CN117209574B - High-toxicity destruxin for transformation of locust pests, and preparation method and application thereof - Google Patents
High-toxicity destruxin for transformation of locust pests, and preparation method and application thereof Download PDFInfo
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Abstract
The application relates to the technical field of pesticides, in particular to high-toxicity destruxins of Metarrhizium anisopliae specially modified for locust pests, and a preparation method and application thereof. The protein comprises: SEQ ID NO:1, and/or a fusion protein thereof, and/or a homologous protein thereof. The toxin protein or the coding gene thereof discovered by the application is a key virulence factor of the metarhizium anisopliae for causing high pathogenicity to the locust, and the toxin protein only has toxicity to the locust pests, but does not affect other insects. The recombinant entomopathogenic fungi effectively solves the defect that original broad-spectrum entomopathogenic fungi have insufficient pathogenicity to locust pests, so that the recombinant entomopathogenic fungi have high toxicity to the locust pests while effectively preventing and controlling other various pests, improve pest prevention and control efficiency, realize the effects of one fungus for multiple purposes and saving use cost, and can not cause food and ecological safety problems.
Description
Technical Field
The application relates to the technical field of pesticides, in particular to high-toxicity destruxins of Metarrhizium anisopliae specially modified for locust pests, and a preparation method and application thereof.
Background
The chemical pesticide consumption of China is about 180 ten thousand tons per year, and the chemical pesticide is the first place in the world. Excessive use of chemical pesticides can cause organic pollution to soil and seriously damage food safety. In addition, the abuse of chemical pesticides can also lead to the improvement of the drug resistance of pests, so that the embarrassing situation that no drug is available for the pests is faced. Therefore, the reduction of the use of chemical pesticides is one of the primary problems to be solved in the process of advancing to the strong agriculture country in China.
Entomopathogenic fungi are a widely used class of biopesticides that are environmentally friendly and difficult to render resistant to pests. Therefore, the entomopathogenic fungi preparation has good application prospect when being matched with chemical pesticides. At present, the entomopathogenic fungi preparations which are widely used at home and abroad are beauveria bassiana, metarhizium anisopliae, metarhizium rotiferum and metarhizium locust. Wherein beauveria bassiana, metarhizium anisopliae and metarhizium anisopliae can infect and kill more than 200 agricultural pests, and the high toxicity of metarhizium anisopliae is specific to locust pests.
At present, the destruxin product on the market cannot achieve both the universality and the high efficiency. The broad-spectrum Metarrhizium anisopliae preparation has high toxicity to insects such as Lepidoptera, diptera and Coleoptera, but has low toxicity to pests of the family locust (such as migratory locust, desert locust and small car locust). For example: studies have shown that the Metarrhizium anisopliae preparation has a half-Life (LT) to anopheles (diptera), pisum sativum aphid (hemiptera), yellow meal worm (coleoptera), chilo suppressalis (lepidoptera) 50 ) The time period for half killing the migratory locust is 3.2+/-0.1 days, 5.11+/-0.23 days, 5.5+/-0.79 days and 5.917 +/-0.261 days respectively, but is about 8.60+/-0.15 days. While the metarhizium anisopliae preparation can effectively kill various locust pests, the metarhizium anisopliae preparation is only effective on the locusts, and cannot give consideration to other pests. If the two are used together, the use cost is increased, meanwhile, competition among different fungi can be caused, and the pest control effect is weakened.
Genetic engineering is the dominant method for improving the insecticidal efficiency of entomopathogenic fungi. For example, researchers have introduced the neurotoxin gene of scorpion (Androctonus australis) into the metarhizium anisopliae genome and stably expressed the gene, thereby significantly improving the insecticidal efficiency of metarhizium anisopliae; and researchers express the beta-neuropeptide (beta-NP) toxin gene of the solenopsis invicta (Solenopsis invicta) in the beauveria bassiana, so that the insecticidal efficiency of the beauveria bassiana is remarkably improved. These genetically engineered techniques, while improving the pathogenicity of the strain against pests, can cause genetic and ecological contamination during application and raise food and environmental safety concerns as these toxins are also harmful to humans and animals.
Meanwhile, unmodified broad-spectrum metarhizium anisopliae and beauveria bassiana preparations have little influence on some hymenoptera pollinating insects (such as bees and bumblebees), but genetically engineered high-virulence strains often have no selectivity on hosts, and meanwhile, the pathogenicity of agricultural pests is improved, and meanwhile, the pathogenicity of the metarhizium anisopliae and beauveria bassiana preparations on pollinating insects is also higher. Thus, the reduction of pollinating insects also does not favor the growth of some crop fruits, which in turn affects yield.
Disclosure of Invention
In view of this, the invention provides a high virulence Metarhizium anisopliae specifically modified against locust pests, and a preparation method and application thereof. The protein is a key virulence factor of the metarhizium anisopliae for causing high pathogenicity to the locust, and has toxicity to the locust pests only, but does not affect other insects. The recombinant entomopathogenic fungi can realize the effects of one fungus with multiple purposes and use cost saving, and cannot cause food and ecological safety problems.
In order to achieve the above object, the present invention provides the following technical solutions:
in a first aspect, the present invention provides the use of a protein in the preparation of a biopesticide, the protein comprising at least one of the following proteins:
(11) SEQ ID NO: 1;
(12) SEQ ID NO:1 and/or C terminal of the protein shown in the specification is connected with a label;
(13) SEQ ID NO:1 through substitution and/or deletion and/or addition of one or more amino acid residues;
(14) And SEQ ID NO:1 and having at least 75% identity and the same function.
In a specific embodiment of the present invention, the above protein is used for controlling locust type pests.
In an embodiment of the invention, the locust family pest (Acrididae) belongs to one of the class of entomoidae, orthoptera, the general family of locust, commonly known as locust or locust. Such as migratory locust (east asia migratory locust, chinese tibetan migratory locust, etc.), desert locust, dolly locust, earth locust, rice locust, cotton locust, bamboo locust, etc.
In a second aspect, the present invention provides the use of a gene comprising at least one of the following genes in the preparation of a biopesticide:
(21) SEQ ID NO: 2;
(22) SEQ ID NO:2, a degenerate sequence or a homologous sequence of the gene set forth in SEQ ID NO:2, the gene has a sequence of at least 75% identity;
(23) Under stringent conditions with SEQ ID NO:2 or the complementary sequence thereof.
"identity" refers to sequence similarity to a native nucleic acid sequence or amino acid sequence. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.
The term "at least 75%" is, for example, any one of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or any one of the values in the range of values defined above.
In the application of the protein or the gene in preparing the biological pesticide, the biological pesticide is at least the biological pesticide for controlling locust pests.
In a third aspect, the present invention provides a biomaterial for expressing the above gene, the biomaterial comprising at least one of an expression cassette, a vector, a recombinant cell, and a recombinant microorganism.
In an embodiment of the invention, the vector comprises at least one of a plasmid, a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a linear polynucleotide.
In an embodiment of the invention, the recombinant cells include plant cells and/or animal cells not intended for propagation.
In an embodiment of the invention, the recombinant microorganism comprises at least one of fungi, bacteria, algae.
In an embodiment of the invention, the fungus comprises at least one of yeast, aspergillus niger, trichoderma reesei.
In an embodiment of the invention, the bacteria include at least one of agrobacterium, escherichia coli, bacillus, pseudomonas, and lactobacillus.
In an embodiment of the invention, the algae comprises at least one of blue algae, synechococcus, anabaena, chlamydomonas reinhardtii.
In a fourth aspect, the present invention provides a recombinant entomopathogenic fungus comprising the above gene and/or the above biological material.
In an embodiment of the invention, the recombinant entomopathogenic fungi comprise a broad spectrum of metarhizium anisopliae and/or beauveria bassiana.
In an embodiment of the invention, the broad-spectrum metarhizium anisopliae comprises one or two of metarhizium anisopliae and metarhizium anisopliae.
In the embodiment of the invention, the broad-spectrum metarhizium anisopliae has broad insecticidal spectrum and can infect more than 200 pests of lepidoptera, coleoptera, homoptera, hemiptera, orthoptera and diptera.
In a fifth aspect, the present invention provides the use of the recombinant entomopathogenic fungi described above for the preparation of a biopesticide.
In a sixth aspect, the present invention provides a method for preparing the recombinant entomopathogenic fungi described above, comprising: introducing the above genes and/or the above biological materials into entomopathogenic fungi to obtain recombinant entomopathogenic fungi.
In the examples provided by the present invention, the means for introducing the above genes into an entomopathogenic fungus include: the gene is connected to a carrier to obtain a recombinant carrier, the recombinant carrier is transferred into agrobacterium, and the agrobacterium-mediated method is utilized to transfer into entomopathogenic fungi.
In addition to the above-described methods for preparing recombinant entomopathogenic fungi, other conventional methods for introducing the above-described genes into entomopathogenic fungi, such as protoplast transformation, gene editing technique, and the like, may be employed.
In a seventh aspect, the present invention provides a biopesticide comprising the recombinant entomopathogenic fungi described above.
In embodiments of the present invention, biopesticides may also include other biopesticide species. Other biopesticide types recognized by those skilled in the art are within the scope of the present invention, and the present invention is not limited thereto.
In an eighth aspect, the present invention provides an insecticidal composition comprising the recombinant entomopathogenic fungus described above and a chemical pesticide.
In an embodiment of the present invention, the biopesticide or insecticidal composition may further comprise an agropharmaceutically acceptable adjuvant.
In an embodiment of the invention, the biopesticide or insecticidal composition is selected from the following dosage forms: solutions, emulsions, wettable powders, particulate wettable powders, suspensions, powders, foams, pastes, tablets, granules, aerosols, natural agents impregnated with active compounds, synthetic agents impregnated with active compounds, microcapsules, seed coatings, formulations equipped with combustion devices, cold fogs, hot fogs and the like.
In an embodiment of the invention, the agropharmaceutically acceptable excipients include one or more of the following: fillers, surfactants, binders, colorants, and the like.
Compared with the prior art, the invention has the following beneficial effects:
1. through histology analysis and functional research, a special toxin protein coding gene is found in the metarhizium anisopliae, which is a key virulence factor of metarhizium anisopliae for causing high pathogenicity to locustae, and the protein coded by the gene only has toxicity to locust pests, but does not affect other insects.
2. After the toxin protein coding gene is integrated into broad-spectrum entomopathogenic fungi (such as the metarhizium anisopliae), the recombinant entomopathogenic fungi strain capable of producing the locust toxins in a large amount is successfully constructed, the defect that the original broad-spectrum entomopathogenic fungi have insufficient pathogenicity to locust pests is effectively overcome, the novel recombinant entomopathogenic fungi have high virulence to the locust pests while other various pests are effectively prevented and treated, the pest prevention and control efficiency is improved, and the effects of multiple purposes and use cost saving are realized.
3. The protein itself in the application is a virulence factor of the metarhizium anisopliae, is effective only on locust pests, and cannot influence human beings and other animals (such as poultry, livestock and the like), so that the recombinant strain prepared by the invention cannot cause food and ecological safety problems, and can be safely used.
4. Because the protein of the application is only effective on locust pests, the recombinant strain prepared by the invention does not have substantial influence on the powdery insects.
Drawings
FIG. 1 is a diagram showing the results of screening Metarhizium Anisopliae (MAC) for genes encoding toxin proteins capable of up-regulating expression in vivo; wherein, FIG. 1-1 shows the gene expression intensity of Metarhizium anisopliae (MAA) in migratory locust; FIGS. 1-2 show the gene expression intensity of Metarhizium Anisopliae (MAC) in migratory locust; log2 (fold) is a measure commonly used in bioinformatics to measure the difference between the gene expression intensity of one sample and the expression intensity of a corresponding control sample;
FIG. 2 is a plasmid construction map; wherein Genomic DNA is translated into Genomic DNA; the bar cassette translates to the bar gene, which is a resistance gene;
FIG. 3 shows the purification result of the encoded protein of MAC_08119;
FIG. 4 shows virulence of wild type strains of Metarrhizium anisopliae (MAC-WT) and strain of Metarrhizium anisopliae deleted from MAC_08119 (DeltaMAC_08119) against migratory locust;
FIG. 5 shows virulence of wild type strain of Metarrhizium anisopliae (MAC-WT), strain of Metarrhizium anisopliae deleted in MAC_08119 (ΔMAC_08119) and strain of Metarrhizium anisopliae deleted in MAC_08119 after injection of purified protein (ΔMAC_08119+ purified protein) against migratory locust;
FIG. 6 shows the virulence of Metarhizium anisopliae (OE: MAC_08119) over-expressing the MAC_08119 gene against migratory locust;
FIG. 7 shows the relative mRNA expression levels of the MAC_08119 gene in wild-type Metarrhizium anisopliae (MAA-WT) and recombinant Metarrhizium anisopliae (OE: MAC_08119), wherein the difference between the two groups is significant, i.e., P <0.05;
FIG. 8 shows virulence of wild type Metarhizium anisopliae (MAA-WT) and recombinant Metarhizium anisopliae strain (OE: MAC_08119) against migratory locust; p <0.01 indicates that the difference between the two groups is very significant;
FIG. 9 shows virulence of wild type Metarhizium anisopliae (MAA-WT) and recombinant Metarhizium anisopliae strain (OE: MAC_08119) against yellow meal worm.
FIG. 10 shows virulence of wild type Metarhizium anisopliae (MAA-WT) and recombinant Metarhizium anisopliae strain (OE: MAC_08119) on bumblebees.
Detailed Description
The invention discloses high-toxicity destruxins of Metarrhizium anisopliae specially modified for locust pests, and a preparation method and application thereof, and a person skilled in the art can refer to the content of the destruxins of Metarrhizium anisopliae, and properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
Reagents, instruments, strains, biological materials, etc. used in the present invention are commercially available.
The invention is further illustrated by the following examples:
EXAMPLE 1 identification of Metarhizium anisopliae specific virulence factors
(1) Screening of potential virulence factor coding genes
The method comprises the steps of screening a toxin protein coding gene capable of up-regulating expression in a migratory locust body in Metarhizium Anisopliae (MAC) through a transcriptome, and then comparing the screened 37 candidate genes with homologous genes of metarhizium anisopliae (MAA), so as to further screen the toxin protein coding gene specifically existing in metarhizium anisopliae. After two screenings, macj08119 was determined as the key candidate gene (fig. 1). Wherein MAA_00392 of the Metarhizium anisopliae is a homologous gene of the Metarhizium anisopliae MAC_08119.
The sequence of the MAC_08119 protein (SEQ ID NO: 1) is as follows: MRWGSIVPVAHLVLLGIGTSSAIPNVKRVIIGRVPKQPAKEVFRGDFRSPAEVAIDGGFRPRGDYANDDRAFSIYRHMGGEFWREDDSELSDEDSEEEWISAFVSVTTDVGTGASYGRWLYRIHATPNMIDPTAENENEPEIFALGGVPWSQVMGWYYVRLDGDGHALNPDAITAADFTPNPAYQGRYDQFALTTVEPRSRWRQHDRSYWTQFMNRPDVGPAVGFTGQFPLQLGTYSLEGLPGPSSANGETQINRPPAAAAQAPALNEDDIVVAAEFLRDHNAPCDVSGADPETQWLTLEADSTIARALARMQREGRPNQTLPSPIEEADEQIRIIIERLQRGQEPSTCQLVGECSGLSFSHKKRAAAPAGLSNLCQRIHRPTKDANGLDDATQNSEKCVLRSPINVKVQLSDATSAGSWDRLFLEIGKNPKYDKTQPHYLLKEAPDAGDLMSEDVDLADAYTMKAVTINDIKRVKLVSRLDTGRVASNELMLQDITITAKCAASPKVAEFHKAANEWFTSWGTEIKPTDWSWKKDCDRFKSLKFKWHLGGAVRAGTWDDLKLGVNRDHKVTAHDIQFAQSPAARDSGEMEINLKDTYGSATVPIEKVDYFRVYSTTRWPDSGEDEWELGGIRFTAQCEGYDKAVIMDKFGSDYNWHSRSNGKEMPVAASDWRWEDENLQTRHPFPREEL
The sequence of the MAC_08119 gene (SEQ ID NO: 2) is as follows: ATGCGCTGGGGCAGCATTGTTCCAGTCGCACATCTTGTGCTCCTTGGGATAGGCACCAGCAGTGCTATCCCAAATGTCAAAAGAGTCATTATTGGCAGAGTTCCCAAGCAACCTGCCAAAGAAGTTTTCCGTGGAGACTTTAGAAGCCCTGCTGAGGTTGCAATAGATGGCGGATTCCGCCCAAGAGGCGACTATGCAAATGATGACAGGGCATTTTCCATCTACCGCCACATGGGAGGCGAGTTTTGGAGAGAGGATGACTCGGAACTTTCGGATGAAGATTCTGAAGAAGAATGGATATCAGCATTTGTTTCTGTAACAACCGACGTGGGAACGGGAGCATCATATGGCCGCTGGCTGTACAGGATTCATGCCACTCCGAATATGATTGACCCAACAGCCGAGAACGAGAATGAACCCGAGATTTTCGCGCTGGGGGGTGTTCCTTGGTCACAAGTCATGGGATGGTACTACGTAAGATTGGACGGAGACGGCCACGCTTTGAATCCCGACGCCATTACAGCTGCCGACTTTACCCCCAACCCAGCCTACCAGGGGAGGTATGACCAGTTCGCTCTGACAACCGTTGAACCAAGAAGCCGATGGCGTCAGCATGATAGAAGCTACTGGACACAATTCATGAACCGCCCAGACGTTGGGCCTGCTGTGGGCTTTACTGGTCAATTCCCACTACAACTTGGGACATATTCTCTTGAAGGACTCCCTGGGCCTTCTTCAGCAAATGGCGAGACTCAGATTAACCGGCCACCAGCAGCGGCAGCTCAAGCTCCTGCGCTCAATGAGGACGACATAGTCGTAGCTGCCGAGTTCCTGCGAGACCACAATGCGCCGTGTGACGTCTCCGGAGCTGATCCCGAAACCCAATGGCTTACTCTTGAGGCAGACTCGACAATTGCCCGCGCCTTGGCCCGAATGCAACGGGAAGGTCGTCCCAATCAAACTCTCCCTAGCCCTATTGAGGAGGCGGACGAGCAGATTCGGATAATTATTGAAAGGCTTCAACGGGGGCAGGAACCCAGCACATGCCAACTTGTTGGCGAATGCAGCGGACTGAGCTTCTCCCACAAGAAGAGGGCGGCGGGTAAGTACCGCGATATTTCTGTAAAACATAAGACGGAACTTGTACGGCTTGAAAAATGCTAACTCCTCTTCGGGAAAGCACCAGCAGGGCTCTCAAACCTATGTCAAAGAATCCACAGGCCAACAAAAGACGCAAACGGCTTGGACGACGCCACGCAAAACTCTGAGAAATGCGTGCTACGGTCTCCAATCAATGTCAAGGTTCAACTGTCGGACGCTACCTCGGCCGGAAGCTGGGATCGATTGTTTTTGGAAATCGGAAAGAATCCCAAATATGACAAAACCCAGCCACACTACCTGCTCAAGGAGGCACCCGACGCCGGAGATCTGATGTCTGAAGACGTAGACCTCGCAGACGCTTATACAATGAAGGCGGTAACTATTAACGACATTAAAAGAGTTAAACTGGTCAGCCGATTAGACACTGGACGGGTAGCTTCAAACGAGCTCATGCTGCAAGGTTAGTGGTTTTCATTTACTTCATCTGCGGTTACGGCAGTGAAGCCTGAAAGGAAATGTGAAAGATCGCTAATTTTCTTGACGTTCCTAGATATTACAATCACAGCGAAATGCGCCGCTTCACCAAAAGTCGCTGAATTTCACAAAGCAGCCAATGAATGGTTTACAAGCTGGGGCACAGAAATTAAACCCACGGACTGGTCCTGGAAGAAGGACTGTGATAGGTTTAAGAGCCTGAAGTTTAAATGGCACTTGGGCGGTGCTGTCCGTGCCGGGACGTGGGATGATCTTAAACTAGGTGTTAATCGCGATCACAAAGTGACGGCTCATGATATTCAATTTGCTCAAAGCCCTGCGGCTAGGGACAGTGGTGAGATGGAGATTAACCTAAAGGATACATACGGTTCAGCAACTGTGCCCATAGAAAAAGTCGATTACTTCCGCGTCTATTCCACTACAAGATGGCCTGATTCTGGCGAGGATGAGTGGGAACTGGGAGGTATGTCTGTTGACCAGAACTTACAATTAAAACATATTCTAACCCTATCTAGGAATAAGATTCACTGCGCAATGTGAGGGTTACGACAAAGCAGTTATAATGGACAAATTTGGAAGTGATTATAACTGGCACTCTCGTAGCAATGGCAAAGAGATGCCCGTCGCAGCTAGCGACTGGCGGTGGGAGGATGAAAATCTTCAAACGCGACATCCTTTCCCGAGAGAGGAACTGTGA
(2) Virulence factor functional verification
The toxicity of the protein coded by the MAC_08119 to the host is verified from the positive aspect and the negative aspect through knockout and overexpression technologies. The principles of knockout and overexpression are both chromosomal homologous substitutions, which are now very mature techniques for genetic manipulation of metarhizium anisopliae. The specific method comprises the following steps:
(a) Construction of a MACj08119 knockout and overexpression plasmid.
(a1) Construction of the MACj08119 knockout plasmid
The plasmid construction map is shown in fig. 2, and the primers "mac_08119LF and mac_08119LR" and "mac_08119RF and mac_08119RR" are used to amplify the upstream and downstream sequences (i.e., left and right arms) of the gene mac_08119, respectively, using the DNA of the wild type MAC genome as a template, and the specific primer sequences are as follows:
TABLE 1 primer sequences
Firstly, the PDHt-Bar plasmid is digested by restriction enzymes EcoRI and BamHI, then the left arm expanded by the primers 'MAC_08119 LF and MAC_08119 LR' is connected to the PDHt-Bar by using a homologous recombination method to construct a plasmid PDHt-MAC_08119L-Bar, and the plasmid is confirmed to be constructed correctly after sequencing verification; then, the PDHt-MAC_08119L-Bar plasmid was digested with restriction enzymes XbaI and SacI, and DNA sequences amplified by using primers "MAC_08119RF and MAC_08119RR" were ligated to PDHt-MAC_08119L-Bar by homologous recombination to construct a knockout plasmid PDHt-Bar-MAC_08119L/R. After sequencing, the plasmid was confirmed to be constructed correctly.
(a2) Construction of a MAC_08119 overexpression plasmid vector
First, the wild type MAC genome is used as a template, and the primers PDHt-8119-F and PDHt-8119-R are used to amplify the DNA fragment of the gene MAC_08119. Then, the PDHt-Gpd-Bar plasmid is digested with restriction enzymes EcoRI and MunI, the digested product is recovered, and the vector recovered by the digestion is recombined with the DNA fragment by using a homologous recombination method. After sequencing, the plasmid was confirmed to be constructed correctly, and the recombinant plasmid was named "PDHt-Gpd-MAC_08119-Bar". Wherein, the primer sequences of PDHt-8119-F and PDHt-8119-R are as follows:
PDHt-8119-F (SEQ ID NO: 7):
ATACACACACGCAAAGAATTCATGCGCTGGGGCAGCATTGTT
PDHt-8119-R (SEQ ID NO: 8):
TCGACGGATCCCCCGGGCAATTGTCACAGTTCCTCTCTCGGGAAA
(b) The agrobacterium tumefaciens mediated method constructs a fungus genetic transformation system. Transforming the knocked-out vector or the super-expression vector plasmid constructed in the above into agrobacterium AGL-1, selecting a positive agrobacterium AGL-1 transformed strain identified to be correct by PCR, shaking the strain to a bacterial liquid concentration OD by using an LB liquid medium (containing 50mg/mL kanamycin Kan) 660 0.8-1.0. The bacterial cells were collected and the resuspended OD was diluted with an appropriate amount of IM+200. Mu.M acetosyringone (As) liquid medium 660 Culturing at 28deg.C in dark to reach bacterial liquid concentration OD of 0.15 660 0.5-0.7 for standby.
(c) Preparation of a metarhizium anisopliae spore suspension: scraping conidium of Metarhizium anisopliae from 1/4SDAY culture medium cultured for 2 weeks to 1.5mL sterile water containing 200 μm acetosyringone (As), vortex shaking, filtering with sterilized mirror paper to remove mycelium, collecting filtrate, and adjusting the concentration of spore suspension of Metarhizium anisopliae to 3×10 6 And (5) conidium/mL for later use.
(d) Mixing the prepared Metarhizium anisopliae spore suspension with the OD 660 AGL-1 bacteria solution with a concentration of 0.5-0.7 is uniformly mixed according to a ratio of 1:1, and 200 mu L is uniformly coated on an IM culture medium plate. After 48 hours of incubation in the dark, the transformants were extracted from the fungal genome and verified by PCR with specific primers by washing with sterile water and plating the wash onto a search agar medium containing 500. Mu.g/mL glufosinate and 300. Mu.g/mL thiamycin and incubating for 7-10 days until resistant colonies appear.
(e) Knocking out and over-expressing the metarhizium anisopliae virulence and verifying. Spores of destruxins of Metarrhizium anisopliae which are deleted or overexpressed the MAC_08119 gene and cultured on the culture medium for 14 days are scraped, and sterile water suspension spores containing 0.05% Tween-80 are added. After vortexing, spores were collected after filtration with a piece of mirror paper. The final concentration of the spore suspension was adjusted to 1X 10 by counting under a microscope using a blood cell counting plate 8 Individual conidia/mL. mu.L of the spore suspension was pipetted down the dorsal plate of the migratory locust larvae in east Asia. Wild type metarhizium anisopliae spores (MAC-WT) were used as a control. The number of dead locust was counted every 12 hours.
(f) Macj08119 encodes protein purification. The macj08119 encoding protein was expressed and purified in vitro using an e.coli in vitro expression system (fig. 3). And then injecting the purified protein into the migratory locust for toxicity effect verification.
(g) The results showed that the deletion of mac_08119 (Δmac_08119) significantly inhibited the pathogenicity of metarhizium anisopliae against migratory locust (fig. 4) relative to wild type metarhizium anisopliae strain (MAC-WT), and that injection of purified protein restored the virulence of the deletion of mac_08119 (Δmac_08119) strain against migratory locust (fig. 5). Finally, the overexpression of the gene (OE: MAC_08119) by the metarhizium anisopliae can significantly improve the pathogenicity of metarhizium anisopliae to migratory locust (figure 6). It can be concluded that the protein encoded by the gene MAC_08119 is a toxin protein secreted by Metarrhizium anisopliae against the host locust.
EXAMPLE 2 genetic engineering of Metarhizium Robertsonii
(a) The procedure for the construction of the overexpressing strain was the same as in steps (a) - (d) of example 1.
(b) And detecting the overexpression effect of the MAC_08119.
The RNA was extracted from a sample of the cultured recombinant Metarhizium rosenbergii using the ultra-pure RNA nucleic acid extraction kit from Kanji, and the extracted RNA was inverted into cDNA using the PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) kit from Takara. The quantitative analysis of gene qPCR was performed by using the Talent fluorescent quantitative detection kit (SYBR Green) of Tiangen biology company, and the expression levels of RNA of wild type Metarrhizium anisopliae (MAA-WT) and recombinant Metarrhizium anisopliae MAC_08119 gene (OE: MAC_08119) were quantified by using the gpd gene of Metarrhizium anisopliae as an internal reference.
As can be seen from FIG. 7, the wild type Metarrhizium anisopliae (MAA-WT) had no metarhizium anisopliae MAC_08119 gene, and the recombinant metarhizium anisopliae (OE: MAC_08119) overexpressed the metarhizium anisopliae MAC_08119 gene.
EXAMPLE 3 detection of the pathogenic Effect of recombinant Metarhizium Luobotrys on various insects
In this example, migratory locust and pollinating insect bumblebee and yellow meal worm were selected as study subjects, and the virulence of the recombinant Metarhizium anisopliae prepared in example 2 was tested. The toxicity detection steps are as follows:
scraping wild-type Row on culture medium for 14 daysSpores of Metarhizium anisopliae (MAA-WT) or recombinant Metarhizium anisopliae (OE: MAC_08119) were added to a sterile aqueous suspension of spores containing 0.05% Tween-80. After vortexing, spores were collected after filtration with a piece of mirror paper. Count under microscope using a blood count plate and separately: the final concentration of the spore suspension is adjusted to be 1 multiplied by 10 8 The conidium/mL is sucked up to 5 mu L of spore suspension and dropped under the dorsal plate of the five-instar larva of the migratory locust in the populated east Asia; the final concentration of the spore suspension is adjusted to be 1 multiplied by 10 7 The conidia/mL are uniformly sprayed on the surface of bumblebee or yellow meal worm. Wild type Metarhizium anisopliae (MAA-WT) treatment was used as a control. The number of dead insects was counted every 12 hours.
The results showed that the lethal efficiency of the recombinant Metarhizium rosenbergii strain (OE: MAC_08119) against migratory locust was significantly higher than that of the wild type strain (MAA-WT), half-death time was about 3 days earlier than that of the wild type strain (FIG. 8), and there was no significant improvement in pathogenicity against yellow mealworms (FIG. 9) and bumblebees (FIG. 10). Therefore, the virulence factor of the beauveria bassiana is introduced into the beauveria bassiana, so that the pathogenicity of the beauveria bassiana to the locust pests can be effectively improved, and the defect of poor locust control effect is overcome. Meanwhile, the characteristic of small damage to the powdery insects is maintained.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. Use of a protein for the preparation of a biopesticide for controlling locust family pests, characterized in that the protein comprises at least one of the following proteins:
(11) SEQ ID NO: 1;
(12) SEQ ID NO:1 and/or C-terminal of the protein shown in the formula 1 is connected with a label.
2. An application of a gene in preparing a biological pesticide for controlling locust pests, which is characterized in that the gene is SEQ ID NO:2, and a gene shown in 2.
3. A biological material for expressing the gene of claim 2, comprising at least one of an expression cassette, a vector, a recombinant cell, and a recombinant microorganism.
4. A recombinant entomopathogenic fungus, characterized in that it comprises the gene of claim 2 and/or the biological material of claim 3;
the entomopathogenic fungi is Metarhizium rosenbergii.
5. The use of the recombinant entomopathogenic fungus according to claim 4 for the preparation of a biopesticide for controlling locust pests.
6. A process for producing a recombinant entomopathogenic fungus according to claim 4, wherein the gene according to claim 2 and/or the biological material according to claim 3 are introduced into an entomopathogenic fungus to obtain a recombinant entomopathogenic fungus.
7. A biopesticide, characterized in that it comprises the recombinant entomopathogenic fungus of claim 4.
8. An insecticidal composition comprising the recombinant entomopathogenic fungus of claim 4 and a chemical pesticide.
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