CN115820439A - Entomopathogenic fungus transgenic strain and screening method and application thereof - Google Patents
Entomopathogenic fungus transgenic strain and screening method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to an entomopathogenic fungus transgenic strain and a screening method and application thereof. Through research on the period from the time when the pathogenic fungi enter the blood cavity of the insect to the time when the host dies (after invasion), the trehalose is found to play an important role in the pathogenic fungi of the insect not only as an important carbon source in the body but also in the environmental stress response of many organisms. In the trehalose anabolism pathway, the second step is to catalyze the trehalose-6-phosphate by trehalose-6-phosphate phosphatase (Tpp) to synthesize trehalose, and researches show that the trehalose anabolism pathway of beauveria bassiana is an important pivot for regulating and controlling the growth, the stress resistance and the pathogenicity of fungi. Therefore, the method is a novel method for improving the insecticidal activity of entomopathogenic fungi by constructing a fungus TPP overexpression vector and transforming the TPP overexpression vector into the beauveria bassiana to obtain the TPP overexpression engineering bacteria through a genetic engineering means.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to an entomopathogenic fungus transgenic strain and a screening method and application thereof.
Background
Pest control is an important component of agricultural production. The chemical pesticide plays an active role in preventing and treating pests and ensuring agricultural production increase. However, the long-term use of chemical pesticides in large quantities raises a number of environmental and food safety issues. Microbial control has been receiving widespread attention because of its advantages of safety and sustainable control. Among them, entomopathogenic fungi are the largest group of entomopathogenic microorganisms, from which 60% of naturally dead insects are caused, and are one of the important factors in nature for controlling the number of insect populations. Entomopathogenic fungi infest insects primarily through the body wall, and have particular advantages in the continued control of pests and in maintaining species diversity. At present, fungal pesticides have been used as a substitute product or supplement preparation of chemical pesticides to control various harmful insects. However, like other microbial pesticides, fungal pesticides have the disadvantages of slow pest killing, unstable control effect and the like, which greatly limits the wide application of the fungal pesticides. The improvement of the insecticidal speed of the entomopathogenic fungi has important significance for expanding the application range, reducing the use of chemical pesticides and protecting the ecological environment.
The measures for improving the insecticidal speed of the entomopathogenic fungi insecticide mainly comprise the following steps: (1) The preparation research provides a proper microenvironment for the fungi, and promotes the rapid growth and infection of the fungi; (2) screening strains to obtain strains with high insecticidal activity; (3) obtaining the strain with high insecticidal activity by strain improvement. The research result of the insecticidal fungus preparation plays a promoting role in promoting the industrialization and the application of the insecticidal fungus, particularly the occurrence of the insecticidal fungus oil suspending agent obviously reduces the environmental humidity dependence of the insecticidal fungus, and expands the application range of the insecticidal fungus from forest and other shading areas to drought and high temperature areas. However, since infestation of an insect by an entomopathogenic fungus is a process of interaction between the two, even under suitable environmental conditions, it takes a long time for the fungus to infect the insect until the insect dies. A series of entomopathogenic fungi strains with high insecticidal activity aiming at various insects are obtained by strain screening, wherein more than 100 kinds of fungi pesticides are registered at home and abroad, and natural strains are evolved through long-term interaction with host insects and reach the balance with the host insects in nature, so that the screening of the entomopathogenic fungi strains with high insecticidal activity from a large number of strains is time-consuming and troublesome. At present, on the basis of the research of pathogenic mechanisms of entomopathogenic fungi, the improvement of strains by using genetic engineering means becomes an important way for improving the insecticidal activity of the entomopathogenic fungi.
There are mainly 3 processes for entomopathogenic fungi to infect host insects: (1) the son is attached to the host epidermis; (2) penetrating insect epidermis to invade the insect body; (3) the insect is bred and secreted toxin in the insect body by utilizing the vegetative growth of the insect until the insect is ill and dies. To date, the pathogenic mechanisms of entomopathogenic fungi have been studied mainly by focusing on some hydrolases produced during penetration of the host epidermis by the fungi and by isolating and identifying a number of pathogenic-related genes, such as the protease Prl, chitinase, etc. St Leger (1996) successfully transfers Prl gene into Metarhizium anisopliae (Metarhizium) through a transgenic technology to obtain a Metarhizium anisopliae engineering strain with high toxicity. The engineering strain activates a phenol oxidase system after constitutive overexpression in haemolymph of tobacco hornworm, so that larval blackening is caused, the death time of insects is shortened by 25%, and the food intake is reduced by 40%. In 2005, fang et al successfully constructed the entomopathogenic fungi virulence factor-chitinase Bbchitl gene downstream of the gpd constitutive promoter and transferred it into beauveria bassiana genome to obtain the over-expression engineering strain. The toxicity of the engineering strain to the crab is obviously enhanced: compared with wild strains, the lethal dose of the engineering strains to the crab insects is reduced by 50%, and the lethal time is shortened by 50%. Fang et al (2007) fused bombyx mori BmChBD to Bbchitl to obtain hybrid chitinase, and the gene of the hybrid chitinase (Bbchitl-BmChBD) was successfully transformed into beauveria bassiana after being linked with a constitutive promoter gpd (glyceraldehyde triphosphate dehydrogenase), so that the epidermal penetration of the fungus was improved, and the insect death time was shortened by 23% by the engineered strain compared with the wild strain.
However, due to the research difficulty, the period (after invasion) from the time when the pathogenic fungi enter the blood cavity of the insect to the time when the host dies is less, which is the key period of the life-death fight between the pathogenic fungi and the host, occupies most of the time in the pathogenic process of fungus infection, and the insecticidal activity of the entomopathogenic fungi can be effectively improved by shortening the infection time in the insect body of the pathogenic fungi.
Disclosure of Invention
Through research on the period (after invasion) from the time when the pathogenic fungi enter the blood cavity of the insect to the time when the host dies, the invention discovers that the trehalose can be used as an important carbon source in the body of the insect pathogenic fungi and also plays an important role in the environmental stress response of a plurality of organisms. In the trehalose anabolism pathway, the second step is to catalyze the trehalose-6-phosphate by trehalose-6-phosphate phosphatase (Tpp) to synthesize trehalose, and researches show that the trehalose anabolism pathway of beauveria bassiana is an important pivot for regulating and controlling the growth, the stress resistance and the pathogenicity of fungi. The method shows that the entomopathogenic fungi 6-trehalose phosphate phosphatase (Tpp) is a possible pathogenic factor, constructs a fungus TPP overexpression vector by means of genetic engineering, converts the beauveria bassiana to obtain the engineering bacteria with the TPP overexpression, and is a new method which is easy to accept and can improve the insecticidal activity of the entomopathogenic fungi.
The technical scheme of the invention is as follows:
an entomopathogenic fungus transgenic strain is a constitutive exogenous gene containing homologous 6-phosphate trehalose phosphatase.
Preferably, the homologous trehalose-6-phosphate phosphatase is constitutively exogenous for pAN52-Tpp-Bar.
Preferably, the entomopathogenic fungus is beauveria bassiana.
The screening method of the entomopathogenic fungi transgenic strain is obtained by constructing a constitutive expression vector of the full-length cDNA of the trehalose-6-phosphate phosphatase gene, then establishing a liquid-borne microspore transformation system and then selectively screening.
Preferably, the trehalose phosphate phosphatase 6 gene in the full-length cDNA constitutive expression vector for the trehalose phosphate phosphatase 6 gene is constructed from beauveria bassiana Bb2860.
Furthermore, the screening method specifically comprises the following steps:
(1) Amplifying a full-length cDNA nucleic acid sequence of beauveria bassiana Bb 28606-trehalose phosphate phosphatase gene by Polymerase Chain Reaction (PCR);
(2) The sequence of the step (1) is cloned to a fungus expression vector pAN52 which takes a herbicide resistance gene as a screening marker, and the Tpp gene is under the control of a promoter from an Aspergillus nidulans 3-glyceraldehyde phosphate dehydrogenase (gpdA) gene or a promoter of a triosephosphate isomerase (tpiA) gene and a terminator of a tryptophan synthase (trpC) gene, so as to obtain a 6-trehalose phosphate phosphatase gene constitutive expression vector pAN52-Tpp-Bar;
(3) Integrating pAN52-Tpp-Bar into a beauveria bassiana genome by utilizing a beauveria bassiana solution bio-spore generating system;
(4) Obtained by resistance screening and polymerase chain reaction screening.
Preferably, the amplification primers in step (1) are:
the base sequence of the primer 1 is shown as SEQ-ID-NO.1, and specifically comprises the following components: 5, -AAAGGATCCATGGCTCGCCGCGAGTCGC-3, wherein a BamHI enzyme cutting site is marked out by a T line;
the base sequence of the primer 2 is shown as SEQ-ID-NO.2, and specifically comprises the following components: 5, -AAACCATGGTCATGCTGTAGGAATGTGCCCCTC-3, underlined is NcoI cleavage site).
Preferably, the method of step (4) is: selecting a normally growing single colony, extracting DNA, verifying a transgenic strain Bar gene by PCR (polymerase chain reaction) by using a specific primer A and a specific primer B, detecting the expression quantity of Tpp in each transformant by qRT-PCR by using a specific primer C and a specific primer D, and taking 18sRNA as an internal reference; selecting a transformant with the highest expression quantity from the strains, namely the screened strain;
the sequence of the specific primer A is shown as SEQ-ID-NO.3, and specifically comprises the following steps: 5 'CGGTCTGCACCATCGTCAA-3';
the sequence of the specific primer B is shown as SEQ-ID-NO.4, and specifically comprises the following steps: 3 '-TCAAATCTCGGTGACGGC-5';
the sequence of the specific primer C is shown as SEQ-ID-NO.5, and specifically comprises the following components: 5' TGCCATCACACTATCCTT-;
the sequence of the specific primer D is shown as SEQ-ID-NO.6, and specifically comprises the following steps: 3 '-ACCTCCACGTCCCATTTTCTT-5'.
The invention also aims to protect the application of the entomopathogenic fungi transgenic strain in preparing fungal pesticides.
Drawings
FIG. 1 is a diagram showing quantitative PCR detection of expression level of trehalose-6-phosphate phosphatase (Tpp) gene in a transgenic strain;
FIG. 2 shows the trehalose content in wild-type and Tpp-highly expressed strains;
FIG. 3 is the semilethal time of conidia infecting galleria mellonella larva through the body wall;
FIG. 4 shows the semi-lethal time of conidia infecting galleria mellonella larva through blood cavity,
The invention has the advantages of
The transgenic strain of the entomopathogenic fungi has high efficiency of expressing the trehalose phosphate phosphatase 6-phosphate; the preparation method is easy to operate and has good repeatability; when the entomopathogenic fungi transgenic strain is applied to a fungal pesticide, the content of trehalose in an insect host is improved by over-expressing a 6-trehalose phosphate phosphatase gene through the transgenic strain when the fungal pesticide acts in an insect body, and the adverse resistance capability and the growth capability of pathogenic fungi in the host insect body are promoted, so that the insecticidal activity of the fungal pesticide beauveria bassiana is effectively improved.
Detailed Description
Example 1
A screening method of entomopathogenic fungi transgenic strains comprises the following steps:
1. construction of constitutive expression vector of trehalose-6-phosphate phosphatase (Tpp) Gene
Firstly, a 2880 bp Tpp gene full-length cDNA sequence is amplified by Polymerase Chain Reaction (PCR) by using a primer 1 (5, -AAAGGATCCATGGCTCGCGCGCGAGTCGC-3) and a primer 2 (5, -AAACCATGGTGTCATGCTGTAGGAATGTGCCCCTC-3) which are designed according to a full-length cDNA nucleic acid sequence (GenBank accession number: EJP 71046) of the beauveria bassiana Bb 2860-phosphate trehalase gene full-length cDNA sequence (T-BAI restriction site) and a NcoI restriction site which is underlined), and the Tpp gene is cloned between BamHI and NcoI sites of a filamentous fungus expression vector pAN52-Bar, so that the Tpp gene is under the control of a promoter from an Aspergillus nidulans 3-glyceraldehyde phosphate dehydrogenase (gpdA) gene and a terminator of a tryptophan (trpC) gene to obtain a trehalase genome constitutive expression vector pAN 52-Tpp; transferring the pAN52-Tpp-Bar expression vector into escherichia coli by a CaCl2 method for amplification, linearizing a product extracted from the vector in a large amount by SpeI, removing phosphoric acid, and extracting by phenol-chloroform to obtain the linearized constitutive expression vector;
2. establishment of spore transformation System
Optionally taking a tube bud spore suspension from a refrigerator at-80 deg.C, thawing on ice, centrifuging at [ x1] and 4 deg.C for 5 min, and removing supernatant. 240 μ L of 50% PEG 4000, 36 μ L of mol/L LiAc,25 μ L of 4 g/L heat-denatured salmon sperm DNA, 10 μ L of 0.1 μ g/μ L SpeI-digested pAN52-Tpp-Bar, and 35 μ L of 1 mol/L dithiothreitol were added to the blastospore pellet in this order. After the suspension is fully and uniformly mixed, the mixture is firstly iced for 30 min and then is heat shocked for 20 min at 42 ℃. Subsequently, blastospores were collected by centrifugation at 4720 Xg and 4 ℃ and suspended in 0.5 mL of double distilled water. The spore suspension was spread evenly on a Chao's plate containing 200. Mu.g/mL of the herbicide glufosinate (PPT) per 100. Mu.L, and incubated at 25 ℃ for 6 days. The obtained plate colonies were transferred to a multi-well plate containing 300. Mu.g/mL PPT in a Czochralski medium, and cultured. Colonies that continued to grow were selected as the pending transformants for further analysis.
3. Transformation and screening
After 10 days, normal growth single colonies were picked, DNA was extracted, the gene of the transgenic strain Bar was verified by PCR using specific primer pairs (5 'CGGTCTGCACCATCGTCGTCAA-3' and 3 '-TCAAATCTCGGTGACGGC-5'), the expression level of Tpp in each transformant was detected by qRT-PCR using specific primer pairs (5 'TGCCATCCCACTATCAATCCTTT-3' and 3 '-ACCTCCCGTCCATTTCTT-5'), and 18s RNA was used as an internal reference. And selecting the transformant with the highest expression quantity from the strains to obtain the screened strain.
Example 2
Effect investigation of expression of trehalose-6-phosphate phosphatase by entomopathogenic fungus transgenic strains
Respectively inoculating the meristematic broomcorn of the beauveria bassiana overexpression transgenic strain BbOETpp and the original strain Bb2860 (BbWT) on a Sasa solid medium (SDA) (40 g/L glucose, 10 g/L peptone, 10 g/L yeast extract and 20 g/L agar) plate, and culturing at the constant temperature of 25 ℃ for 7 days; scraping spore powder with an inoculating shovel, suspending in sterilized 0.02% Tween 80 (w/v) solution, mixing, filtering with four layers of lens wiping paper, counting with a blood counting chamber, and making into 10 7 Spore suspension of each ml; inoculating to basic salt liquid culture medium (ICM) containing trehalose as sole carbon source at an inoculation amount of 1% (v/v) (1.0 g/L KH) 2 PO 4 ，0.5 g/L MgSO 4 KCl 1.0 g/L, ammonium sulfate 2 g/L, trehalose 10 g/L, MES 10 g/L) at 25 ℃ and 250rpm, 3ml of the culture broth was taken every day, and mycelia were collected by centrifugation at 12000g at 4 ℃. Total RNA of the strain was extracted according to the Promega total RNA extraction kit instructions, and then reverse-transcribed into first-strand cDNA using a random primer according to the Promega reverse transcriptase instructions, using this as a template, and quantitative PCR was performed using primers (5 ', TGCCATCCATCACTATCTT 3' and 3 '-ACCTCCCACGTCCCATTTTTCTT-5'), as shown in FIG. 1.
Example 3
Preparation of fungal pesticides and investigation of Activity
1 ml of 10 7 Inoculating one/ml of mature conidium aqueous suspension of the beauveria bassiana transgenic engineering strain BbOETpp into a 250ml triangular flask filled with 100 ml of Sasa liquid culture medium (SDA) (40 g/L glucose, 10 g/L peptone and 10 g/L yeast extract), carrying out shake culture at 25 ℃ and 150rpm for 2 days, and preparing liquid strains; soaking rice at room temperature (25 deg.C) for 4 hr, packaging into mushroom bag, sterilizing at 121 deg.C for 30 min, and cooling to 25 deg.C; according to the weight ratio of 1: inoculating a liquid strain of the transgenic engineering strain BbOETpp at the ratio of 10, uniformly mixing, culturing at 25 ℃ for 7 days, drying with allochroic silica gel in a dryer, collecting the dry spore powder fungus pesticide of the beauveria bassiana transgenic strain BbOETpp by using an 80-mesh screen, and sealing and storing at 4 ℃.
The transgenic strain of the entomopathogenic fungi has high efficiency of expressing the trehalose phosphate phosphatase 6-phosphate; the preparation method is easy to operate and has good repeatability; when the entomopathogenic fungi transgenic strain is applied to the fungus pesticide, the mycose content in an insect host is increased by over-expressing the 6-trehalose phosphate phosphatase gene by the transgenic strain when the fungus pesticide acts in an insect body (figure 2), so that the insecticidal activity of the fungus pesticide beauveria bassiana is effectively improved (figures 3 and 4).
Sequence listing
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<120> entomopathogenic fungus transgenic strain and screening method and application thereof
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Claims (9)
1. An entomopathogenic fungus transgenic strain is characterized in that: the strain contains homologous 6-phosphate trehalose phosphatase constitutive exogenous genes.
2. The transgenic strain of an entomopathogenic fungus according to claim 1, characterized in that: the constitutive exogenous gene of the homologous trehalose-6-phosphate phosphatase is pAN52-Tpp-Bar.
3. The transgenic strain of an entomopathogenic fungus according to claim 1, characterized in that: the entomopathogenic fungi is beauveria bassiana.
4. The method for screening a transgenic strain of an entomopathogenic fungus according to claim 1, wherein: the gene is obtained by constructing a constitutive expression vector of the full-length cDNA of the trehalose-6-phosphate phosphatase gene, then establishing a liquid-borne microspore transformation system and then selectively screening.
5. The screening method according to claim 4, wherein: the trehalose phosphate phosphatase 6 gene in the full-length cDNA constitutive expression vector for the trehalose phosphate phosphatase 6 gene is from beauveria bassiana Bb2860.
6. The screening method according to claim 4, wherein: the method specifically comprises the following steps:
(1) Amplifying a full-length cDNA nucleic acid sequence of beauveria bassiana Bb 28606-trehalose phosphate phosphatase gene by Polymerase Chain Reaction (PCR);
(2) The sequence of the step (1) is cloned to a fungus expression vector pAN52 which takes a herbicide resistance gene as a screening marker, and the Tpp gene is under the control of a promoter from an Aspergillus nidulans 3-glyceraldehyde phosphate dehydrogenase (gpdA) gene or a promoter of a triosephosphate isomerase (tpiA) gene and a terminator of a tryptophan synthase (trpC) gene, so as to obtain a 6-trehalose phosphate phosphatase gene constitutive expression vector pAN52-Tpp-Bar;
(3) Integrating pAN52-Tpp-Bar into a beauveria bassiana genome by utilizing a beauveria bassiana solution bio-spore generating system;
(4) Obtained by resistance screening and polymerase chain reaction screening.
7. The screening method according to claim 6, wherein: the amplification primers in the step (1) are as follows:
base of primer 1The sequence is shown as SEQ-ID-NO.1, and specifically comprises: 5, -AAAGGATCCATGGCTCGCCGCGAGTCGC-3, wherein a BamHI enzyme cutting site is marked out by a T line;
the base sequence of the primer 2 is shown as SEQ-ID-NO.2, and specifically comprises the following components: 5, -AAACCATGGTCATGCTGTAGGAATGTGCCCCTC-3, ncoI cleavage site underlined).
8. The screening method according to claim 6, wherein: the method in the step (4) comprises the following steps: selecting a normally growing single colony, extracting DNA, verifying a transgenic strain Bar gene by PCR (polymerase chain reaction) by using a specific primer A and a specific primer B, detecting the expression quantity of Tpp in each transformant by qRT-PCR by using a specific primer C and a specific primer D, and taking 18sRNA as an internal reference; selecting a transformant with the highest expression quantity from the strains, namely the screened strain;
the sequence of the specific primer A is shown as SEQ-ID-NO.3, and specifically comprises the following steps: 5 'CGGTCTGCACCATCGTCAA-3';
the sequence of the specific primer B is shown as SEQ-ID-NO.4, and specifically comprises the following steps: 3 '-TCAAATCTCGGTGACGGC-5';
the sequence of the specific primer C is shown as SEQ-ID-NO.5, and specifically comprises the following components: 5' TGCCATCACACTATCCTT-;
the sequence of the specific primer D is shown as SEQ-ID-NO.6, and specifically comprises the following steps: 3 '-ACCTCCACGTCCCATTTCTT-5'.
9. Use of the transgenic strain of an entomopathogenic fungus according to claim 1 for the preparation of a fungal pesticide.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1412311A (en) * | 2002-11-26 | 2003-04-23 | 西南农业大学 | Method for raising microbial pesticide effect by utilizing synergistic action of infection related gene |
| CN101886046A (en) * | 2010-06-13 | 2010-11-17 | 重庆大学 | Entomopathogenic fungi acidic trehalase transgenic strain, preparation method and use thereof |
| CN102181472A (en) * | 2011-03-24 | 2011-09-14 | 重庆大学 | Fungus expression vector and construction and screening method thereof |
| CA2981342A1 (en) * | 2015-06-18 | 2016-12-22 | Novozymes A/S | Polypeptides having trehalase activity and the use thereof in process of producing fermentation products |
| WO2019005755A1 (en) * | 2017-06-28 | 2019-01-03 | Novozymes A/S | Polypeptides having trehalase activity and polynucleotides encoding same |
| CN113956986A (en) * | 2021-09-18 | 2022-01-21 | 西南大学 | Method for improving growth and sporulation capacity and toxicity of insect biocontrol fungi |
-
2022
- 2022-07-01 CN CN202210768734.9A patent/CN115820439A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1412311A (en) * | 2002-11-26 | 2003-04-23 | 西南农业大学 | Method for raising microbial pesticide effect by utilizing synergistic action of infection related gene |
| CN101886046A (en) * | 2010-06-13 | 2010-11-17 | 重庆大学 | Entomopathogenic fungi acidic trehalase transgenic strain, preparation method and use thereof |
| CN102181472A (en) * | 2011-03-24 | 2011-09-14 | 重庆大学 | Fungus expression vector and construction and screening method thereof |
| CA2981342A1 (en) * | 2015-06-18 | 2016-12-22 | Novozymes A/S | Polypeptides having trehalase activity and the use thereof in process of producing fermentation products |
| WO2019005755A1 (en) * | 2017-06-28 | 2019-01-03 | Novozymes A/S | Polypeptides having trehalase activity and polynucleotides encoding same |
| CN113956986A (en) * | 2021-09-18 | 2022-01-21 | 西南大学 | Method for improving growth and sporulation capacity and toxicity of insect biocontrol fungi |
Non-Patent Citations (1)
| Title |
|---|
| 魏晓宇: "6-磷酸海藻糖磷酸酯酶在球孢白僵菌抗逆和侵染过程中的功能和机制", 中国优秀硕士学位论文全文数据库农业科技辑, no. 09, 15 September 2019 (2019-09-15), pages 043 - 166 * |
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