WO2022144020A1 - 蚊虫性信息素及其在蚊虫防治上的应用 - Google Patents

蚊虫性信息素及其在蚊虫防治上的应用 Download PDF

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WO2022144020A1
WO2022144020A1 PCT/CN2022/070053 CN2022070053W WO2022144020A1 WO 2022144020 A1 WO2022144020 A1 WO 2022144020A1 CN 2022070053 W CN2022070053 W CN 2022070053W WO 2022144020 A1 WO2022144020 A1 WO 2022144020A1
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mosquitoes
competitiveness
gene
desat1
male
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PCT/CN2022/070053
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English (en)
French (fr)
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王四宝
王官栋
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中国科学院分子植物科学卓越创新中心
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Publication of WO2022144020A1 publication Critical patent/WO2022144020A1/zh

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    • 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
    • A01N27/00—Biocides, pest repellants or attractants, or plant growth regulators containing hydrocarbons
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01M—CATCHING, TRAPPING OR SCARING OF ANIMALS; APPARATUS FOR THE DESTRUCTION OF NOXIOUS ANIMALS OR NOXIOUS PLANTS
    • A01M1/00—Stationary means for catching or killing insects
    • A01M1/02—Stationary means for catching or killing insects with devices or substances, e.g. food, pheronones attracting the insects
    • 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
    • A01P19/00—Pest attractants
    • 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
    • A01P23/00—Chemosterilants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5085—Supracellular entities, e.g. tissue, organisms of invertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01M—CATCHING, TRAPPING OR SCARING OF ANIMALS; APPARATUS FOR THE DESTRUCTION OF NOXIOUS ANIMALS OR NOXIOUS PLANTS
    • A01M2200/00—Kind of animal
    • A01M2200/01—Insects
    • A01M2200/012—Flying insects
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention belongs to the field of biotechnology; more particularly, the present invention relates to mosquito sex pheromone and its application in mosquito population control.
  • Mosquitoes are important disease vectors. In tropical and subtropical countries, malaria, dengue fever and other mosquito-borne diseases have caused serious public health problems. Malaria transmitted by Anopheles mosquitoes seriously endangers human life and health. Although countries have invested a lot of manpower, material and financial resources to prevent and control malaria in recent years, the situation is still not optimistic. According to the survey, in 2017 alone, in the south of the Sahara Desert in Africa, malaria still had a mortality rate of 435,000 people. threaten people's health. Worldwide, nearly half of the population is at risk of contracting malaria.
  • Wolbachia is an insect endosymbionts that form endosymbionts in insects, and previous studies have shown that Wolbachia can inhibit the spread of mosquito-borne infectious diseases.
  • Wolbachia-induced phenotype - cytoplasmic incompatibility so that after mating of Wolbachia-carrying male mosquitoes with wild female mosquitoes, the eggs produced cannot develop and the next generation cannot be reproduced, which is important for mosquito population control. is an effective method.
  • population control strategies for Anopheles mosquitoes still face challenges due to the lack of an effective method for mosquito sex sorting and for improving the mating competitiveness of Wolbachia-carrying male mosquitoes.
  • the purpose of the present invention is to provide mosquito sex pheromone and its application in mosquito control.
  • the alkane is: C 27 H 56 , which is used as a pheromone to promote the mating competitiveness (mating ability), reproductive competitiveness of male mosquitoes or female mosquitoes Mosquito attraction.
  • the alkane is: C 27 H 56 , which is used as a pheromone to promote the mating competitiveness and reproductive competitiveness of male mosquitoes or attract female mosquitoes Ability composition.
  • the promotion of male mosquito mating competitiveness, reproductive competitiveness or attracting ability to female mosquitoes is a significant promotion, for example, according to a statistical calculation method (for example, based on comparing the test group with the Compared with the control group (statistics), the promotion is more than 5%, more than 10%, more than 20%, more than 30%, more than 50%, more than 80% or higher.
  • the derivatives of the alkanes include (but are not limited to): isomers, solvates, precursors or salts of the alkanes; preferably, the alkanes are n-heptacosane .
  • the mosquitoes include: Anopheles mosquitoes.
  • the male mosquitoes include: male sterile mosquitoes, genetically modified mosquitoes (including genetically modified mosquitoes or mosquito symbiotic microorganisms), and wild-type mosquitoes.
  • the alkane is included in a composition, and the composition further includes: a functional compound, an adjuvant, and a pharmaceutically acceptable carrier; preferably, the adjuvant includes an adjuvant .
  • a method of modulating the mating competitiveness, reproductive competitiveness, or attraction to female mosquitoes of male mosquitoes comprising: (a) treating the male mosquitoes with an alkane or a derivative thereof; or, (b) ) regulates the amount of alkane or its derivatives in male mosquitoes; the alkane is: C 27 H 56 .
  • the "method for adjusting the mating competitiveness, reproductive competitiveness or attraction to female mosquitoes of male mosquitoes" includes “preparing a method for adjusting the mating competitiveness, reproductive competitiveness or attraction ability to female mosquitoes” methods of male mosquitoes", the two expressions are used interchangeably.
  • the derivatives of the alkanes include (but are not limited to): isomers, solvates, precursors or salts of the alkanes; preferably, the alkanes are n-heptacosane .
  • the mosquitoes include: Anopheles mosquitoes.
  • the male mosquitoes include: male sterile mosquitoes, genetically modified mosquitoes (including genetically modified mosquitoes or mosquito symbiotic microorganisms), and wild-type mosquitoes.
  • regulating the presence of alkanes or derivatives thereof in male mosquitoes comprises: promoting (significantly promoting, such as promoting 10%, 20%, 40%, 60%, 80%) , 90% or higher) the expression of the desat1 gene in mosquitoes, thereby increasing the presence of the alkane, thereby promoting the mating competitiveness, reproductive competitiveness or attraction to female mosquitoes of male mosquitoes.
  • regulating the presence of alkanes or derivatives thereof in male mosquitoes comprises: reducing (significantly reducing, such as reducing 10%, 20%, 40%, 60%, 80%) , 90% or lower) the expression of the desat1 gene in mosquitoes, thereby reducing the presence of the alkanes, thereby inhibiting the mating competitiveness, reproductive competitiveness or attraction to female mosquitoes of male mosquitoes.
  • the promoting the expression of the desat1 gene in mosquitoes comprises: regulating with an up-regulated molecule comprising the following group: an expression cassette or an expression construct overexpressing the desat1 gene; or, with the desat1 gene or Regulatory molecules that interact with their expressed proteins to increase their expression or activity.
  • the reducing the expression of the desat1 gene in mosquitoes includes: regulating with a down-regulated molecule including the following group: an agent for knocking out or silencing the desat1 gene, an agent for inhibiting the activity of the protein encoded by the desat1 gene; preferably , the down-regulated molecules include: gene editing reagents, homologous recombination reagents or site-directed mutagenesis reagents for the desat1 gene, the reagents carry out loss-of-function mutation of the protein encoded by the desat1 gene, or interference molecules that specifically interfere with the expression of the desat1 gene
  • the down-regulated molecule comprises a double-stranded RNA (dsdesat1) that silences the desat1 gene; more preferably, the double-stranded RNA has a nucleotide sequence shown in SEQ ID NO: 1.
  • composition or kit for promoting the mating competitiveness, reproductive competitiveness or attraction to female mosquitoes of male mosquitoes comprising: alkane; and adjuvants such as n-hexane;
  • the alkane is: C 27 H 56 .
  • a method for screening substances that modulate the mating competitiveness, reproductive competitiveness or attracting ability of female mosquitoes of male mosquitoes comprising: (1) adding candidate substances to a system expressing desat1 gene (2) Detecting the system, and observing the expression or activity of the desat1 gene, if its expression or activity is increased (significantly increased, such as increased by 10%, 20%, 40%, 60%, 80%, 90% or more) ), it indicates that the candidate substance is a substance that can be used to promote the mating competitiveness, reproductive competitiveness or attraction to female mosquitoes of male mosquitoes; if its expression or activity is reduced (significantly reduced, such as 10%, 20%, 40% , 60%, 80%, 90% or lower), it indicates that the candidate substance is a substance (such as a chemical substance, such as a pheromone, which can be used to reduce the mating competitiveness, reproductive competitiveness or attracting ability of female mosquitoes of male mosquitoes) compound).
  • a substance such as a chemical substance, such as a pheromone
  • the "reduction" in the reduction in the mating competitiveness, reproductive competitiveness or attracting ability of female mosquitoes of male mosquitoes, the "reduction" is a significant reduction, for example, according to a statistical calculation method (for example, based on comparing the test group with Compared with the control group, it can be reduced by more than 5%, more than 10%, more than 20%, more than 30%, more than 50%, more than 80% or lower.
  • the method further includes: setting a control group without adding the candidate substance, so as to clearly distinguish the difference between the expression or activity of the protein encoded by the desat1 gene in the test group and the control group.
  • the candidate substances include (but are not limited to): small molecule compounds, regulatory molecules (such as up-regulated molecules, down-regulated molecules, gene editing) designed for the desat1 gene or its upstream or downstream proteins or genes constructs, etc.).
  • regulatory molecules such as up-regulated molecules, down-regulated molecules, gene editing
  • the small molecule compounds include compounds isolated from mosquitoes; preferably, they include mosquito body wall hydrocarbons.
  • the candidate substance when it is a small molecule compound, it is identified or analyzed by methods including (but not limited to) gas chromatography and/or mass spectrometry (GC/MS) identification.
  • GC/MS mass spectrometry
  • alkanes or derivatives thereof as semiochemicals for identifying male mosquito mating competitiveness, reproductive competitiveness or attracting ability to female mosquitoes, or for preparing and identifying male mosquito mating competition
  • a reagent for detecting force, reproductive competitiveness or attracting ability to female mosquitoes; the alkane is: C 27 H 56 .
  • the desat1 gene or its encoded protein as a marker for identifying the mating competitiveness, reproductive competitiveness or attraction ability of female mosquitoes of male mosquitoes.
  • the identification of male mosquito mating competitiveness, reproductive competitiveness or attracting ability to female mosquitoes is a qualitative/quantitative method, for example, according to a certain mosquito population, obtain the average of its mating and reproductive ability value, based on which the individual or subpopulation under the population is analyzed and judged.
  • a long-chain alkane can act as a semaphore (pheromone) to promote the mating competitiveness, reproductive competitiveness or attraction to female mosquitoes of male mosquitoes.
  • the long-chain alkane of the present invention is isolated from the epidermal hydrocarbons of mosquitoes, has a significant effect on promoting the mating competitiveness of mosquitoes, and can be applied to improve the effect of mosquito population control such as male sterility technology or genetic control technology.
  • the present invention first provides a compound having the parent nucleus structure shown in structural formula (I):
  • the present invention also includes derivatives of the above-mentioned compounds having the core structure represented by formula (I).
  • Said derivatives include: isomers, solvates, precursors of said compounds, or their biologically/pharmaceutically acceptable salts, as long as they also have the same parent nucleus structure represented by formula (I)
  • the compounds have the same or substantially the same function. In particular, they have the same or similar functions of heptacosane described in the examples of the present invention.
  • biologically/pharmaceutically acceptable salt refers to the salt formed by the reaction of the compound with inorganic acid, organic acid, alkali metal or alkaline earth metal.
  • these salts include (but are not limited to): (1) salts formed with the following inorganic acids: such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid; (2) salts formed with the following organic acids, such as acetic acid, oxalic acid, succinic acid, tartaric acid , methanesulfonic acid, maleic acid, or arginine.
  • salts include salts with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, in the form of esters, carbamates, or other conventional "precursors", which have The same or similar functions as described for heptadecane.
  • the "precursor of a compound” refers to a compound whose precursor of the compound undergoes a metabolic or chemical reaction in the mosquito body wall or in vivo to transform into a parent nucleus structure represented by structural formula (I) when administered by an appropriate method, or Derivatives of the compounds with the core structure represented by the chemical structural formula (I), they have the same or similar functions of heptacosane described in the examples of the present invention.
  • the compound of the present invention can be obtained by various methods well-known in the art and using well-known raw materials, such as chemical synthesis or from biological (such as insects or plants) ), or the methods for transforming on the basis of extraction, these methods are all included in the present invention.
  • the compounds of the present invention can be synthesized by known methods; the synthesized compounds can be further purified by column chromatography, high performance liquid chromatography, and the like. In addition, the compounds of the present invention can also be obtained commercially.
  • the present invention also provides a composition (eg, a pharmaceutical composition or a pesticide composition), containing an effective amount of the compound described in the parent core structure represented by formula (I), or its derivatives (eg, isomers, solvents, etc.) compounds, precursors). That is, the present invention provides a composition containing the compound described in the parent core structure represented by formula (I) or an analog thereof.
  • a composition eg, a pharmaceutical composition or a pesticide composition
  • the "comprising”, “having” or “including” includes “comprising”, “mainly consisting of”, “substantially consisting of”, and “consisting of”; “ Consists essentially of”, “consisting essentially of” and “consisting of” are subordinate concepts of “contains”, “has” or “includes”.
  • a “biologically/pharmaceutically acceptable salt” ingredient is one that is suitable for administration to (eg, sprayed or painted on) mosquitoes without undue harm to the environment (environmentally friendly), ie, there is a reasonable benefit / Hazard ratio of substances.
  • a “biologically/pharmaceutically acceptable salt” is a biologically/pharmaceutically acceptable solvent, suspending agent or excipient used to deliver a compound of the present invention to an animal.
  • the carrier can be liquid or solid.
  • the composition contains 0.001-50% by weight of the compound of the parent core structure represented by formula (I) or a biologically/pharmaceutically acceptable salt thereof.
  • the composition contains 0.05-30% by weight of the compound of the parent core structure represented by formula (I) or a biologically/pharmaceutically acceptable salt thereof;
  • the composition contains 0.01-20% by weight of the compound of the parent core structure represented by formula (I) or its salt.
  • the dosage form of the composition of the present invention can be various, as long as the dosage form can make the active ingredient reach the mosquito body effectively.
  • it can be selected from: gels, aerosols, tablets, capsules, powders, granules, syrups, solutions, or suspensions.
  • preferred pharmaceutical compositions are liquid (eg, suspension, solution) compositions or aerosol compositions.
  • the compounds of the present invention or compositions thereof may also be stored in sterile devices suitable for spraying, injection, smearing or instillation.
  • the present invention also provides a composition for promoting the mating competitiveness, reproductive competitiveness or attracting ability of female mosquitoes of male mosquitoes, which comprises: alkane; agent (eg, an adjuvant) or a pharmaceutically acceptable carrier.
  • agent eg, an adjuvant
  • the functional compound, adjuvant (eg, adjuvant) or pharmaceutically acceptable carrier has characteristics suitable for mosquitoes, and can assist the function or effect of the alkane on mosquitoes.
  • compositions or compounds described in the present invention can also be placed in kits to facilitate application by those skilled in the art.
  • the kit may further include instructions for use by those skilled in the art to perform appropriate applications.
  • the mosquitoes include Anopheles mosquitoes.
  • the present inventors identified the compounds of the present invention as pheromones for the first time.
  • Pheromones also known as pheromones, are generally chemical substances that can be secreted by animal individuals into the body or outside the body, and are detected by other individuals of the same species through sensory organs, causing the latter to express certain behaviors, emotions, psychology or Substances that alter physiological mechanisms.
  • the pheromone has a communication function.
  • the present invention provides compounds of the core structure represented by formula (I) or derivatives thereof, such as isomers, solvates, precursors thereof, or their biologically/pharmaceutically acceptable Use of an accepted salt for promoting male mosquito mating competitiveness, reproductive competitiveness, or attraction to female mosquitoes; or, for the preparation of a combination that promotes mating competitiveness, reproductive competitiveness, or attraction to female mosquitoes thing.
  • the compound of the parent nucleus structure represented by the formula (I) of the present invention or its derivatives can be applied to combine with male sterility technology.
  • Male sterility or genetic control techniques can be applied to control the population of mosquitoes and other sanitary pests.
  • the key to the ability to compete with sterile males is the ability of such males to compete with male mosquitoes in the wild. compete.
  • the compound of the parent nucleus structure represented by the formula (I) of the present invention or its derivatives can endow male mosquitoes with improved mating competitiveness, reproductive competitiveness or attracting ability to female mosquitoes, and this characteristic is especially suitable for such male mosquitoes Sterile or genetically modified male mosquitoes, when their mating competitiveness, reproductive competitiveness or competitiveness against female mosquitoes are enhanced and released into the wild, allowing male mosquitoes (with reproductive capacity) in the wild to mate with female mosquitoes Significant reduction, thereby effectively controlling the number of offspring mosquitoes.
  • heptacosane or its derivatives naturally exist in the mosquito body wall, as an embodiment of the present invention, it also includes regulation related to the production of the heptacosane or its derivatives.
  • heptacosane or its derivatives After knowing the function of heptacosane or its derivatives, it can be used as a molecular marker to identify the reproductive capacity of male mosquitoes.
  • One of skill in the art can use any of a variety of techniques known or being developed in the art for qualitative/quantitative identification of compounds to determine the amount of heptacosane or derivatives thereof, such as chromatography and mass spectrometry.
  • a method for identifying body wall pheromones comprising how to collect mosquito body wall hydrocarbons, and identifying mosquito epidermis hydrocarbons by gas chromatography and mass spectrometry (GC/MS). Methods of analysis of components.
  • the desat1 gene can affect the synthesis of the mosquito epidermis hydrocarbon n-heptacosane, and then affect the mating of the mosquitoes. Therefore, the desat1 gene can be used as a target gene for regulating pheromone synthesis.
  • the activity of the desat1 gene can be up-regulated using an up-regulator of the expression or activity of the desat1 gene.
  • the up-regulators of the expression or activity of the desat1 gene include promoters, agonists, and activators.
  • the "up-regulation” and “promotion” include “up-regulation” and “promotion” of protein activity or "up-regulation” and “promotion” of protein expression.
  • the down-regulators of desat1 gene or its encoding gene refer to some agents that can reduce the activity of desat1 gene, reduce the stability of desat1 gene or its encoding gene, down-regulate the expression of desat1 gene, reduce the effective time of desat1 gene, Substances that inhibit the transcription and translation of the desat1 gene or reduce the phosphorylation/activation level of the protein can be used in the present invention as useful substances for down-regulating the desat1 gene. They can be chemical compounds, small chemical molecules, biomolecules. The biomolecules can be at the nucleic acid level (including DNA, RNA) or at the protein level.
  • the down-regulating agents are: interfering RNA molecules or antisense nucleotides that specifically interfere with the expression of the desat1 gene or other signaling pathway genes; or a gene editing agent that specifically edits the desat1 gene, and the like.
  • a method for down-regulating the mosquito desat1 gene including targeted mutation, gene editing, or gene interference of the desat1 gene, so as to realize the down-regulation of its expression.
  • the desat1 gene is transformed into its mutant, so that it no longer functions.
  • RNAi interference or CRISPR/Cas9 system is used for gene editing. Appropriate sgRNA target sites will bring higher gene editing efficiency, so before proceeding with gene editing, suitable target sites can be designed and found. After designing specific target sites, in vitro cell activity screening can also be performed to obtain effective target sites for subsequent experiments.
  • a method for down-regulating the activity of the protein expressed by the mosquito desat1 gene comprising using a binding molecule, such as an antibody, that specifically binds to the protein expressed by the desat1 gene.
  • the binding molecules can be formulated into anti-insect formulations.
  • the binding molecules are, for example, monoclonal or polyclonal antibodies, such monoclonal antibodies can be prepared using hybridoma technology. Polyclonal antibodies can be prepared by a variety of methods well known to those skilled in the art.
  • the methods can be carried out using any suitable conventional means, including reagents, temperature, pressure conditions, and the like.
  • the present invention provides a method for screening substances that regulate the mating competitiveness, reproductive competitiveness or attracting ability of female mosquitoes of male mosquitoes, the method comprising: (1) adding a candidate substance to a system expressing the desat1 gene; (2) Detecting the system, and observing the expression or activity of the desat1 gene, if its expression or activity is increased, it indicates that the candidate substance can be used to promote the mating competitiveness of male mosquitoes, reproductive competitiveness or attraction to female mosquitoes. Substance; if its expression or activity is reduced, it indicates that the candidate substance is a substance that can be used to reduce the mating competitiveness, reproductive competitiveness or attracting ability of female mosquitoes of male mosquitoes.
  • a control group can also be set.
  • the control group can be a system without the addition of the candidate substance.
  • candidate substances there is no particular limitation on the candidate substances, and it can be a wide variety of substances of interest.
  • small molecule compounds, genes or proteins from compound libraries or gene libraries or small molecule compounds, agonists, up-regulators, promoters designed against the desat1 gene or its encoded proteins or their upstream or downstream molecules agents, gene editing agents, interfering molecules, nucleic acid inhibitors, binding molecules (eg, antibodies or ligands).
  • the method further includes: conducting further cell experiments and/or animal experiments on the obtained potential substances to further select and determine the substances that are really useful for regulating the mating performance of male mosquitoes.
  • Nucleic acid analysis or protein analysis can be performed by one of skill in the art using any of a variety of techniques known or being developed in the art, all of which are encompassed by the present invention. Said methods include, but are not limited to, sequencing, PCR amplification, probe methods, hybridization methods, restriction enzyme digestion analysis methods, and allelic polymorphism analysis methods (such as melting curve methods) for nucleic acid sequence analysis. identification, etc.
  • dsdesat1 microinjected double-stranded RNA
  • dsGFP Double-stranded RNA
  • dsdesat1 DNA sequence of double-stranded RNA (dsdesat1) is as follows (SEQ ID NO:1):
  • RNA-injected male mosquitoes and 20 unmated and untreated female mosquitoes were taken and placed in cups and allowed to mate freely. After overnight mating, the seminal vesicles of female mosquitoes were dissected under a stereo microscope to observe whether the seminal vesicles had sperm. The presence or absence of sperm is the criterion for judging mating.
  • desat1 can affect the synthesis of epidermal hydrocarbons and thus affect mating. Further, in order to identify the key communication information substances that regulate the mating of Anopheles mosquitoes, the present inventors extracted hydrocarbons from the epidermis of Anopheles mosquitoes for differential comparative analysis, specifically identified by the following methods:
  • the newly emerged male Anopheles mosquitoes were collected, microinjected with desat1 double-stranded RNA, and injected with dsGFP as a control. After rearing for 3 days under normal conditions, 80 Anopheles mosquitoes were taken from the experimental group and the control group, and put into pre-added 800 ⁇ L hexane. In a 1.5ml centrifuge tube, Anopheles mosquitoes were soaked and extracted in hexane for 5 minutes. The hexane extraction supernatant was transferred to a new 1.5 mL centrifuge tube, and the sample was concentrated to 30 ⁇ L in a vacuum desiccator as the sample to be tested.
  • the instrument used is Agilent 7890B GC system coupled with high-resolution Agilent 7200 Q-TOF MS, and the analytical column used for GC/MS is HP-5MS column (Agilent, 30m ⁇ 0.25mm, 0.25 ⁇ m film thickness, 5% phenyl methyl siloxane stationary phase).
  • Set the oven temperature program to increase from 80 °C to 200 °C, the temperature increase rate is 20 °C growth rate, maintain at 200 °C for 2 min, and then increase from 200 °C to 320 °C, the temperature increase rate is 5 °C growth rate.
  • the sample was injected in 5 ⁇ L at a flow rate of 1.0 mL/min.
  • Mass spectral QTOF was recorded at 5 scans per second with a mass-to-charge ratio of 30 to 550 m/z.
  • the ion source temperature and emission current were set to 230 °C and 6.8 ⁇ A, respectively, keeping the electron energy at 70 eV, and the QTOFMS was operated in 2 GHz-EDR mode (2 GHz extended dynamic range) to extend the linear dynamic range.
  • Data collection and evaluation were performed by MassHunter acquisition, MassHunter Quantitative and qualitative analysis (version B07.00, Agilent Technologies, CA), respectively. Peak identification was determined by comparison to standard solutions, American National Standards, and NIST14 mass spectra. Mass spectrometry analysis showed that a total of 16 hydrocarbons were isolated and identified in the body wall of Anopheles mosquitoes, as shown in Figure 3.
  • Embodiment 3 The test that the pheromone n-heptacosane affects the mating of Anopheles mosquitoes
  • Freshly emerged male and female mosquitoes were collected separately and reared under normal conditions for 2 days. Prepare 75 ⁇ g/mL n-heptane-heptane solution in n-hexane, and apply the prepared solution on the abdomen of male Anopheles mosquitoes with a micro brush. Each treatment is divided into three groups with 20 mice in each group, and n-hexane is used as the control. . After rearing under normal conditions for 2 days, 20 unmated female mosquitoes were added to each group and allowed to mate freely.
  • n-heptadecane is a sex pheromone that can promote the mating competitiveness of male Anopheles mosquitoes.
  • n-heptadecane can improve the mating competitiveness of a variety of male Anopheles mosquitoes, including wild-type, sterile or genetically engineered male Anopheles mosquitoes.

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Abstract

本发明提供了一种新型的蚊虫性信息素及其在蚊虫防治上的应用。本发明的新型信息素分离自蚊虫，具有促进雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的功能。所述性信息素可通过调控雄性蚊虫(尤其是雄性不育蚊虫或遗传修饰蚊虫)交配竞争力和生殖能力，为蚊虫种群控制提供新的有效途径。

Description

蚊虫性信息素及其在蚊虫防治上的应用 技术领域
本发明属于生物技术领域；更具体地，本发明涉及蚊虫性信息素及其在蚊虫种群防治上的应用。
背景技术
蚊虫是重要的疾病传播媒介，在热带和亚热带国家，疟疾、登革热和其它依靠蚊虫作为媒介传播的疾病造成严重的公共健康问题，其中按蚊传播的疟疾严重危害人类的生命和健康。虽然近几年各国投入了大量的人力、物力和财力来防控疟疾，但情况仍不容乐观，据调查仅2017年，在非洲沙哈拉沙漠以南，疟疾仍以43,5000人的死亡率威胁着人们的生命健康。在世界范围内，还有将近一半的人面临着被疟疾感染的风险。
现今，对于疟疾传播媒介按蚊的防治仍然缺乏有效的方法。室内滞留喷洒和经长效杀虫剂处理的蚊帐来控制媒介传播疟疾仍然是防治疟疾的主要方法。但是，由于疟疾媒介昆虫对大部分的杀虫剂如除虫菊酯，有机磷酸酯类等产生抗性，使得以上的方法控制媒介昆虫的有效性受到了减弱。近年来，涌现出一些新的媒介控制技术，包括雄蚊不育、基因改造技术、基于共生菌的方法。然而受制于对蚊虫交配和生殖生物学机制的了解，这些新兴的媒介控制策略仍然存在一些不足从而影响种群控制的有效性。
昆虫不育技术：雄性不育技术取得成功的关键在于不育的雄虫释放到环境中的交配竞争力，它们需要有足够的能力与野外的雄性竞争。然而，对于蚊子的交配的化学生态学，尤其是雄蚊在交配过程中的化学通讯信号，本领域中知之甚少。迄今，有关疟疾传播媒介按蚊交配活动的调控机制，以及求偶信号和性信息素了解甚少，需要进一步的研究才能更加了解信息素在蚊子交配中的作用，从而有助于本领域技术人员对蚊子的行为进行控制和干预，进一步控制蚊子种群数量。
昆虫不相容技术：Wolbachia是一种昆虫内共生菌，在昆虫体内形成内共生体，以往的研究表明Wolbachia能够抑制蚊媒传染病的传播。Wolbachia诱导的表型——胞质不相容，使得携带有沃尔巴克氏体的雄蚊与野生的雌蚊交配后，所产的卵不能发育，无法繁育下一代，这对于蚊虫的种群控制是一个有效的方法。但是由于缺乏一种有效的蚊虫性别分选以及提高携带Wolbachia雄性 蚊虫的交配竞争力的方法，所以对于疟蚊的种群控制的策略仍然面临挑战。
携带抗病原体效应基因的种群替代技术：以往的研究表明通过遗传操作使蚊虫缺失或者插入某个特定的基因可以抑制蚊体内病原体的发育来阻断蚊媒病原体的传播，或通过遗传改造肠道共生菌表达效应分子或天然抗病原体共生菌来抑制或杀灭蚊体内病原体从而阻止蚊子传播疾病。这些新型遗传控制技术已被作为控制蚊媒传染病的新策略。种群替代技术控制蚊媒传染病最重要的是对蚊虫或蚊虫共生菌进行基因改造或利用天然抗病原体的共生菌，而这些控制技术的应用效率取决于释放的转基因雄蚊在野外的交配竞争能力，但至今仍然缺乏一种有效提高雄性蚊虫交配竞争能力的方法。
综上所述，提高雄性蚊虫(包括遗传改造或不育雄性蚊虫)的交配竞争能力对于基于昆虫不育技术或种群替代技术的种群遗传控制的有效性至关重要。发掘提高雄性蚊虫的交配竞争力的物质对于提高蚊媒种群控制效率至关重要，对于蚊媒传染病防控具有重要的应用价值。
发明内容
本发明的目的在于提供蚊虫性信息素及其在蚊虫防治上的应用。
在本发明的第一方面，提供烷烃或其衍生物的应用，所述的烷烃为：C 27H 56，用于作为信息素促进雄性蚊虫交配竞争力(交配能力)、生殖竞争能力或对雌性蚊虫的吸引能力。
在本发明的第一方面，提供烷烃或其衍生物的应用，所述的烷烃为：C 27H 56，用于作为信息素制备促进雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的组合物。
在一个优选例中，所述的促进雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力，该“促进”为显著性促进，例如根据统计学计算方法(例如基于将受试组与对照组相比来统计)，促进5％以上，10％以上，20％以上，30％以上，50％以上，80％以上或更高。
在另一优选例中，所述烷烃的衍生物包括(但不限于)：所述烷烃的异构体、溶剂合物、前体或盐；较佳地，所述烷烃为正二十七烷。
在另一优选例中，所述的蚊虫包括：按蚊属蚊虫。
在另一优选例中，所述的雄性蚊虫包括：雄性不育的蚊虫、遗传改造的蚊虫(包括遗传改造蚊虫或蚊虫共生微生物)、野生型蚊虫。
在另一优选例中，所述烷烃被包含于组合物中，所述组合物还包含有：功能性化合物、辅剂、药剂学可接受的载体；较佳地，所述辅剂包括佐剂。
在本发明的另一方面，提供一种调节雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的方法，包括：(a)利用烷烃或其衍生物处理雄性蚊虫；或，(b)在雄性蚊虫体内调节烷烃或其衍生物的存在量；所述的烷烃为：C 27H 56。
在一个优选例中，所述的“调节雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的方法”包括“制备交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力被调节的雄性蚊虫的方法”，该两种表述可互换使用。
在另一优选例中，所述烷烃的衍生物包括(但不限于)：所述烷烃的异构体、溶剂合物、前体或盐；较佳地，所述烷烃为正二十七烷。
在另一优选例中，所述的蚊虫包括：按蚊属蚊虫。
在另一优选例中，所述的雄性蚊虫包括：雄性不育的蚊虫、遗传改造的蚊虫(包括遗传改造蚊虫或蚊虫共生微生物)、野生型蚊虫。
在另一优选例中，(b)中，所述在雄性蚊虫体内调节烷烃或其衍生物的存在量包括：促进(显著促进，如促进10％、20％、40％、60％、80％、90％或更高)蚊虫体内desat1基因的表达，从而提高所述烷烃的存在量，进而促进雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力。
在另一优选例中，(b)中，所述在雄性蚊虫体内调节烷烃或其衍生物的存在量包括：降低(显著降低，如降低10％、20％、40％、60％、80％、90％或更低)蚊虫体内desat1基因的表达，从而降低所述烷烃的存在量，进而抑制雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力。
在另一优选例中，所述促进蚊虫体内desat1基因的表达包括：利用包括下组的上调分子进行调控：过表达所述desat1基因的表达盒或表达构建物；或，与所述desat1基因或其表达的蛋白相互作用、从而提高其表达或活性的调控分子。
在另一优选例中，所述降低蚊虫体内desat1基因的表达包括：利用包括下组的下调分子进行调控：敲除或沉默desat1基因的试剂，抑制desat1基因编码蛋白的活性的试剂；较佳地，所述下调分子包括：针对所述desat1基因的基因编辑试剂、同源重组试剂或定点突变试剂，所述试剂将desat1基因编码蛋白进行功能丧失性突变，或特异性干扰desat1基因表达的干扰分子；较 佳地，所述下调分子包括沉默desat1基因的双链RNA(dsdesat1)；更佳地，该双链RNA具有SEQ ID NO:1所示核苷酸序列。
在本发明的另一方面，提供一种用于促进雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的组合物或试剂盒，其包括：烷烃；以及辅剂，如正己烷；所述的烷烃为：C 27H 56。
在本发明的另一方面，提供一种筛选调节雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的物质的方法，包括：(1)将候选物质加入到表达desat1基因的体系中；(2)检测所述体系，观测其中desat1基因的表达或活性，若其表达或活性提高(显著提高，如提高10％、20％、40％、60％、80％、90％或更高)，则表明该候选物质为可用于促进雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的物质；若其表达或活性降低(显著降低，如降低10％、20％、40％、60％、80％、90％或更低)，则表明该候选物质为可用于降低雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的物质(如化学物质，具体如信息素化合物)。
在一个优选例中，所述降低雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力中，该“降低”为显著性降低，例如根据统计学计算方法(例如基于将受试组与对照组相比来统计)，降低5％以上，10％以上，20％以上，30％以上，50％以上，80％以上或更低。
在另一优选例中，所述方法还包括：设置不添加所述候选物质的对照组，从而明确分辨测试组中所述desat1基因编码的蛋白表达或活性与对照组的差异。
在另一优选例中，所述的候选物质包括(但不限于)：小分子化合物、针对所述desat1基因或其上游或下游蛋白或基因设计的调控分子(如上调分子、下调分子、基因编辑构建物等)。
在另一优选例中，所述的小分子化合物包括从蚊虫分离的化合物；较佳地包括蚊虫体壁碳氢化合物。
在另一优选例中，所述候选物质为小分子化合物时，通过包括(但不限于)气相色谱和/或质谱(GC/MS)鉴定的方法进行鉴定或分析。
在本发明的另一方面，提供烷烃或其衍生物的应用，用于作为鉴定雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的信息化合物，或用于制备鉴定雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的检测 试剂；所述的烷烃为：C 27H 56。
在本发明的另一方面，提供desat1基因或其编码的蛋白的应用，用于作为鉴定雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的标志物。
在一个优选例中，所述的鉴定雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力为一种可以定性/定量的方法，例如根据某蚊虫的种群，获得其交配生殖能力的平均值，基于该平均值来对个体或该种群下的亚群体进行分析评判。
本领域的技术人员可对前述的技术方案和技术特征进行任意组合而不脱离本发明的发明构思和保护范围。本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
图1、干扰desat1的双链RNA序列与干扰效率验证。
图2、干扰desat1影响按蚊交配能力的结果。
图3、干扰desat1按蚊体壁信息素差异的质谱鉴定结果。
图4、干扰desat1信息素二十七烷的气相色谱和质谱定量结果。
图5、信息素二十七烷提高雄性按蚊交配竞争力。
具体实施方式
本发明人经过深入的研究，首次揭示了一种长链烷烃可以作为信号素(信息素)，发挥促进雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的作用。本发明所述的长链烷烃分离自蚊虫的表皮碳氢化合物，对于促进蚊虫交配竞争力具有显著效果，可应用于提高雄性不育技术或遗传控制技术等蚊虫种群控制效果。
化合物及组合物
本发明首先提供了一种具有结构式(I)所示的母核结构的化合物：
C 27H 56    (I)。
其结构式如下：
Figure PCTCN2022070053-appb-000001
本发明还包括上述具有式(I)所示母核结构的化合物的衍生物。所述的衍生物包括：所述化合物的异构体、溶剂合物、前体，或它们的生物学/药剂学上可接受的盐，只要它们也具有与式(I)所示母核结构的化合物具有相同或基本相同的功能。特别是，它们具有本发明实施例中记载的二十七烷相同或近似的功能。
所述的“生物学/药剂学上可接受的盐”是指化合物与无机酸、有机酸、碱金属或碱土金属等反应生成的盐。这些盐包括(但不限于)：(1)与如下无机酸形成的盐：如盐酸、硫酸、硝酸、磷酸；(2)与如下有机酸形成的盐，如乙酸、草酸、丁二酸、酒石酸、甲磺酸、马来酸、或精氨酸。其它的盐包括与碱金属或碱土金属(如钠、钾、钙或镁)形成的盐，以酯、氨基甲酸酯，或其它常规的“前体”的形式，它们具有本发明实施例中记载的二十七烷相同或近似的功能。
所述的“化合物的前体”指当用适当的方法施用后，该化合物的前体在蚊虫体壁或体内进行代谢或化学反应而转变成结构式(I)所示母核结构的化合物，或化学结构式(I)所示母核结构的化合物的衍生物，它们具有本发明实施例中记载的二十七烷相同或近似的功能。
本领域人员应理解，在得知了本发明化合物的结构以后，可通过多种本领域熟知的方法、利用公知的原料，来获得本发明的化合物，比如化学合成或从生物(如昆虫或植物)中提取或在提取基础上进行改造的方法，这些方法均包含在本发明中。
可以利用公知的方法来合成本发明的化合物；合成的化合物可以进一步通过柱层析法、高效液相色谱法等方式进一步纯化。此外，也可以通过商购的方式获得本发明的化合物。
本发明还提供了一种组合物(如，药物组合物或农药组合物)，含有有效量的式(I)所示母核结构所述的化合物、或其衍生物(如异构体、溶剂合物、前体)。也即，本发明提供了含有式(I)所示母核结构所述的化合物或其类似物的组合物。
本发明中，所述的“含有”，“具有”或“包括”包括了“包含”、“主要由……构成”、“基本上由……构成”、和“由……构成”；“主要由……构成”、“基本上由……构成”和“由……构成”属于“含有”、“具有”或 “包括”的下位概念。
如本文所用，“生物学/药剂学上可接受的盐”成分是适用于给药于(如喷洒于或涂抹于)蚊虫且对环境没有过度坏处(环境友好)的物质，即有合理的效益/风险比的物质。“生物学/药剂学上可接受的盐”是用于将本发明的化合物传送给动物的生物学/药剂学上可接受的溶剂、悬浮剂或赋形剂。载体可以是液体或固体。
在本发明中，所述的组合物含有按照重量比例为0.001-50％的式(I)所示母核结构的化合物或其生物学/药剂学上可接受的盐。较佳的，所述的组合物含有按照重量比例为0.05-30％的式(I)所示母核结构的化合物或其生物学/药剂学上可接受的盐；更佳地，所述的组合物含有按照重量比例为0.01-20％的式(I)所示母核结构的化合物或其盐。
本领域人员应理解，根据临床实际需求或制药学中的设计方式，其它的重量比例也是可行的。
本发明所述的组合物的剂型可以是多种多样的，只要是能够使活性成分有效地到达蚊虫机体的剂型都是可以的。比如可选自：凝胶剂、气雾剂、片剂、胶囊、粉末、颗粒、糖浆、溶液、或悬浮液。根据本发明的化合物所欲施用的方式，本领域人员可以选择方便应用的剂型。从易于制备和给药的立场看，优选的药物组合物是液态(如混悬液、溶液)组合物或气雾态组合物。本发明的化合物或其组合物也可储存在适宜于喷洒、注射、涂抹或滴注的消毒器具中。
在本发明的一个优选方式中，本发明还提供了一种用于促进雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的组合物，其包括：烷烃；以及功能性化合物、辅剂(如佐剂)或药剂学可接受的载体。所述的功能性化合物、辅剂(如佐剂)或药剂学可接受的载体具有适用于蚊虫的特点，能够辅助所述烷烃对于蚊虫的功能或效果。
本发明所述的组合物或化合物还可被置于试剂盒中，以便于本领域技术人员的应用。较佳地，所述的试剂盒中还可包括指导本领域技术人员进行适当应用的使用说明书。
应用
本发明人在研究中发现，本发明的式(I)所示母核结构的化合物可以作为信息素，具有促进雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力。 本发明中，所述的蚊虫包括按蚊属蚊虫。
本发明人首次将本发明的化合物确定为信息素。信息素，也称做外激素，其一般为化学物质，可以由动物个体分泌到体外或体表，被同物种的其他个体通过感觉器官察觉，使后者表现出某种行为，情绪，心理或生理机制改变的物质。所述信息素具有通讯功能。
基于本发明人的新发现，本发明提供了式(I)所示母核结构的化合物或其衍生物，如其异构体、溶剂合物、前体，或它们的生物学/药剂学上可接受的盐的用途，用于促进雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力；或，用于制备促进雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的组合物。
作为本发明的一种优选方式，本发明的式(I)所示母核结构的化合物或其衍生物可应用于与雄性不育技术进行结合。雄性不育技术或遗传控制技术可以应用于控制蚊子及其它卫生害虫的种群数量的关键在于不育的雄虫的竞争能力，前提是：此类雄虫需要有足够的能力与野外的雄性蚊虫进行竞争。而本发明的式(I)所示母核结构的化合物或其衍生物则能够赋予雄性蚊虫提高的交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力，这一特性尤其适用于此类雄性不育或基因改造的雄蚊，当其交配竞争力、生殖竞争能力或对雌性蚊虫的竞争能力增强并被释放于野外环境后，使得野外环境的雄性蚊虫(具有生殖能力)与雌性蚊虫的交配显著性减少，从而有效控制后代蚊虫的数量。
鉴于本发明所述的化合物的功能，且二十七烷或其衍生物在蚊虫体壁天然存在，作为本发明的一种实施方式，还包括调节与产生该二十七烷或其衍生物有关的基因来调节该化合物在蚊虫体内的存在量，从而调节雄性蚊虫的交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力。
在得知了二十七烷或其衍生物的功能以后，可以以其为分子标记物，来进行雄性蚊虫的生殖能力的鉴定。本领域技术人员可以采用任何本领域公知的或正在发展的多种技术来进行化合物的定性/定量鉴定，以确定二十七烷或其衍生物的量，例如色谱和质谱等技术。
作为本发明的优选方式，提供了一种体壁信息素鉴定的方法，所述方法包括如何收集蚊虫体壁碳氢化合物，以及利用气相色谱和质谱(GC/MS)鉴定蚊虫表皮碳氢化合物的组分的分析方法。
本发明人的研究中已显示，desat1基因能够影响蚊虫表皮碳氢化合物正二十七烷的合成、进而影响蚊虫的交配。因此，desat1基因可以作为实现调控信息素合成的靶基因。
应理解，在得知了所述desat1基因在对于所述化合物的产生量(体内产生量)的调控方面的作用后，可以根据实际所需，采用本领域人员熟知的多种方法来调节所述的desat1基因的表达或活性，这些方法均被包含在本发明中。
可以利用desat1基因的表达或活性的上调剂来上调desat1基因的活性。所述desat1基因的表达或活性的上调剂包括了促进剂、激动剂、激活剂。所述的“上调”、“促进”包括了蛋白活性的“上调”、“促进”或蛋白表达的“上调”、“促进”。一些可提高desat1基因编码的蛋白的活性、提高desat1基因基因或蛋白的稳定性、上调desat1基因的表达、增加desat1基因编码的蛋白的有效作用时间的物质，这些物质均可用于本发明，作为对于上调desat1基因或其编码的蛋白有用的物质。它们可以是化合物、化学小分子、生物分子。所述的生物分子可以是核酸水平(包括DNA、RNA)的，也可以是蛋白水平的。
本发明中，所述的desat1基因或其编码基因的下调剂是指一些可降低desat1基因的活性、降低desat1基因或其编码基因的稳定性、下调desat1基因的表达、减少desat1基因有效作用时间、抑制desat1基因的转录和翻译的物质、或降低蛋白的磷酸化/激活水平，这些物质均可用于本发明，作为对于下调desat1基因有用的物质。它们可以是化合物、化学小分子、生物分子。所述的生物分子可以是核酸水平(包括DNA、RNA)的，也可以是蛋白水平的。例如，所述的下调剂是：特异性干扰desat1基因或其它信号通路基因表达的干扰RNA分子或反义核苷酸；或是特异性编辑desat1基因的基因编辑试剂，等等。
作为本发明的一种可选方式，提供一种下调蚊虫desat1基因的方法，包括对desat1基因进行靶向性地突变、基因编辑或基因干扰等，从而实现其表达下调。作为一种更为具体的实施例方式，藉由上述任一的方法，使desat1基因转变为其突变体，从而使其不再发挥作用。作为一种更为具体的实施例方式，采用RNAi干扰或CRISPR/Cas9系统进行基因编辑。合适的sgRNA靶位点，会带来更高的基因编辑效率，所以在着手进行基因编辑前，可以设计并找到合适的靶位点。在设计特异性靶位点后，还可以进行体外细胞活性筛选，以获得有效的靶位点用于后续实验。
作为本发明的一种可选方式，提供一种下调蚊虫desat1基因表达的蛋白的活性的方法，包括运用特异性结合desat1基因表达的蛋白的结合分子，例如抗体。所述的结合分子可以制成抗昆虫的制剂。所述结合分子例如单克隆抗体或多克隆抗体，此类单克隆抗体可以利用杂交瘤技术来制备。多克隆抗体可用本领域技术人员熟知的各种方法来制得。
可采用任何适当的常规手段，包括试剂、温度、压力条件等来实施所述的方法。
在得知了本发明所披露的desat1基因调控蚊虫信息素合成的分子机制之后，可以基于该特征来筛选通过在该分子机制中起作用，从而调控雄性蚊虫的交配性能的物质(包括潜在物质)。
因此，本发明提供一种筛选调节雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的物质的方法，所述方法包括：(1)将候选物质加入到表达desat1基因的体系中；(2)检测所述体系，观测其中desat1基因的表达或活性，若其表达或活性提高，则表明该候选物质为可用于促进雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的物质；若其表达或活性降低，则表明该候选物质为可用于降低雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的物质。
在本发明的优选方式中，在进行筛选时，为了更易于观察到desat1基因的表达或活性或其所在的信号通路、基因或蛋白的表达或活性的改变，还可设置对照组，所述的对照组可以是不添加所述候选物质的体系。
对于候选物质没有特别的限制，可以是感兴趣的、广泛的物质。例如，但不限于：来自化合物库或基因库的小分子化合物、基因或蛋白；或针对desat1基因或其编码的蛋白或它们的上游或下游分子设计的小分子化合物、激动剂、上调剂、促进剂、基因编辑试剂、干扰分子、核酸抑制物、结合分子(如抗体或配体)。在得知了本发明所设计的信号通路信息后，本领域技术人员可以作出多种多样的设计，这些均包含在本发明中。
作为本发明的优选方式，所述的方法还包括：对获得的潜在物质进行进一步的细胞实验和/或动物试验，以进一步选择和确定对于调节雄性蚊虫的交配性能真正有用的物质。
在得知了desat1基因的功能以后，可以以其为分子标记物，来进行雄性蚊虫的生殖能力的鉴定。本领域技术人员可以采用任何本领域公知的或正在发 展的多种技术来进行核酸分析或蛋白分析，这些技术均可被包含在本发明中。所述的方法例如包括但不限于：测序法，PCR扩增法，探针法，杂交法，限制性酶切分析法，等位基因多态性分析法(如溶解曲线法)进行核酸序列的鉴定，等等。
综上，运用本发明的技术方案，可以有效地调控如促进蚊虫如按蚊交配，达到提高控制蚊虫种群的目的。
以下实施例进一步说明本发明内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，中所述的条件，或按照制造厂商所建议的条件。
实施例1、干扰desat1影响按蚊交配
为了研究desat1对斯氏按蚊交配的影响，本发明人收集刚羽化的斯氏按蚊雄蚊，显微注射靶向干扰desat1的双链RNA(dsdesat1)，以注射干扰绿色荧光蛋白基因GFP的双链RNA(dsGFP)为对照。注射dsdesat1后显著干扰了desat1基因的表达，如图1。
其中，双链RNA(dsdesat1)相应的DNA序列如下(SEQ ID NO:1)：
Figure PCTCN2022070053-appb-000002
在正常条件下饲养3天后，取20只注射过双链RNA的雄蚊和20只未交配且未处理过的雌蚊，放在杯中任其自由交配。交配过夜后，在体视显微镜下解剖雌蚊的储精囊，观察储精囊有无精子。以有无精子为判断交配与否的标准。
统计结果发现，干扰desat1后显著抑制按蚊交配，如图2。证实了desat1能够调控按蚊交配。
实施例2、体壁碳氢化合物的分离与鉴定
如前所述，desat1能够影响表皮碳氢化合物的合成进而影响交配。进一步地，为了鉴定调控按蚊交配的关键通讯信息物质，本发明人从按蚊表皮提取了碳氢化合物进行差异比较分析，具体通过如下方法鉴定：
1、表皮碳氢化合物的提取
收集刚羽化的雄性按蚊，显微注射desat1双链RNA，以注射dsGFP为对照，在正常条件下饲养3天后，实验组和对照组分别取80只按蚊，放入预先加入800μL己烷的1.5ml的离心管中，按蚊在己烷中浸泡萃取5分钟。将己烷萃取上清转移至新的1.5mL离心管中，在真空蒸干机中将样品浓缩至30μL，作为待检测的样本。
2、化合物质谱鉴定
使用的仪器为Agilent 7890B GC system耦合高分辨率Agilent 7200 Q-TOF MS，GC/MS使用的分析柱为HP-5MS column(Agilent，30m×0.25mm，0.25μm film thickness，5％phenyl methyl siloxane stationary phase)。设定柱箱温度程序为80℃增长到200℃，温度增长速率为20℃增长速率，在200℃维持2min，接着从200℃增长到320℃，温度增长速率为5℃增长速。样本注射5μL，流速为1.0mL/min。质谱QTOF以每秒5次扫描的速度记录，质荷比为30～550m/z。将离子源温度和发射电流分别设置为230℃和6.8μA，将电子能量保持在70ev，QTOFMS在2GHz-EDR模式下运行(2GHz扩展动态范围)，以扩展线性动态范围。数据采集和评价分别采用MassHunter acquisition、MassHunter Quantitative和定性分析(version B07.00，Agilent Technologies，CA)。峰的鉴定是通过与标准溶液、美国国家标准和NIST14的质谱比较而确 定的。质谱分析结果显示，在按蚊体壁中总共分离鉴定出16种碳氢化合物，如图3。
3、受desat1调控的体壁碳氢化合物的鉴定
为了鉴定调控按蚊交配求偶的关键信息素物质，本发明人比较了dsGFP对照组和desat1干扰组按蚊体壁碳氢化合物谱的差异来发现这些物质，而干扰desat1后，正二十七烷的组分比例显著下降，如图4。
该结果提示，正二十七烷受desat1调控。
实施例3、信息素正二十七烷影响按蚊交配的测试
为了验证正二十七烷是否调控了按蚊的交配，本发明人开展了测试实验，流程如下：
分开收集刚羽化的雄蚊和雌蚊，在正常条件下饲养2天。配制75μg/mL的正二十七烷的正己烷溶液，将配制好的溶液用微型毛笔涂在雄性按蚊的腹部，每个处理分为三组，每组20只，以涂正己烷为对照。在正常条件下饲养2天后，每组加入20只未交配过的雌蚊任其自由交配，交配过夜后解剖储精囊，统计交配率。
实验结果显示，涂有正二十七烷的雄性按蚊的交配竞争力显著提高，如图5。该结果说明，正二十七烷是能够促进雄性按蚊交配竞争力的性信息素。
同样地，正二十七烷也能提高各种雄性按蚊交配竞争力，包括野生型、不育或遗传改造的雄性按蚊。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 烷烃或其衍生物的应用，所述的烷烃为：C 27H 56，用于作为信息素：
    促进雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力；或
    制备促进雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的组合物。
  2. 如权利要求1所述的应用，其特征在于，所述烷烃的衍生物包括：所述烷烃的异构体、溶剂合物、前体或盐；较佳地，所述烷烃为正二十七烷。
  3. 如权利要求1所述的应用，其特征在于，所述的蚊虫包括：按蚊属蚊虫；和/或
    所述的雄性蚊虫包括：雄性不育的蚊虫、遗传改造的蚊虫、野生型蚊虫。
  4. 如权利要求1所述的应用，其特征在于，所述烷烃被包含于组合物中，所述组合物还包含有：功能性化合物、辅剂、药剂学可接受的载体；较佳地，所述辅剂包括佐剂。
  5. 一种调节雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的方法，包括：(a)利用烷烃或其衍生物处理雄性蚊虫；或，(b)在雄性蚊虫体内调节烷烃或其衍生物的存在量；所述的烷烃为：C 27H 56。
  6. 如权利要求5所述的方法，其特征在于，所述烷烃的衍生物包括：所述烷烃的异构体、溶剂合物、前体或盐；较佳地，所述烷烃为正二十七烷。
  7. 如权利要求5所述的方法，其特征在于，所述的蚊虫包括：按蚊属蚊虫；和/或
    所述的雄性蚊虫包括：雄性不育的蚊虫、遗传改造的蚊虫、野生型蚊虫。
  8. 如权利要求5所述的方法，其特征在于，(b)中，所述在雄性蚊虫体内调节烷烃或其衍生物的存在量包括：
    促进蚊虫体内desat1基因的表达，从而提高所述烷烃的存在量，进而促进雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力；或
    降低蚊虫体内desat1基因的表达，从而降低所述烷烃的存在量，进而抑制雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力。
  9. 如权利要求5所述的方法，其特征在于，所述促进蚊虫体内desat1基因的表达包括：利用包括下组的上调分子进行调控：过表达所述desat1基因的表达盒或表达构建物；或，与所述desat1基因或其表达的蛋白相互作用、从而提高其表达或活性的调控分子；或
    所述降低蚊虫体内desat1基因的表达包括：利用包括下组的下调分子进行调控：敲除或沉默desat1基因的试剂，抑制desat1基因编码的蛋白的活性的试剂；较佳地，所述下调分子包括：针对所述desat1基因的基因编辑试剂、同源重组试剂或定点突变试剂，所述试剂将desat1基因编码的蛋白进行功能丧失性突变，或特异性干扰desat1基因表达的干扰分子；较佳地，所述下调分子包括沉默desat1基因的双链RNA；更佳地，该双链RNA具有SEQ ID NO:1所示核苷酸序列。
  10. 一种用于促进雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的组合物或试剂盒，其包括：烷烃；以及辅剂，如正己烷；所述的烷烃为：C 27H 56。
  11. 一种筛选调节雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的物质的方法，包括：
    (1)将候选物质加入到表达desat1基因的体系中；
    (2)检测所述体系，观测其中desat1基因的表达或活性，若其表达或活性提高，则表明该候选物质为可用于促进雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的物质；若其表达或活性降低，则表明该候选物质为可用于降低雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的物质。
  12. 如权利要求11所述的方法，其特征在于，所述方法还包括：设置不添加所述候选物质的对照组，从而明确分辨测试组中所述desat1基因编码的蛋白 表达或活性与对照组的差异；和/或
    所述的候选物质包括：小分子化合物、针对所述desat1基因或其上游或下游蛋白或基因设计的调控分子。
  13. 如权利要求11所述的方法，其特征在于，所述的小分子化合物包括从蚊虫分离的化合物；较佳地包括蚊虫体壁碳氢化合物；和/或
    所述候选物质为小分子化合物时，通过包括气相色谱和/或质谱鉴定的方法进行鉴定或分析。
  14. 烷烃或其衍生物的应用，用于作为鉴定雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的信息化合物，或用于制备鉴定雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的检测试剂；所述的烷烃为：C 27H 56。
  15. desat1基因或其编码的蛋白的应用，用于作为鉴定雄性蚊虫交配竞争力、生殖竞争能力或对雌性蚊虫的吸引能力的标志物。
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