06 protein producing bacteria or transgenic leguminous plants, and said soybean leaf roller pest is contacted with at least said WBY-7.06 protein by feeding said bacteria or said transgenic leguminous plant tissue, wherein said soybean leaf roller pest growth is inhibited and/or caused to die after said contacting to effect control of soybean leaf roller pest damage to leguminous plants.
4. A method according to claim 3, wherein the tissue of the transgenic leguminous plant is a root, leaf, stem, fruit, tassel, female ear, anther or filament.
5. A method according to claim 3, wherein the WBY-7.06 protein has the nucleotide sequence set forth in SEQ ID No. 2 in bacteria.
The application of WBY-7.06 protein in controlling soybean leaf roller pests is characterized in that the amino acid sequence of the WBY-7.06 protein is shown as SEQ ID NO. 1.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022246153A1 (en) * | 2021-05-21 | 2022-11-24 | Syngenta Crop Protection Ag | Compositions and methods for controlling insects |
| 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 |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022246153A1 (en) * | 2021-05-21 | 2022-11-24 | Syngenta Crop Protection Ag | Compositions and methods for controlling insects |
| 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 |
|---|
| Convergence of Bar and Cry1Ac Mutant Genes in Soybean Confers Synergistic Resistance to Herbicide and Lepidopteran Insects;Tien Dung Nguyen等;Front Plant Sci;第12卷;第1-13页 * |
| 作物抗虫育种的研究进展;李艳茹等;延边大学农学学报;第27卷(第4期);第306-310页 * |
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