WO2023137785A1 - 苜蓿烯的应用、一种重组生防真菌和一种僵虫及其应用 - Google Patents

苜蓿烯的应用、一种重组生防真菌和一种僵虫及其应用 Download PDF

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WO2023137785A1
WO2023137785A1 PCT/CN2022/074175 CN2022074175W WO2023137785A1 WO 2023137785 A1 WO2023137785 A1 WO 2023137785A1 CN 2022074175 W CN2022074175 W CN 2022074175W WO 2023137785 A1 WO2023137785 A1 WO 2023137785A1
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recombinant
mellonella
alfalene
larvae
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方卫国
唐丹
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浙江大学
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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
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/10—Animals; Substances produced thereby or obtained therefrom
    • A01N63/14—Insects
    • 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
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
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    • A01P19/00—Pest attractants
    • A—HUMAN NECESSITIES
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    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
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    • C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
    • C12Y402/03058—Longifolene synthase (4.2.3.58)
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    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the invention belongs to the technical field of genetic engineering, and in particular relates to the application of alfalene, a recombinant biocontrol fungus, a zombie and the application thereof.
  • Fungi are the most common insect pathogens, and more than 1,000 fungi have been found to infect insects. Among them, fungi of the genus Metarhizium, Beauveria, Paecilomyces and Verticillium in the Hyphomycetes have been developed into a variety of fungal agents for the prevention and control of agricultural, forestry and health pests, as well as the diseases they transmit (Zhao H, Lovett B, Fang W. Genetically Engineering Entomopathogenic Fungi. Adv Genet. 2016, 94:137-63.). At present, there are more than 60 kinds of filamentous fungal insecticides registered in the world, which are the new technology and product source of pest biological control.
  • the factors that determine the efficiency of fungal insecticides in controlling pests in the field mainly include four aspects: the inoculation rate (the proportion of pests with spores attached to the body wall) when fungal insecticides are applied, the tolerance to environmental adversity before the spores invade the pests, the killing speed, and the diffusion speed of newly formed spores on the dead insects.
  • the inoculation rate the proportion of pests with spores attached to the body wall
  • the tolerance to environmental adversity before the spores invade the pests the killing speed
  • the diffusion speed the diffusion speed of newly formed spores on the dead insects.
  • fungal insecticides According to the life activity characteristics of pests, people use fungal insecticides in different ways.
  • pests with weak activity such as Lepidoptera larvae
  • the existing methods are to produce a large number of spores by means of fermentation, etc., and make fungal insecticide preparations such as oils, powders and granules, which are mainly released in the field by spraying and soil application; during the application process, the fungal spores may directly contact the pests, or the pests themselves are active and stained with spores sprayed in the environment (leaves or soil) and inoculated.
  • fungicides mainly spraying fungicides on a solid surface (such as black cotton cloth, mosquito nets, walls, etc.) St Leger RJ.
  • Transgenic Metarhizium rapidly kills mosquitoes in a malaria-endemic region of Burkina Faso. Science. 2019 May 31; 364(6443):894-897.
  • the inoculation efficiency determines the effect of fungal insecticides on the control of flying pests such as mosquitoes, and the various strategies adopted in the existing application methods have improved the inoculation rate to a certain extent, but they still cannot meet the requirements of pest control, and the inoculation efficiency needs to be further improved.
  • the purpose of the present invention is to provide the application of alfalene, a recombinant biocontrol fungus and a botworm and the application thereof.
  • the alfalene, recombinant biocontrol fungus and botworm can attract insects and improve the insect inoculation rate.
  • the present invention provides the application of alfalene in attracting insects and/or controlling pests, the alfalene has the structure shown in formula I;
  • the insects include one or more of fruit flies, wax moths and mosquitoes.
  • the present invention also provides a recombinant biocontrol fungus, which comprises a recombinant fungal expression plasmid, and a pine longifolene synthesis gene is inserted into the recombinant fungal expression plasmid.
  • the nucleotide sequence of the pine longifolene synthesis gene is shown in SEQ ID NO.1.
  • the original bacteria of the recombinant biocontrol fungi include Metarhizium anisopliae.
  • the present invention also provides a zombie infected with the recombinant biocontrol fungus described in the above scheme.
  • the present invention also provides the application of the recombinant bio-control fungus described in the above scheme or the described zombie insects in attracting insects and/or controlling insect pests.
  • the insects include one or more of fruit flies, wax moths and mosquitoes.
  • the present invention also provides the application of the recombinant biocontrol fungus described in the above scheme or the zombella in the preparation of alfalene; the alfalene has a structure shown in formula I;
  • the present invention also provides an insect attractant, the active ingredient of which includes alfalene or the recombinant biocontrol fungus described in the above-mentioned scheme or the described dead insect; the chemical structural formula of the alfalene is shown in formula I.
  • the invention provides the application of alfalene in attracting insects and/or controlling insect pests.
  • Alfalene is attractive to insects and can increase the rate of insect inoculation.
  • the research of the present invention finds that the higher the concentration of alfalene, the stronger the attracting effect on mosquitoes and fruit fly larvae, and when the content of alfalene is as low as 10 -11 g, the fruit fly larvae will still react to it. Alfalene is also attractive to the larvae of the greater wax moth.
  • the present invention also provides a recombinant biocontrol fungus, which comprises a recombinant fungal expression plasmid, and a pine longifolene synthesis gene is inserted into the recombinant fungal expression plasmid.
  • the synthetic amount of longifolene and alfalfaene of the recombinant biocontrol fungus is high, and the amount of spore volatilization of longifene and alfalfaene is large, so that the attractiveness to insects is enhanced, and the insect inoculation rate can be improved.
  • the invention provides a new solution for the low inoculation efficiency of fungal insecticides for preventing mosquitoes and other pests, and shows broad application prospects.
  • Fig. 1 is the apparatus and test result that the dead worms obtained by Roberts Metarhizium anisopliae infecting the older larvae of G. mellonella in Example 1 are attractive to healthy insects
  • a in Fig. 1 is a schematic diagram of the device for Two-way choice method to detect the attractiveness of G. mellonella worms to insects, wherein the gray and white insects represent the dead worms and contrast frozen dead insects respectively
  • B in Fig. 1 is a schematic diagram of the device for the two-way choice method to detect the attractiveness of G.
  • the middle is the place where healthy fruit fly larvae are released, and the healthy insects entering the left and right are affected by dead insects and control dead insects respectively;
  • C in Figure 1 is the response index (Response index) of larvae, fruit fly larvae and adults of Aedes albopictus to the worms formed by Metarhizium anisopliae Roberts infection;
  • D in Figure 1 is a schematic diagram of the two-way choice method for detecting the attractiveness of worms to adults of Aedes albopictus, in which gray and white insects Represent dead insects and control frozen dead insects respectively, and healthy mosquitoes are placed in tube No. 1 in the middle;
  • Figure 2 is the effect of different concentrations of longifene and alfalene on insect behavior; wherein A is the effect of different concentrations of longifene on the behavior of fruit fly larvae; B is the effect of different concentrations of longifene on the behavior of fruit fly larvae; C is the effect of different concentrations of longifene on the behavior of Aedes albopictus adults; D is the effect of different concentrations of alfalene on the behavior of Aedes albopictus adults;
  • Figure 3 is the effect of longifolene and alfalene on the behavior of the larvae of Mellonella mellonella
  • Figure 4 is the construction of the transgenic strain Mr-Tps; wherein, A is the vector map of pPK2-bar-gpd-GFP-Tps; B is the transcribed expression of the TPS gene, wherein Act is the internal reference gene;
  • Figure 5 shows the volatilization of longifolene and alfalene in WT and transgenic strain Mr-Tps zombies under different culture conditions and their effects on insect behavior; where, A is the WT and the transgenic strain Mr-Tps cultivated on the zombie; B is the WT and the transgenic strain Mr-Tps cultivated on the PDA medium; C is the WT and the transgenic strain Mr-Tps cultivated on the fermentation sporulation medium; D is the zombie formed by Mr-Tps and WT infection
  • the invention provides the application of alfalene in attracting insects and/or controlling pests, the chemical structure of the alfalene is shown in formula I;
  • alfalene is attractive to insects; and the insects preferably include one or more of fruit flies, mosquitoes and Mellonella mellonella.
  • the present invention also provides a recombinant biocontrol fungus, which comprises a recombinant fungal expression plasmid, and a pine longifolene synthesis gene is inserted into the recombinant fungal expression plasmid.
  • the recombinant biocontrol fungus has stronger insect attraction and insecticidal efficacy than the original biocontrol fungus.
  • the nucleotide sequence of the pine longifolene synthesis gene is as shown in SEQ ID NO.1, specifically:
  • the protein encoded by the pine longifolene synthesis gene is longifolene synthase Tps; the amino acid sequence of the longifolene synthase Tps is as shown in SEQ ID NO.2, specifically:
  • the present invention inquired the protein sequence (Genbank accession number: ABV44454) of Pinus sylvestris longifolene synthase Tps on NCBI (https://www.ncbi.nlm.nih.gov/). According to the service provided by Codon Usage Database (http://www.kazusa.or.jp/codon/), the codon preference of Metarhizium robertsii was obtained, and the codon type with the highest frequency was selected to obtain the coding sequence of Tps protein.
  • the original bacteria of the recombinant biocontrol fungi preferably include Metarhizium anisopliae, more preferably include Metarhizium anisopliae.
  • the original plasmid of the recombinant fungal expression plasmid is preferably pPK2-bar-gpd-GFP; the insertion site of the pine longifolene synthesis gene on the recombinant fungal expression plasmid is preferably BamHI and EcoR V.
  • the present invention has no special limitation on the construction method of the recombinant fungal expression plasmid, and conventional methods in the art can be used.
  • the present invention has no special limitation on the construction method of the recombinant biocontrol fungus, and conventional methods in the field can be used.
  • the present invention also provides a zombie infected with the recombinant biocontrol fungus described in the above scheme.
  • the zombie is preferably prepared by the following method:
  • the recombinant biocontrol fungi described in the above scheme are used to infect the recipient larvae. After the recipient larvae are infected and die, the corpses are disinfected and cultured in moisture to form zombies covered with spores.
  • the recipient larvae are preferably the last instar larvae of Mellonella mellonella.
  • the infection of recipient larvae with the recombinant bio-control fungus described in the above scheme includes infecting the recipient larvae with the spore suspension of the recombinant bio-control fungus described in the above-mentioned scheme; the concentration of the recombinant bio-control fungus in the spore suspension of the recombinant bio-control fungus is preferably 1 ⁇ 10 7 spores/ml.
  • the disinfectant used in the disinfection is preferably sodium hypochlorite solution; the mass concentration of sodium hypochlorite in the sodium hypochlorite solution is preferably 0.05%.
  • the present invention also provides the application of the recombinant bio-control fungus described in the above scheme or the described zombie insects in attracting insects and/or controlling insect pests.
  • the insects may include one or more of Drosophila mellonella, Mellonella mellonella and mosquitoes.
  • the present invention also provides the application of the recombinant biocontrol fungus described in the above scheme or the zombella in the preparation of alfalene; the alfalene has a structure shown in formula I;
  • the present invention also provides an insect attractant, the active ingredient includes alfalene or the recombinant biocontrol fungus described in the above scheme or the described dead insect; the alfalene has a structure shown in formula I;
  • the effective amount of alfalene used on fruit fly larvae in a space of 64 cm is preferably 10 -11 g to 10 -5 g, of which 10 -5 g has the strongest attracting effect on fruit fly larvae, and the effective amount of alfalene used on adults of Aedes albopictus in a space of 640 cm is 10 -9 g to 10 -5 g, of which 10 -5 g has the strongest attracting effect on mosquitoes.
  • the present invention also provides an insecticide or an insecticide device, comprising the insect attractant described in the above scheme.
  • Example 1 Metarhizium anisopliae Roberts infects older larvae of Mellonella mellonella, and the dead worms obtained are attractive to healthy insects
  • spore suspension (1 ⁇ 10 7 spores/ml) was prepared with Triton-X-100 solution (0.01%) and used to infect older larvae of Mellonella mellonella. After the larvae were infected and died, the corpses were disinfected with sodium hypochlorite solution (0.05%) and kept moist for cultivation to form zombies covered with spores.
  • the two-way choice method was used to detect the attractiveness of the larvae of the mellonella mellonella to the larvae of Drosophila mellonella.
  • Place the G. mellonella larvae (control) that had just been frozen to death and had been prevented at room temperature for 20 minutes and the above-mentioned G. mellonella larvae on both sides of a 9cm petri dish (containing 2% water agar), then placed 20 healthy 3rd instar Canton-S fruit fly larvae in the middle of the petri dish, and took pictures after 10 minutes to record the selection of the Drosophila larvae, and calculate Response index (response index) [(the number of fruit fly larvae that selected the G. mellonella larvae-selected the control G. mellonella larva number of larvae)/total number of fruit fly larvae (20)]. This experiment was repeated 6 times.
  • the improved Two-way choice method was used to detect the attractiveness of the larvae of the larvae of the larvae of the mellonella mellonella.
  • Three of the above-mentioned frozen-to-dead control G. mellonella larvae and three of G. mellonella larvae were placed in a petri dish with a diameter of 9 cm, and then the petri dish was placed diagonally in the device, and 40 healthy G. mellonella larvae were released at the center of the device, and then placed in the dark at room temperature. After 1 hour, the selection of G. mellonella was recorded.
  • Calculation Response index [the number of the greater wax moth selected from dead worms of the greater wax moth - the number of greater wax moths selected to freeze to death of the greater wax moth]/total number of larvae of the greater wax moth]. This experiment was repeated 6 times.
  • the larvae formed by Metarhizium anisopliae Roberts infecting the larvae of Mellonella mellonella produced 13 kinds of volatile substances (Table 1).
  • Table 1 The larvae formed by Metarhizium anisopliae Roberts infecting the larvae of Mellonella mellonella produced 13 kinds of volatile substances (Table 1).
  • four were known compounds longifene, alfalene, ⁇ -farnesene and geosmin
  • the predecessors have clarified the ability of ⁇ -farnesene and geosmin to attract insects, and the mechanism of insects' perception of these two types of compounds.
  • the present invention has carried out in-depth research on the insect attraction of longifolene and alfalene.
  • the detection of the attractiveness of longifolene and alfalene to Drosophila larvae by Two-way choice method is similar to the above-mentioned detection of the attractiveness of zygote to Drosophila larvae.
  • Zombies were changed to a circular filter paper sheet with a diameter of 5 mm (containing 10 ⁇ l of longifolene or alfalene solutions with different concentrations), and the control was a filter paper sheet containing 10 ⁇ l of solvent n-hexane.
  • the attractiveness of longifene and alfalene to these two insects was also analyzed by using the method of detecting the attractiveness of zomboids to the larvae of Melonella mellonella and adults of mosquitoes.
  • the dead worms were replaced by cotton balls with a diameter of about 2 cm (containing 100 ⁇ l of solutions containing different concentrations of longifolene or alfalene), and the control was cotton balls containing 100 ⁇ l of the solvent n-hexane.
  • cotton balls with a diameter of about 1 cm containing 100 ⁇ l of solutions containing longifolene or alfalene at different concentrations
  • the control was cotton balls containing 100 ⁇ l of the solvent n-hexane.
  • Example 4 Construction of a recombinant strain of Metarhizium anisopliae with increased volatilization of alfalene and longifolene 1) Method:
  • the codon type with the highest frequency was selected to obtain the coding sequence of the TPS protein, the sequence information is shown in SEQ ID NO.1, and was synthesized by Hangzhou Youkang Biotechnology Co., Ltd. and connected to the puc57-simple-TOPO vector to obtain a plasmid containing the coding sequence of longifolene synthase, which was named puc57-simple-TOPO-PsTPS.
  • the PCR product was digested with restriction endonucleases BamH I and EcoR V (Thermo Scientific), and connected with the vector pPK2-bar-gpd-GFP cut with the same enzyme, the vector information is shown in A in Figure 4, and the expression vector pPK2-bar-gpd-GFP-Tps of Tps was obtained.
  • plasmid pPK2-bar-gpd-GFP-PsTps was transferred into the Agrobacterium tumefaciens strain AGL1, it was then transferred into the Metarhizium anisopliae ARSEF 2575 strain.
  • Transformants were preliminarily screened with herbicide resistance selection and green fluorescent protein (GFP) observations, and further PCR was used to prove (primers used were PsTps-CDS-FP-BamHI and PsTps-CDS-RP-EcoRV) that the Tps expression cassette was successfully integrated into the genome of Metarhizium anisopliae, and RT-PCR proved that the Tps encoding gene was transcribed and expressed (B in Figure 4), and the Metarhizium anisopliae strain Mr-Tps with heterologous expression of the Tps gene was obtained.
  • GFP green fluorescent protein
  • RT-PCR proves that the Tps coding gene is transcribed and expressed
  • the analysis of the volatile substances produced by the worms formed by the transgenic strain Mr-Tps infecting the larvae of Mellonella mellonella found that it produced 11 kinds of volatile substances, of which the compound with the highest proportion was longifolene, the second was farnesene, the third was longifolene, and the fourth was alfalene.
  • the larvae infected with the transgenic strain Mr-Tps volatilized more kinds of sesquiterpenes, including longifolene, longifolene and cedrene.
  • the strain Mr-Tps produced 6 kinds of volatile substances, among which the compound with the highest proportion was longifolene (Table 2). Compared with the wild type strain, the ability of the strain Mr-Tps to volatilize longifolene and alfalfaene increased by 98 times and 4 times, respectively, but farnesene and geosmin were not detected (B in Figure 5).
  • Example 5 Expression of the Tps gene improves the attractiveness of Metarhizium anisopliae to insects
  • the attractiveness of the transgenic strain Mr-Tps and the wild-type strain WT grown on PDA and fermentation medium to mosquitoes was compared.
  • Embodiment 6 the transgenic bacterial strain control mosquito method based on the black cloth method
  • the mosquitoes were crushed with a sterilized grinding rod, they were evenly spread on the Metarhizium anisopliae screening medium (containing 100 ⁇ g/mL ampicillin, 100 ⁇ g/mL kanamycin, 80 ⁇ g/mL streptomycin; 4 ⁇ g/mL dodine; 10 ⁇ g/mL benomyl’s PDA), the plates were incubated upside down at 26°C in the dark, and after 5 days, the number of colonies (CFUs) on each plate was counted to calculate the inoculation rate and inoculum size.
  • Metarhizium anisopliae screening medium containing 100 ⁇ g/mL ampicillin, 100 ⁇ g/mL kanamycin, 80 ⁇ g/mL streptomycin; 4 ⁇ g/mL dodine; 10 ⁇ g/mL benomyl’s PDA

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Abstract

一种苜蓿烯的应用、一种重组生防真菌和一种僵虫及其应用。提供了苜蓿烯在昆虫引诱和/或防治虫害中的应用。苜蓿烯对昆虫有引诱力。苜蓿烯浓度越高，对蚊子和果蝇幼虫的引诱效果越强，当苜蓿烯的含量低至10-11g时，果蝇幼虫仍会对其产生反应。苜蓿烯对大蜡螟幼虫也具有吸引力。还提供了一种重组生防真菌，包含重组真菌表达质粒，所述重组真菌表达质粒上插入有松树长叶烯合成基因。所述重组生防真菌的长叶烯和苜蓿烯的合成量提高，孢子挥发长叶烯和苜蓿烯的量提高，从而对昆虫的吸引力增强。

Description

苜蓿烯的应用、一种重组生防真菌和一种僵虫及其应用 技术领域
本发明属于基因工程技术领域，具体涉及苜蓿烯的应用、一种重组生防真菌和一种僵虫及其应用。
背景技术
真菌是最常见的昆虫病原物，目前已发现了1000多种真菌可感染昆虫，其中丝孢菌纲中的绿僵菌(Metarhizium)、白僵菌(Beauveria)、拟青霉(Paecilomyces)和轮枝菌(Verticillium)等属的真菌，已被开发成多种菌剂用于防治农业、林业和卫生害虫，以及它们所传播的疾病(Zhao H,Lovett B,Fang W.Genetically Engineering Entomopathogenic Fungi.Adv Genet.2016,94:137-63.)。目前全世界注册登记的丝状真菌杀虫剂达60余种，是害虫生物防治新技术及产品来源。现有研究发现，基于丝孢菌纲的真菌杀虫剂主要通过体壁接触感染而杀死害虫。这一过程包括孢子在昆虫体壁上吸附、萌发、穿透昆虫体壁，然后真菌在寄主血腔中通过繁殖和分泌毒素等方式导致寄主僵硬死亡，最后，真菌从僵虫内部长出并在僵虫体表产生大量的孢子。孢子可能在环境中传播，感染其它健康的害虫，进一步甚至长期控制害虫。决定真菌杀虫剂田间防治害虫效率的因素主要包括4个方面：施用真菌杀虫剂时的接种率(体壁粘上孢子的害虫比例)，孢子侵入害虫之前对环境逆境的耐受力，杀虫速度，以及僵虫上新形成孢子的扩散速度。前人在深入研究昆虫病原真菌致病和抗逆机制的基础上，利用基因工程技术提高它们的杀虫速度或抗逆能力，为增强真菌杀虫剂的效率提供了多个遗传改良菌株(Zhao H,Lovett B,Fang W.Genetically Engineering Entomopathogenic Fungi.Adv Genet.2016,94:137-63.)。
根据害虫的生命活动特点，人们采用不同方式来使用真菌杀虫剂。对于活动能力较弱的害虫如鳞翅目幼虫，现有办法都是通过发酵等方式生产大量的孢子，并制成油剂\粉剂和颗粒等真菌杀虫剂制剂，主要采用喷施和土壤施放等方式在田间释放；在施用的过程中，真菌孢子可能直接接触到害虫，或者害虫自身活动而沾上喷洒在环境中(叶片或土壤)的孢子而被接种。人们也尝试利用其它方法提高接种率，如把真菌杀虫剂装载在昆 虫天敌(如捕食螨)身上，然后天敌将孢子传播给目标害虫，达到真菌杀虫剂和天敌共同防治害虫的目的(吴圣勇,杨清坡,徐长春,徐学农,雷仲仁.昆虫病原真菌和捕食螨间的互作关系及二者联合应用研究进展.中国生物防治学报.2019,35:127-133.)。对于活动能力强，通过飞舞等方式活动的害虫如蚊虫，通过喷洒的方法接种效率低下。人们根据蚊虫活动的特点，设计了一些施用真菌杀虫剂的方法，主要有将真菌杀虫剂预先喷施在一个固体表面(如黑色棉布、蚊帐和墙壁等)，并将这些固体介质安放在室内和蚊子入室的通道(如非洲传统房子的屋檐)里(Lovett B,Bilgo E,Millogo SA,Ouattarra AK,Sare I,Gnambani EJ,Dabire RK,Diabate A,St Leger RJ.Transgenic Metarhizium rapidly kills mosquitoes in a malaria-endemic region of Burkina Faso.Science.2019 May 31；364(6443):894-897.)。此外，还有人尝试在户外安置一些含有蚊虫引诱剂和真菌杀虫剂的装置来提高蚊虫接种率。
总体来说，接种效率决定了真菌杀虫剂防治飞舞害虫如蚊虫的效果，而现有施用方式所采用的多样的策略，一定程度上提高了接种率，但是还不能满足害虫防控的要求，接种效率有待进一步提高。
发明内容
有鉴于此，本发明的目的在于提供苜蓿烯的应用、一种重组生防真菌和一种僵虫及其应用，所述苜蓿烯、重组生防真菌和僵虫能够引诱昆虫，提高昆虫接种率。
本发明提供了苜蓿烯在昆虫引诱和/或防治虫害中的应用，所述苜蓿烯具有式I所示结构；
Figure PCTCN2022074175-appb-000001
优选的，所述昆虫包括果蝇、大蜡螟和蚊子中的一种或几种。
本发明还提供了一种重组生防真菌，包含重组真菌表达质粒，所述 重组真菌表达质粒上插入有松树长叶烯合成基因。
优选的，所述松树长叶烯合成基因的核苷酸序列如SEQ ID NO.1所示。
优选的，所述重组生防真菌的原始菌包括绿僵菌。
本发明还提供了一种僵虫，感染有上述方案所述的重组生防真菌。
本发明还提供了上述方案所述的重组生防真菌或者所述的僵虫在昆虫引诱和/或防治虫害中的应用。
优选的，所述昆虫包括果蝇、大蜡螟和蚊子中的一种或几种。
本发明还提供了上述方案所述的重组生防真菌或者所述的僵虫在制备苜蓿烯中的应用；所述苜蓿烯具有式I所示结构；
Figure PCTCN2022074175-appb-000002
本发明还提供了一种昆虫引诱剂，活性成分包括苜蓿烯或者上述方案所述的重组生防真菌或者所述的僵虫；所述苜蓿烯的化学结构式如式I所示。
本发明提供了苜蓿烯在昆虫引诱和/或防治虫害中的应用。苜蓿烯对昆虫有引诱力，能够提高昆虫接种率。本发明研究发现，苜蓿烯浓度越高，对蚊子和果蝇幼虫的引诱效果越强，当苜蓿烯的含量低至10 -11g时，果蝇幼虫仍会对其产生反应。苜蓿烯对大蜡螟幼虫也具有吸引力。
本发明还提供了一种重组生防真菌，包含重组真菌表达质粒，所述重组真菌表达质粒上插入有松树长叶烯合成基因。在本发明中，所述重组生防真菌的长叶烯和苜蓿烯的合成量高，孢子挥发长叶烯和苜蓿烯的量大，从而对昆虫的吸引力增强，进而能够提高昆虫接种率。本发明为真菌杀虫剂防蚊虫等害虫时存在的接种效率低提供了新的解决方案，展示了广阔的应用前景。
附图说明
图1为实施例1中罗伯茨绿僵菌感染大蜡螟高龄幼虫所得僵虫对健康 昆虫有吸引力采用的装置和试验结果，其中，图1中的A为Two-way choice法检测大蜡螟僵虫对昆虫的吸引力的装置示意图，其中灰色和白色昆虫分别代表僵虫和对照冻死昆虫；图1中的B为Two-way choice法检测大蜡螟僵虫对果蝇幼虫的吸引力的装置示意图；其中灰色和白色昆虫分别代表僵虫和对照冻死昆虫，中间为健康果蝇幼虫释放处，进入左边和右边的健康昆虫分别表示受僵虫和对照死虫影响；图1中的C为大蜡螟幼虫、果蝇幼虫和白纹伊蚊成虫对罗伯茨绿僵菌感染大蜡螟形成的僵虫的响应指数(Response index)；图1中的D为Two-way choice法检测大蜡螟僵虫对白纹伊蚊成虫的吸引力的装置示意图，其中灰色和白色昆虫分别代表僵虫和对照冻死昆虫，健康的蚊虫放在中间的1号管中；
图2为不同浓度的长叶烯和苜蓿烯对昆虫行为的影响；其中A为不同浓度的长叶烯对果蝇幼虫行为的影响；B为不同浓度的苜蓿烯对果蝇幼虫行为的影响；C为不同浓度的长叶烯对白纹伊蚊成虫行为的影响；D为不同浓度的苜蓿烯对白纹伊蚊成虫行为的影响；
图3为长叶烯和苜蓿烯对大蜡螟幼虫行为的影响；
图4为转基因菌株Mr-Tps的构建；其中，A为pPK2-bar-gpd-GFP-Tps载体图谱；B为TPS基因被转录表达，其中Act为内参基因；
图5为WT和转基因菌株Mr-Tps僵虫长叶烯、苜蓿烯在不同培养条件下的挥发量及对昆虫行为的影响；其中，A为在僵虫上培养的WT和转基因菌株Mr-Tps；B为在PDA培养基上培养的WT和转基因菌株Mr-Tps；C为在发酵产孢培养基上培养的WT和转基因菌株Mr-Tps；D为Mr-Tps和WT感染形成的僵虫对昆虫行为的影响，**代表存在极显著性差异(n＝6，P<0.01，Wilcoxon signed-ranktest)；
图6为白纹伊蚊和果蝇幼虫对不同培养条件下的WT和Mr-Tps偏好性分析；其中，A为白纹伊蚊在PDA、虫尸(cadavers)以及发酵产孢培养基(fermentation medium)上生长的WT和Mr-Tps的偏好性，**代表存在极显著性差异(n＝6，P<0.01，Wilcoxon signed-rank test)；B为果蝇幼虫在PDA培养基、虫尸(cadavers)以及发酵产孢培养基(fermentation medium)上生长的WT和Mr-Tps的偏好性，**代表存在极显著性差异(n＝6，P<0.01，Wilcoxon signed-rank test)；
图7为WT和Mr-Tps对白纹伊蚊的接种率和接种量；其中A表示总量为1×10 8个孢子的WT和Mr-Tps对白纹伊蚊的接种率，接种率＝被接种蚊子的数量/蚊子总数，**代表存在极显著性差异(n＝6，P<0.01，Wilcoxon signed-rank test)；B表示总量为1×10 8个孢子的WT和Mr-Tps对白纹伊蚊的接种率，接种量为白纹伊蚊被接种的孢子数量，*代表存在显著性差异(n＝6，P<0.05，Wilcoxon signed-rank test)。
具体实施方式
本发明提供了苜蓿烯在昆虫引诱和/或防治虫害中的应用，所述苜蓿烯的化学结构式如式I所示；
Figure PCTCN2022074175-appb-000003
本发明对所述苜蓿烯的来源没有特殊限制，采用本领域的常规方法制备得到或者来源于市售。在本发明中，苜蓿烯对昆虫有引诱力；所述昆虫优选的包括果蝇、蚊子和大蜡螟中的一种或几种。
本发明还提供了一种重组生防真菌，包含重组真菌表达质粒，所述重组真菌表达质粒上插入有松树长叶烯合成基因。
在本发明中，所述重组生防真菌比原始生防真菌的的昆虫吸引力和杀虫效力更强。
在本发明中，所述松树长叶烯合成基因的核苷酸序列如SEQ ID NO.1所示，具体为：
Figure PCTCN2022074175-appb-000004
Figure PCTCN2022074175-appb-000005
在本发明中，所述松树长叶烯合成基因编码的蛋白质为长叶烯合酶Tps；所述长叶烯合酶Tps的氨基酸序列如SEQ ID NO.2所示，具体为：
Figure PCTCN2022074175-appb-000006
Figure PCTCN2022074175-appb-000007
本发明在NCBI(https://www.ncbi.nlm.nih.gov/)上查询了樟子松(Pinus sylvestris)长叶烯合酶Tps的蛋白序列(Genbank accession number:ABV44454)。根据Codon Usage Database(http://www.kazusa.or.jp/codon/)提供的服务得到罗伯茨绿僵菌(Metarhizium robertsii)的密码子偏好性，选择了频率最高的密码子类型得到Tps蛋白的编码序列。
在本发明中，所述重组生防真菌的原始菌优选的包括绿僵菌，更优选的包括罗伯茨绿僵菌。
在本发明中，所述重组真菌表达质粒的原始质粒优选为pPK2-bar-gpd-GFP；所述松树长叶烯合成基因在所述重组真菌表达质粒上的插入位点优选为BamHI和EcoR V。本发明对所述重组真菌表达质粒的构建方法没有特殊限制，采用本领域的常规方法即可。
本发明对所述重组生防真菌的构建方法没有特殊限制，采用本领域的常规方法即可。
本发明还提供了一种僵虫，感染有上述方案所述的重组生防真菌。
在本发明中，所述僵虫优选的采用以下方法制备得到：
采用上述方案所述的重组生防真菌感染受体幼虫，待受体幼虫被感染死亡后，对虫尸消毒后进行保湿培养，形成被孢子覆盖的僵虫。
在本发明中，所述受体幼虫优选为大蜡螟末龄幼虫。在本发明中，所述采用上述方案所述的重组生防真菌感染受体幼虫包括采用上述方案所述的重组生防真菌的孢子悬浮液感染受体幼虫；所述重组生防真菌的孢子悬浮液中重组生防真菌的浓度优选为1×10 7spores/ml。在本发明中，所述消毒采用的消毒剂优选为次氯酸钠溶液；所述次氯酸钠溶液中次氯酸钠的质量浓度优选为0.05％。
本发明还提供了上述方案所述的重组生防真菌或者所述的僵虫在昆虫引诱和/或防治虫害中的应用。
在本发明的实施例中，所述昆虫可以包括果蝇、大蜡螟和蚊子中的一种或几种。
本发明还提供了上述方案所述的重组生防真菌或者所述的僵虫在制备苜蓿烯中的应用；所述苜蓿烯具有式I所示结构；
Figure PCTCN2022074175-appb-000008
本发明还提供了一种昆虫引诱剂，活性成分包括苜蓿烯或者上述方案所述的重组生防真菌或者所述的僵虫；所述苜蓿烯具有式I所示结构；
Figure PCTCN2022074175-appb-000009
在本发明中，苜蓿烯在64cm 3空间里对果蝇幼虫的有效使用量优选为10 -11g～10 -5g，其中10 -5g对果蝇幼虫引诱效果最强，苜蓿烯在640cm 3空间里对白纹伊蚊成虫的有效使用量为10 -9g到10 -5g，其中10 -5g对蚊子引诱效果最强。
本发明还提供了一种杀虫剂或杀虫装置，包括上述方案所述的昆虫引诱剂。
下面将结合本发明中的实施例，对本发明中的技术方案进行清楚、完整地描述。
实施例1：罗伯茨绿僵菌感染大蜡螟高龄幼虫所得僵虫对健康昆虫有吸引力
1.检测方法
(1)准备罗伯茨绿僵菌感染大蜡螟高龄幼虫而形成的僵虫
罗伯茨绿僵菌在PDA上培养14d后，用Triton-X-100溶液(0.01％)制备孢子悬浮液(1×10 7spores/ml)，并用于感染大蜡螟高龄幼虫，待幼虫被感染死亡后，用次氯酸钠溶液(0.05％)消毒虫尸并保湿培养，形成 被孢子覆盖的僵虫。
(2)Two-way choice法检测大蜡螟僵虫对果蝇健康幼虫的吸引力
利用如图1中的A所示的装置，用Two-way choice法检测大蜡螟僵虫对果蝇幼虫的吸引力。将刚冻死并在室温防止了20分钟的大蜡螟(对照)和上述一头大蜡螟僵虫放置在9cm培养皿(内含2％的水琼脂)的两侧，然后将20头健康3龄Canton-S果蝇幼虫置于培养皿正中间，10min后拍照记录果蝇幼虫的选择，并计算Response index(反应指数)＝【(选择大蜡螟僵虫的果蝇幼虫数目-选择对照大蜡螟幼虫的果蝇幼虫数目)/果蝇幼虫总数目(20头)】。该实验重复6次。
(3)检测大蜡螟僵虫对大蜡螟健康幼虫行为学的影响
利用图1中的B所示的装置，用改良的Two-way choice法检测大蜡螟僵虫对大蜡螟幼虫的吸引力。将上述冻死的对照大蜡螟幼虫和大蜡螟僵虫各三头放在直径为9cm的培养皿中，然后将培养皿对角放置于装置中，将40头健康大蜡螟幼虫释放于装置的中心点，然后于室温黑暗放置，1h后记录大蜡螟的选择。计算Response index(反应指数)＝【选择大蜡螟僵虫的大蜡螟数目-选择冻死大蜡螟的大蜡螟数目)/大蜡螟幼虫总数目】。该实验重复6次。
(4)检测大蜡螟僵虫对蚊子成虫的吸引力
僵虫对蚊子(白纹伊蚊)成虫吸引力的检测按有关文献进行(Robinson Ailie,Busula Annette O,Voets Mirjam A,Beshir Khalid B,Caulfield John C,Powers Stephen J,Verhulst Niels O,Winskill Peter,Muwanguzi Julian,Birkett Michael A,Smallegange Renate C,Masiga Daniel K,Mukabana W Richard,Sauerwein Robert W,Sutherland Colin J,Bousema Teun,Pickett John A,Takken Willem,Logan James G,de Boer Jetske G.Plasmodium-associated changes in human odor attract mosquitoes.Proc Natl Acad Sci USA 2018；115:4215.)，所用装置如如图1中的D所示。将上述冻死的对照大蜡螟幼虫和大蜡螟僵虫各一头放在直径为3.5cm的小安杯中，并放置在装置两侧的圆筒中。将10头羽化后3-5day未进行血餐的雌性蚊子收集在最中间1号的管子中，用纱布蒙住1号管子两端的口后于4℃冰箱放置3min以降低蚊子活动力。然后将1号管子与2号和3号管连接。最后将整个装 置放置在26℃黑暗的培养箱中，10h后记录蚊子的选择，并计算response index(反应指数)＝【选择大蜡螟僵虫的蚊子数目-选择冻死大蜡螟的蚊子数目)/蚊子总数目】。
2.结果
上述Two-way choice法行为学测定表明，罗伯茨绿僵菌感染的大蜡螟幼虫形成的僵虫对果蝇幼虫、大蜡螟幼虫和白纹伊蚊成虫均具有引诱作用(图1中的C)。
实施例2：僵虫产生对害虫有引诱力的挥发性化合物
1.僵虫挥发性化合物分析方法
(1)萃取方法。将5头罗伯茨绿僵菌感染大蜡螟幼虫形成的僵虫置于体积为20ml的进样瓶中，将50/30μm DVB/CAR/PDMS萃取头插入进样瓶中吸附萃取50min，整个过程45℃水浴加热。
(2)SPME-GC-MS分析方法。萃取结束后，手动进样，进样口250℃解析3min。色谱柱为30m×0.25mm，0.25μm的DB－5MS色谱柱；柱温起始温度为35℃，保持5min，然后以2℃/min升至145℃，再以15℃/min升至250℃(保留10min)。根据总离子流图，结合每个色谱峰的质谱特征数据进行分析，各挥发性组分通过与质谱库(NIST05)进行比对鉴别，初步鉴定物质类型。按照质谱库提供的物质的CAS号购买标品，进一步通过GC-MS分析标品的保留时间和质谱，将被鉴定物质和标品的保留时间和质谱特征进行对比，若它们的特征一致，则确认被鉴定化合物与标准品为同一物质。
2.结果
罗伯茨绿僵菌感染大蜡螟幼虫形成的僵虫产生13种挥发性物质(表1)，通过与标准品比较扥等方法发现，其中占比最高的5类化合物中，4个是已知化合物(长叶烯，苜蓿烯，β-金合欢烯和土臭素)，其中占比最高的化合物是一种未知的倍半萜烯。前人已经阐明了β-金合欢烯和土臭素引诱昆虫的能力，以及昆虫感知这两类化合物的机制，本发明对长叶烯和苜蓿烯的昆虫引诱力进行了深入研究。
表1 罗伯茨绿僵菌感染的大蜡螟产生的挥发性物质及各自占比
Figure PCTCN2022074175-appb-000010
Figure PCTCN2022074175-appb-000011
实施例3：苜蓿烯和长叶烯的昆虫引诱力
1)分析方法
长叶烯和苜蓿烯标准品购自Sigma-Aldrich，纯度为99％。
Two-way choice法检测长叶烯和苜蓿烯对果蝇幼虫引诱力的检测与上述检测僵虫对果蝇幼虫引诱力类似。其中僵虫改为直径为5mm的圆形滤纸片(含10μl含不同浓度的长叶烯或者苜蓿烯溶液)，对照为含10微升溶剂正己烷的滤纸片。类似地，也用僵虫对大蜡螟幼虫和蚊子成虫引诱力的检测方法来分析长叶烯和苜蓿烯对这两种昆虫的引诱力。在大蜡螟引诱装置中，僵虫被直径约被2cm的棉球(含100μl含不同浓度长叶烯或者苜蓿烯的溶液)替换，对照为含100微升溶剂正己烷的棉球。在蚊子引诱装置中，用直径约为1cm的棉球(含100μl含不同浓度长叶烯或者苜蓿烯的溶液)替代僵虫，对照为含100微升溶剂正己烷的棉球。
2)结果
进一步探究果蝇幼虫对长叶烯和苜蓿烯产生反应的浓度范围，设置了7个长叶烯和苜蓿烯的含量梯度即10 -5g、10 -6g、10 -7g、10 -8g、10 -9g、10 -9g、10 -10g、10 -11g进行行为学实验。结果参见图2中的A和图2中的B，结果显示，长叶烯和苜蓿烯浓度越高，对果蝇幼虫的引诱效果越强，当长叶烯和苜蓿烯的含量低至10 -11g时，果蝇幼虫仍会对其产生反应。
类似地，设置了5个长叶烯和苜蓿烯的含量梯度即10 -5g、10 -6g、10 -7g、10 -8g、10 -9g来探究蚊子对长叶烯和苜蓿烯产生反应的浓度范围。结果参见图2中的C和图2中的D，结果显示，10 -6g长叶烯对蚊子引诱效果最 强，蚊子对10 -9g长叶烯基本没有反应。对于苜蓿烯而言，苜蓿烯的浓度越高，对蚊子的引诱效果越强。同时，长叶烯和苜蓿烯对大蜡螟幼虫也具有吸引力，结果参见图3。
实施例4：构建苜蓿烯和长叶烯挥发量提高的罗伯茨绿僵菌重组菌株1)方法：
1)基因的发现和合成
在NCBI(https://www.ncbi.nlm.nih.gov/)上查询了樟子松(Pinus sylvestris)长叶烯合酶Tps的蛋白序列(Genbank accession number:ABV44454)，序列信息如SEQ ID NO.2所示。根据Codon Usage Database( http://www.kazusa.or.jp/codon/)提供的服务得到罗伯茨绿僵菌
(Metarhizium robertsii)的密码子偏好性，选择频率最高的密码子类型得到TPS蛋白的编码序列，序列信息如SEQ ID NO.1所示，并由杭州有康生物科技公司合成并连接到puc57-simple-TOPO载体上，得到含长叶烯合酶编码序列的质粒，命名为puc57-simple-TOPO-PsTPS。
2)TPS表达载体的构建和重组菌株的构建
以puc57-simple-TOPO-PsTps为模板，用高保真DNA聚合酶KOD Plus Neo(TOYOBO)进行PCR克隆Tps编码序列，所用引物为PsTps-CDS-FP-BamHI(ATGCCC，SEQ ID NO.3)和PsTps-CDS-RP-EcoRV(ACTGGGG，SEQ ID NO.4)。PCR产物用限制性核酸内切酶BamH I和EcoR V(Thermo Scientific)酶切，并与用同样酶切的载体pPK2-bar-gpd-GFP连接，载体信息见图4中的A，得到Tps的表达载体pPK2-bar-gpd-GFP-Tps。
将质粒pPK2-bar-gpd-GFP-PsTps转入根癌农杆菌菌株AGL1中后，然后转入罗伯茨绿僵菌ARSEF 2575菌株中。用除草剂抗性选择和绿色荧光蛋白(GFP)观察初步筛选到转化子，进一步用PCR证明(所用引物为PsTps-CDS-FP-BamHI和PsTps-CDS-RP-EcoRV)Tps表达盒成功整合进罗伯茨绿僵菌的基因组，RT-PCR证明Tps编码基因被转录表达(图4中的B)，得到异源表达Tps基因的罗伯茨绿僵菌菌株Mr-Tps。
2)结果
(1)RT-PCR证明Tps编码基因被转录表达
凝胶电泳检测Tps编码基因被转录表达，结果如图4中的B所示。
(2)转基因菌株Mr-Tps挥发更多的长叶烯和苜蓿烯
按照上述方法分析由转基因菌株Mr-Tps感染大蜡螟幼虫所形成的僵虫产生的挥发性物质发现(表2)，它产生11种挥发性物质，其中占比最高的化合物是长叶烯，第二是金合欢烯，第三是长叶环烯，第四是苜蓿烯。与野生型菌株WT感染形成的僵虫相比，转基因菌株Mr-Tps感染大蜡螟幼虫形成的僵虫挥发更多种类的倍半萜烯，包括长叶环烯，长叶蒎烯和雪松烯。
表2 WT和转基因菌株Mr-Tps僵虫在不同培养条件产生的挥发性物质分析结果
Figure PCTCN2022074175-appb-000012
进一步分析发现，与野生型菌株相比，菌株Mr-Tps的僵虫长叶烯和苜蓿烯的挥发量分别提高193倍和28倍，土臭素提高了1.09倍，金合欢烯提高了1.48倍(图5中的A)。
在PDA培养基上，菌株Mr-Tps产生6种挥发性物质，其中占比最高的化合物是长叶烯(表2)，与野生型菌株相比，菌株Mr-Tps挥发长叶烯和苜蓿烯的能力分别提高了98倍和4倍，但是未检测到金合欢烯和土臭素(图5中的B)。
当前，人们利用基于大米和麦麸的发酵培养基上工业化大规模生产绿 僵菌孢子。在类似的发酵培养基上，转基因菌株Mr-Tps产生5种挥发性物质，其中占比最高的是长叶烯，占比第二的是1，3-辛二烯(表2)，与野生型菌株相比，菌株Mr-Tps挥发长叶烯和苜蓿烯的能力分别提高了38.4倍和17.6倍，但是未检测到金合欢烯和土臭素(图5中的C)。
实施例5：表达Tps基因提高了绿僵菌对昆虫的吸引力
按照上述的装置，比较了转基因菌株Mr-Tps和野生型菌株感染形成的僵虫对蚊虫成虫，以及果蝇和大蜡螟幼虫的吸引力，其中转基因菌株和野生型菌株感染形成的僵虫分别代替上述装置的僵虫和对照冻死昆虫，用preference percentage(偏好百分率)＝【选择转基因菌株(或野生型菌株)感染形成的僵虫的昆虫数目/昆虫总数目】表示两类菌株对昆虫的吸引力。
结果如下图5中的D所示，相较于WT僵虫，果蝇幼虫、大蜡螟幼虫、蚊子成虫显著偏好转基因菌株Mr-Tps僵虫。
按照上述的装置，比较了PDA上、发酵培养基上生长的转基因菌株Mr-Tps和野生型菌株WT对蚊虫的吸引力。称取鲜重为0.3g在PDA和发酵培养基上生长14d的转基因菌株Mr-Tps和野生型菌株WT菌丝体分别放在装置的两侧，处理一段时间后，计算preference percentage(偏好百分率)＝选择转基因菌株和野生型菌株菌丝体的昆虫数目/昆虫总数目。
结果如图6中的A和6中的B所示，相较于WT菌丝体，蚊子成虫和果蝇幼虫显著偏好转基因菌株Mr-Tps菌丝体。
实施例6：以黑布方法为基础的转基因菌株防治蚊子方法
1)方法
如技术背景中所述，在防治蚊子成虫时，真菌孢子主要放在黑布等固体表面。为此，本发明检测了上述转基因菌株是否在黑布上也会有更强的吸引力。罗伯茨绿僵菌在PDA上培养14d后，用Triton-X-100溶液(0.01％)和植物油制备成孢子总量为1×10 8含8％植物油的孢子悬液油剂。将孢子悬液油剂均匀地喷洒在黑纱布表面，静置晾干后放入经过酒精消毒1m×1m×1m的笼子中，再放入15头5到8天且未进行血餐的白纹伊蚊雌蚊，该实验在黑暗条件下进行。12h后逐只收集蚊子并分别放入装有200μl0.01％Triton-X-100的1.5ml离心管中，用灭菌的研磨棒将蚊子碾碎后，均匀涂到绿僵菌筛选培养基上(含100μg/mL ampicillin，100μg/mL kanamycin， 80μg/mL streptomycin；4μg/mL dodine；10μg/mLbenomyl的PDA)，将平板在26℃条件下倒置避光培养，5天后，统计每个平板上的菌落数(CFUs)，计算接种率和接种量。
2)结果，接种率和死亡率。
结果参见图7中的A和7中的B，结果显示，在1m 3的笼子中，转基因菌株Mr-Tps对蚊子的接种率，和每头蚊子上的接种量显著高于野生型菌株(WT)。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。

Claims (10)

  1. 苜蓿烯在昆虫引诱和/或防治虫害中的应用，所述苜蓿烯具有式I所示结构；
    Figure PCTCN2022074175-appb-100001
  2. 根据权利要求1所述的应用，其特征在于，所述昆虫包括果蝇、大蜡螟和蚊子中的一种或几种。
  3. 一种重组生防真菌，包含重组真菌表达质粒，所述重组真菌表达质粒上插入有松树长叶烯合成基因。
  4. 根据权利要求3所述的重组生防真菌，其特征在于，所述松树长叶烯合成基因的核苷酸序列如SEQ ID NO.1所示。
  5. 根据权利要求3所述的重组生防真菌，其特征在于，所述重组生防真菌的原始菌包括绿僵菌。
  6. 一种僵虫，感染有权利要求3～5任意一项所述的重组生防真菌。
  7. 权利要求3～5任意一项所述的重组生防真菌或者权利要求5所述的僵虫在昆虫引诱和/或防治虫害中的应用。
  8. 根据权利要求5所述的应用，其特征在于，所述昆虫包括果蝇、大蜡螟和蚊子中的一种或几种。
  9. 权利要求3～5任意一项所述的重组生防真菌在制备苜蓿烯中的应用；所述苜蓿烯具有式I所示结构；
    Figure PCTCN2022074175-appb-100002
  10. 一种昆虫引诱剂，活性成分包括苜蓿烯或者权利要求3～5任意一项所述的重组生防真菌或者权利要求6所述的僵虫；所述苜蓿烯具有式I所示结构；
    Figure PCTCN2022074175-appb-100003
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