CN116396872A - Recombinant beauveria bassiana and preparation method and application thereof - Google Patents
Recombinant beauveria bassiana and preparation method and application thereof Download PDFInfo
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- CN116396872A CN116396872A CN202211726778.1A CN202211726778A CN116396872A CN 116396872 A CN116396872 A CN 116396872A CN 202211726778 A CN202211726778 A CN 202211726778A CN 116396872 A CN116396872 A CN 116396872A
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- beauveria bassiana
- recombinant
- gene encoding
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- parasitic wasp
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- 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
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- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses recombinant beauveria bassiana and a preparation method and application thereof. The invention firstly discloses a recombinant beauveria bassiana which can express parasitic wasp venom protein. Further discloses a preparation method of the recombinant beauveria bassiana and application thereof in disinsection. According to the invention, the gene sequence of the parasitic wasp venom protein is optimized according to the codon preference of the beauveria bassiana, the virulence factor resource library for biological control is expanded, and the sequence is integrated into the beauveria bassiana to obtain the recombinant beauveria bassiana, so that the parasitic wasp venom protein can be efficiently expressed, and the defects of slow effect and weak toxicity of the biocontrol bacteria are overcome. In addition, the preparation method of the recombinant beauveria bassiana is simple and convenient to operate, and the obtained recombinant beauveria bassiana is very stable and can continuously express exogenous virulence factors.
Description
Technical Field
The invention relates to the technical field of biological control. In particular to recombinant beauveria bassiana and a preparation method and application thereof.
Background
Parasitic wasps are important natural enemy resources for controlling many pests in the agriculture and forestry ecological system, and the venom protein is a naturally-occurring biopesticide library, so that the parasitic wasps have very wide application prospects in biological control of the pests. The venom proteins of parasitic wasps have the following advantages: firstly, the insecticidal efficiency is high, parasitic wasps and host pests are subjected to long-term co-evolution, certain venom proteins of the parasitic wasps form very efficient virulence factors under the pressure of natural selection, and the hosts are not easy to generate drug resistance. Secondly, the specificity is strong, and it is different from bee venom of bees, wasps and the like. The venom protein of parasitic wasps is completely harmless to higher animals such as human beings and animals, does not harm beneficial insects in ecological systems such as bees, butterflies, dragonflies and the like, and has obvious lethal effect only on host pests. Finally, the bee venom is environment-friendly, is derived from insects, does not pollute the environment and can be degraded. The venom of parasitic wasps can be used as a synergist in biological pest control. The sensitivity of host pests to the biological pesticides is obviously improved by using the venom of parasitic wasps to treat entomopathogenic fungi, insect baculoviruses and microsporidia, and better insecticidal effect is obtained. The midge side cocoon bee (Microplitis mediator) is a parasitic wasp of lepidoptera larva, and the host comprises more than forty important agricultural pests such as cotton bollworms (Helicoverpa armigera), armyworms (Pseudaletia separata), cabbage loopers (Mamestra brassicae) and the like. The biological control effect is better when the biological control agent is applied in the open field in Xinjiang, hebei and other places. Recently, the major components of venom proteins from mid-red side hornet have been resolved by a combination of transcriptome and proteome, and from this, a snake venom-like metalloprotease MmVRF1 has been identified. Emitter in hostIts micro dose (1×10) -4 ng) of the MmVRF1 recombinant protein, the host larvae can be directly killed. However, parasitic wasps are smaller in size and the difficulty of obtaining venom from the parasitic wasps and applying it on a large scale is greater.
Beauveria bassiana (Beauveria bassiana) is an entomopathogenic fungus, and the host comprises more than 750 insects of 15 meshes and 149 families and 13 mites of 6 families, is a common biocontrol fungus, and has the advantages of wide insecticidal spectrum, easiness in large-scale production, no harm to the environment and the like. However, beauveria bassiana still has some defects in the application process of biological control, mainly slow insecticidal effect and unstable bacterial strain control effect.
Therefore, by combining the advantages and disadvantages of beauveria bassiana and parasitic wasps and utilizing the technical means of modern molecular biology, a novel pesticide which is efficient, low in toxicity, green and stable is developed to meet the development requirements of the country, and has profound significance.
Disclosure of Invention
The first aim of the invention is to provide a recombinant beauveria bassiana which can efficiently express parasitic wasp venom protein MmVRF1 and overcomes the defects of slow onset of action and weak toxicity of the beauveria bassiana.
The second object of the invention is to provide the application of the recombinant beauveria bassiana.
The third object of the invention is to provide the preparation method of the recombinant beauveria bassiana, which is simple and convenient to operate, and the obtained beauveria bassiana is very stable and can continuously express exogenous virulence factors.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a recombinant beauveria bassiana capable of expressing a parasitic wasp venom protein.
Illustratively, the recombinant beauveria bassiana is integrated with an exogenous gene encoding a parasitic wasp venom protein, wherein the gene encoding the parasitic wasp venom protein is a nucleotide sequence as set forth in any one of the following:
a1 Nucleotide sequence of SEQ ID NO.1 at positions 67-1449;
b1 Nucleotide sequence of SEQ ID NO.2 at positions 67-1449;
c1 A) the complement of a 1) or b 1) above;
d1 A polynucleotide derived from the nucleotide sequence of SEQ ID NO.1 or SEQ ID NO.2 due to the degeneracy of the genetic code.
Preferably, in the recombinant beauveria bassiana, an endogenous gene encoding a beauveria bassiana chitinase chit1 signal peptide is integrated before an exogenous gene encoding a parasitic wasp venom protein, and the gene encoding the beauveria bassiana chitinase chit1 signal peptide has a nucleotide sequence as follows:
a2 Nucleotide sequence of SEQ ID NO. 3;
b2 A) the complement of a 2) above; or (b)
c2 A polynucleotide derived from the nucleotide of SEQ ID NO.3 due to the degeneracy of the genetic code.
The parasitic wasp venom protein of the invention is specifically a middle red side cocoon bee venom protein, and a gene encoding the parasitic wasp venom protein and a gene encoding beauveria bassiana chitinase chip 1 signal peptide can be artificially synthesized or amplified by using a PCR technology. The invention removes and replaces the signal peptide of the parasitic wasp venom protein itself with the beauveria bassiana chitinase chit1 signal peptide, namely, integrates the endogenous gene for encoding beauveria bassiana chitinase chit1 signal peptide before the gene for encoding the parasitic wasp venom protein, so that the recombinant beauveria bassiana can secrete and express the parasitic wasp venom protein in a heterologous way.
The recombinant beauveria bassiana of the present invention has a preservation number of CGMCC No.23043, is classified and named as beauveria bassiana Beauveria bassiana, and is preserved in China general microbiological culture Collection center (address: north West road No.1, 3 in the Beijing area of Chaoyang) at day 7 and day 14 of 2021.
In a second aspect, the present invention provides an insecticide comprising the recombinant beauveria bassiana described above, or a progeny thereof, or a conidium produced thereby, or a mycelium produced thereby, or any combination thereof.
Preferably, the pesticide further comprises an agropharmaceutically acceptable carrier; the agropharmaceutically acceptable carrier can be one or more of mica powder, light calcium carbonate, clay, talcum powder, kaolin, diatomite, attapulgite, bentonite, sepiolite, urea, potassium chloride, sodium sulfate, ammonium sulfate, sodium nitrate, ammonium nitrate and ammonium chloride.
In a specific embodiment of the invention, the insecticide is used for controlling cotton bollworms and/or spodoptera frugiperda.
The application of the recombinant beauveria bassiana, or the offspring of the recombinant beauveria bassiana, or the conidium produced by the recombinant beauveria bassiana, or the mycelium produced by the conidium, or any combination of the conidium and the mycelium in the preparation of the pesticide is also within the protection scope of the invention.
Preferably, the insecticide of the present invention may further comprise other active ingredients capable of killing insects. Exemplary are one or more of destruxins, pyrethroids, carbamates, nicotinoids, nerve sodium channel blockers, insecticidal macrolides, gamma-aminobutyric acid (GABA) antagonists, insecticidal ureas, and juvenile hormone mimics.
In a third aspect, the invention provides a method for preparing recombinant beauveria bassiana, comprising the following steps: the gene encoding the beauveria bassiana chitinase chip 1 signal peptide and the gene encoding the parasitic wasp venom protein are operably introduced into beauveria bassiana.
Preferably, the introduction is performed by agrobacterium-mediated methods.
In a specific embodiment of the invention, the preparation method of the recombinant beauveria bassiana comprises the following steps:
the gene encoding the beauveria bassiana chitinase chip 1 signal peptide and the gene encoding the parasitic wasp venom protein are connected to an expression vector, and introduced into beauveria bassiana by an agrobacterium-mediated method to integrate the gene encoding the beauveria bassiana chitinase chip 1 signal peptide and the gene encoding the parasitic wasp venom protein onto the chromosome of the beauveria bassiana, so as to obtain the beauveria bassiana engineering strain capable of expressing the parasitic wasp venom protein, namely the recombinant beauveria bassiana.
In a preferred embodiment of the invention, the preparation method of the recombinant beauveria bassiana comprises the following steps:
connecting a gene (Bbsp) encoding a beauveria bassiana chitinase chip 1 signal peptide and a gene (VRF 1) encoding a parasitic wasp venom protein to a plasmid pBARGPE1 to obtain pBARGPE1-Bbsp-VRF1-FLAG, and obtaining a cassette "gpdA promoter-Bbsp-VRF 1-FLAG-trpC Terminator" (abbreviated as gBVft) comprising a fungal over-expression promoter gpdA promoter and a Terminator trpC Terminator to initiate expression of the gene of the exogenous parasitic wasp venom protein;
ligating gBVFt to plasmid pPK2-Bar to obtain pPK2-Bar-gBVFt;
the pPK2-Bar-gBVft is introduced into beauveria bassiana by an agrobacterium-mediated method to obtain a recombinant beauveria bassiana engineering strain capable of expressing parasitic bee venom proteins.
The invention also provides an insecticidal method, which comprises the step of applying the recombinant beauveria bassiana, or the offspring of the recombinant beauveria bassiana, or the generated conidia or the generated mycelium or any combination between the conidia and the mycelium, or the insecticide, or the recombinant beauveria bassiana prepared by the preparation method.
Preferably, the application comprises spraying the crop, such as corn, wheat, etc., with the recombinant beauveria bassiana of the present invention.
Compared with wild beauveria bassiana strain, the recombinant beauveria bassiana engineering strain reduces the lethal medium dose of the beauveria bassiana strain to cotton bollworms by 1.67 times (experimental dose gradient: 1 multiplied by 10) 2 、1×10 3 、1×10 4 The time in death was shortened by 1.55 times (1X 10 in experimental dose) 4 Individual spores), the insecticidal efficacy is significantly improved.
The beneficial effects of the invention are as follows:
1. according to the invention, the gene sequence of the parasitic wasp venom protein is optimized according to the codon preference of the beauveria bassiana, the virulence factor resource library for biological control is expanded, and the sequence is integrated into the recombinant beauveria bassiana obtained by the beauveria bassiana, so that the parasitic wasp venom protein can be efficiently expressed, and the defects of slow effect and weak toxicity of the biocontrol bacteria are overcome.
2. The preparation method of the recombinant beauveria bassiana is simple and convenient to operate, and the obtained recombinant beauveria bassiana is very stable and can continuously express exogenous virulence factors.
Drawings
The following describes the embodiments of the present invention in further detail with reference to the drawings.
FIG. 1 is a schematic diagram of the use of parasitic wasp venom to enhance biocontrol microbial virulence; wherein A is an anatomical schematic diagram of parasitic bollworms of the mid-red side cocoon bees and related organs of toxic glands; b is a schematic diagram of constructing a vector and enhancing the virulence of engineering bacteria by using an agrobacterium transformation method.
FIG. 2 shows the result of electrophoresis detection of fragments required for constructing pBARGPE1-Bbsp-VRF1-FLAG vector; wherein A is a band of a gene fragment of chitinase chip 1 signal peptide amplified by using a primer Bbsp-F/R (represented by "Bbsp" in the figure) by using beauveria bassiana genomic DNA as a template; b is a band of a gene fragment of a venom protein of the Zhonghong side cocoon bee (without self signal peptide) amplified by using a primer VRF1-F/R (denoted by "VRF1" in the figure) by using a cDNA of a venom gland of the Zhonghong side cocoon bee as a template; c is a band of linearized plasmid pBARGPE1 (denoted by "linearized pBARGPE1" in the figure) linearized with the plasmid pBARGPE1 as template using primers L-pBARGPE 1-F/R.
FIG. 3 is a schematic representation of the structure of the FLAG-tag containing vector pBARGPE1-Bbsp-VRF 1-FLAG.
FIG. 4 shows the result of electrophoresis detection of fragments required for constructing the pPK2-Bar-VRF1-FLAG vector; wherein A is a band of linearization plasmid pPK2-Bar (represented by "linearization pPK2-Bar" in the figure) which is linearized by using a primer L-pPK2-F/R by taking the plasmid pPK2-Bar as a template; b is a band of gBVft carrying a linker amplified using the primer O-gBVft-F/R (denoted by "gBVft" in the figure) using the plasmid pBARGPE1-Bbsp-VRF1-FLAG as a template.
FIG. 5 is a schematic representation of the structure of the fungal transformation vector pPK 2-Bar-gBVft.
FIG. 6 shows 1/4SDAY medium (PPT + Chloramphenicol (Chloramphenicol) + ) Results of screening transformants.
FIG. 7 shows the result of electrophoresis detection of PCR-verified positive transformants.
FIG. 8 shows the Western-blot detection results of the medium red side cocoon bee venom protein MmVRF1 secreted in the beauveria bassiana positive transformant; wherein 1-8 are different positive transformants.
FIG. 9 shows the results of spore yield detection and comparison of the deepest 3 positive transformants (Bb-VRF 1-1,2, 7) and wild type beauveria bassiana (Beauveria bassiana ARSEF 2860, abbreviated as Bb 2860) in FIG. 7.
FIG. 10 shows the results of germination rate detection and comparison of 3 positive transformants (Bb-VRF 1-1,2, 7) with the germination rate detection and comparison of wild beauveria bassiana (Beauveria bassiana ARSEF 2860, abbreviated as Bb 2860).
FIG. 11 shows the results of colony expansion rate detection and comparison of 3 positive transformants (Bb-VRF 1-1,2, 7) and wild-type beauveria bassiana (Beauveria bassiana ARSEF 2860, abbreviated as Bb 2860).
FIG. 12 shows colony morphology of wild beauveria bassiana (Beauveria bassiana ARSEF 2860, abbreviated as Bb 2860) and Bb-VRF1 transformed into a parasitic wasp venom protein expression gene on three media ((1) CDM (Czapek-Dox Medium), (2)1/4 SDAY, SDAY Medium containing only 1/4 components), (3) PDA potato dextrose Medium, potato Dextrose Agar Medium).
FIG. 13 shows the results of insecticidal efficacy against 3-year-old cotton bollworms by the insect dipping method.
FIG. 14 shows the results of measurement of insecticidal efficacy against 5-year-old cotton bollworms by injection.
FIG. 15 shows the results of insecticidal efficacy against Spodoptera frugiperda at age 6 by injection.
FIG. 16 shows the result of measuring the insecticidal efficiency against the 3-year-old plutella xylostella by the insect dipping method.
FIG. 17 shows a disease state of a test insect after infection with Bb-VRF1, A being cotton bollworm, B being Spodoptera frugiperda, C being Plutella xylostella.
Detailed Description
In order to more clearly illustrate the present invention, the present invention will be further described with reference to preferred embodiments and the accompanying drawings. Like parts in the drawings are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The gene sequence of the venom protein of the medium red side cocoon bee in the following example is shown as SEQ ID NO. 2. The gene sequence of the parasitic wasp venom protein is optimized according to the codon preference of beauveria bassiana and is shown as SEQ ID NO. 1.
The primers used in the following examples are shown in Table 1:
TABLE 1 primer sequences
Note that: 1. the primer named at the beginning of L is used as a linearization primer; 2. the primer named at the beginning of O is a primer with a linker, the linker comprising an overlap region, the underlined part being the overlap region.
3. FLAG tag was included in the underlines of L-pBARGPE1-R and O-VRF1-R and introduced at the completion of recombinant cloning. The method comprises the following steps: FLAG tag sequence 5'-GATTACAAGGACGACGATGACAAG-3'
The underlined part of O-VRF1-R is 5')GTCGTCCTTGTAATC3 'and its reverse complement 5'-GATTACAAGGACGAC-3'is 1-15bp at the 5' end of the FLAG tag.
The underlined portion 5'-GACGACGATGACAAG-3' of L-pBARGPE1-R is 1-15bp at the 3' -end of the FLAG tag. When the recombinant clone is performed, the two can be recombined at the complementary sequence to form a FLAG tag.
The invention is described in detail below in connection with specific embodiments, which are intended to be illustrative rather than limiting.
EXAMPLE 1 preparation of recombinant beauveria bassiana
A schematic diagram of the use of venom by the red-medium side cocoon bee to regulate host cotton bollworm is shown in FIG. 1A, and the parasitic wasp venom protein is secreted by the venom gland, stored in the venom sac, and injected into the host through the ovipositor during the parasitic wasp.
A schematic diagram of the recombinant beauveria bassiana is shown as a B in figure 1, and the specific steps are as follows:
vector construction
1.1 Construction of pBARGPE1-Bbsp-VRF1-FLAG
The genome DNA extracted from beauveria bassiana (Beauveria bassiana ARSEF 2860) is used as a template, and a primer Bbsp-F/R is used for PCR amplification to obtain a gene fragment (a nucleotide sequence shown in SEQ ID NO. 3) of beauveria bassiana chitinase chip 1 signal peptide. Because beauveria bassiana codons are weak in use preference, the gene expression level is low, and sequence optimization only needs to adjust rare codons. The cDNA (nucleotide sequence shown as SEQ ID NO. 2) extracted from the male venom glands of the medium red side cocoon bees after two days of eclosion is used as a template to refer to a kit Mut Express II Fast Mutagenesis Kit V (Vazyme, C214-01) instruction book for point mutation, according to the codon preference of beauveria bassiana, the CGC of the rare codons at 916-918 of the SEQ ID NO.2 is replaced with the CGT (namely, the optimized gene sequence of the medium red side cocoon bee venom protein is the nucleotide sequence shown as SEQ ID NO. 1), and PCR amplification is carried out by using a primer VRF1-F/R to obtain a gene fragment (the nucleotide sequence shown as 67-1449 of the SEQ ID NO. 1) containing the optimized medium red side cocoon bee venom protein (without self signal peptide). The plasmid pBARGPE1 is used as a template, and is linearized by using a primer L-pBARGPE1-F/R to obtain a linearized plasmid pBARGPE1.
Wherein, the high-fidelity PCR reaction system (50 mu L) is shown in the table 2, and the high-fidelity PCR reaction conditions are as follows: pre-denaturation at 98℃for 3min;98℃for 30sec,55 or 60℃for 30sec,68℃for 1kb/min, cycle number 35;68 ℃ for 3min and 12 ℃ for infinity.
Table 2 high fidelity PCR reaction system (50 μl):
| 5 XPimerSTAR GXL buffer | 10μL |
| dNTP mixture (2.5 mM) | 4μL |
| Upstream primer (10. Mu.M) | 2μL |
| Downstream primer (10. Mu.M) | 2μL |
| Template | 5ng-100ng |
| Primer STAR GXL DNA polymerase | 1μL |
| ddH 2 O | To 50 mu L |
The specificity and the band size of the PCR products are verified by agarose gel electrophoresis, and the result is shown in FIG. 2, wherein A is a band of a gene fragment of chitinase chip 1 signal peptide (shown as 'Bbsp' in the figure) amplified by using a primer Bbsp-F/R by taking beauveria bassiana genome DNA as a template; b is a band of a gene fragment of a venom protein of the Zhonghong side cocoon bee (without self signal peptide) amplified by using a primer VRF1-F/R (denoted by "VRF1" in the figure) by using a cDNA of a venom gland of the Zhonghong side cocoon bee as a template; c is a band of linearized plasmid pBARGPE1 (denoted by "linearized pBARGPE1" in the figure) linearized with the plasmid pBARGPE1 as template using primers L-pBARGPE 1-F/R. After the sequencing verification result is correct, the primer O-Bbsp-F/R with the joint (the joint contains an overlapping region) is used for PCR amplification to obtain a gene fragment of the beauveria bassiana chitinase chit1 signal peptide with the joint, the primer O-VRF1-F/R with the joint (the joint contains an overlapping region) is used for PCR amplification to obtain a gene fragment of the medium red side cocoon bee venom protein (without the self signal peptide) with the joint, and the Dpn I is used for digesting the linearized plasmid pBARGPE1 to obtain the digested linearized plasmid pBARGPE1 so as to eliminate the influence of a template chain.
The concentrations of each PCR product (namely, the gene fragment carrying the beauveria bassiana chitinase chip 1 signal peptide of the joint and the gene fragment carrying the venom protein of the midred side cocoon bee (without the self signal peptide) of the joint) and the digestion product (namely, the digested linearization plasmid pBARGPE 1) are measured by using a micro ultraviolet spectrophotometer, a connection reaction system is configured by referring to a kit ClonExpress MultiS One Step Cloning Kit (Vazyme Norpran, cat# C113-01) instruction book, and a carrier pBARGPE1-Bbsp-VRF1-FLAG (the structural schematic diagram is shown in FIG. 3) containing a fungus overexpression promoter and Terminator is obtained, so that a cassette "gpdA promoter-Bbsp-VRF 1-FLAG-trpC Terminator" containing a fungus overexpression promoter is used for constructing engineering bacteria for expressing exogenous virulence factors.
1.2 construction of vector pPK2-Bar-gBVft
Hereafter, the cassette "gpdA Promoter-Bbsp-VRF 1-FLAG-trpC Terminator" will be abbreviated as gBVFt. PCR amplification was performed using the vector pBARGPE1-Bbsp-VRF1-FLAG as template and the primer O-gBVF-F/R with the linker to obtain gBVFt with the linker. The plasmid pPK2-Bar is used as a template, and is linearized by using a primer L-pPK2-F/R to obtain a linearized plasmid pPK2-Bar. The specificity and the band size of the PCR products were verified by agarose gel electrophoresis, and the result is shown in FIG. 4, wherein A is a band of linearized plasmid pPK2-Bar (shown as "linearized pPK2-Bar" in the figure) which is linearized by using the plasmid pPK2-Bar as a template and the primer L-pPK 2-F/R; b is a band of gBVft carrying a linker amplified using the primer O-gBVft-F/R (denoted by "gBVft" in the figure) using the plasmid pBARGPE1-Bbsp-VRF1-FLAG as a template.
After the sequencing verification result is correct, the linear plasmid pPK2-Bar is digested by using Dpn I to eliminate the influence of a template strand. The concentration of each PCR product (gBVft carrying linker) and digestion product (linearized plasmid pPK 2-Bar) was determined by using a micro-UV spectrophotometer, and a ligation reaction system was prepared by referring to the kit ClonExpress MultiS One Step Cloning Kit (Vazyme Norpran, cat. No. C113-01) instructions, to construct the fungal expression vector pPK2-Bar-gBVft (structural schematic diagram is shown in FIG. 5).
Construction of engineering bacteria
Construction of fungal transformation systems using Agrobacterium-mediated method (Agrobacterium tumefaciens mediated transformation, ATMT): plasmid pPK2-Bar-gBVft was transformed into AGL-1 Agrobacterium tumefaciens competent cells in LB plates (kana + ) And (3) selecting bacteria to perform colony PCR verification, and obtaining the AGL-1 agrobacterium tumefaciens transferred into the plasmid pPK2-Bar-gBVft after confirming that the strip is correct and the sequencing of the PCR product is correct. Preparing AGL-1 agrobacterium tumefaciens bacteria liquid transferred into plasmid pPK2-Bar-gBVft and co-culturing with beauveria bassiana conidium suspension, preferentially picking out first-growing and rapid-growing colony to 1/4SDAY plate (PPT) + Chloramphenicol (Chloramphenicol) + ) Two screenings were performed (as shown in figure 6). After 4-7 days of the second screening, the transformant mycelium genomic DNA was extracted and used as a template, PCR detection was performed using the primer gBVft-F/R, the bands were detected by agarose gel electrophoresis and after sequencing was completed, positive transformants were determined.
The method comprises the following specific steps:
2.1 transformation of plasmids into AGL-1 Agrobacterium tumefaciens competent cells
1) AGL-1 Agrobacterium tumefaciens competent cells stored at-70℃were thawed on ice.
2) Under aseptic conditions, 1. Mu.g of plasmid pPK2-Bar-gBVFt was added to a centrifuge tube containing AGL-1 Agrobacterium tumefaciens competent cells, gently mixed, and ice-bathed for 5min.
3) The centrifuge tube is placed in liquid nitrogen for 5min of freezing, and the centrifuge tube is rapidly placed in a water bath at 37 ℃ for 5min and an ice bath for 5min.
4) Under aseptic condition, 800. Mu.L of LB liquid medium (without antibiotics) is added, and the culture is carried out by shaking at 150rpm for 2-3h at a constant temperature of 28 ℃, centrifugating at 5000rpm for 1min, and collecting the bacterial cells.
5) Adding 100 μl of LB liquid medium (without antibiotic) to resuspend the bacterial cells, and applying appropriate amount of bacterial liquid to LB solid medium (kana) + ) On the plate, the plate is inversely cultured for 2-3d at 28 ℃.
6) And (3) selecting single colony of the transformant to perform colony PCR, performing sequencing verification after the size of the strip is confirmed by electrophoresis detection, and performing the sequencing verification to obtain AGL-1 agrobacterium tumefaciens transferred into plasmid pPK2-Bar-gBVFt correctly, preparing bacterial liquid into glycerinum (15% glycerol), and freezing and storing at-80 ℃.
2.2 Agrobacterium tumefaciens mediated genetic transformation of beauveria bassiana
2.2.1 preparation of a conidium suspension of beauveria bassiana
1) Scraping a proper amount of beauveria bassiana (Beauveria bassiana ARSEF 2860) thalli on an SDAY plate after culturing for about 10 days to 5mL of sterile water containing 0.05% Tween-20, and performing vortex dispersion for 2-3min.
2) An appropriate amount of sterile glass wool is filled at the bottom of a sterile syringe, the filtered suspension is collected in a sterile centrifuge tube to remove mycelium, the supernatant is discarded after centrifugation at 8000rpm for 3min, and spores are collected.
3) Spores were resuspended using sterile water, centrifuged at 8000rpm for 3min, and the supernatant was discarded and spores were collected again.
4) The spores were resuspended using IM liquid medium to give a beauveria bassiana conidium suspension and the spore concentration was calculated using a hemocytometer.
2.2.2 Beauveria bassiana transformation and transformant selection
1) AGL-1 Agrobacterium tumefaciens transformed with plasmid pPK2-Bar-gBVft was added to 3mL of LB liquid medium (kana + ) The culture is carried out for 16 to 24 hours at a constant temperature of 28 ℃ and 200 rpm.
2) Determination of bacterial liquid OD 660 After centrifugation at 5000rpm for 3min, the supernatant was discarded to collect the cells, which were resuspended in IM liquid medium and OD was adjusted 660 To 0.15; culturing for 6h at a constant temperature of 28 ℃ and 200rpm to obtain AGL-1 agrobacterium tumefaciens bacterial liquid transferred into plasmid pPK 2-Bar-gBVft.
3) AGL-1. Tumefaciens bacterial solution and beauveria bassiana conidium suspension (concentration 10) transferred into plasmid pPK2-Bar-gBVft are taken 4 mu.L/mL) of each of the above-mentioned materials was filled into a centrifuge tubeAnd (5) uniformly mixing to obtain a mixture.
4) The mixture was spread on a plate of IM solid medium with cellophane (autoclaved cellophane was immersed in sterile water in advance and then spread on the medium) and co-cultured at 26 ℃ for 48h.
5) The cellophane was removed from the IM solid medium, plated on 1/4SDAY medium (containing 100. Mu.g/mL glufosinate, 25. Mu.g/mL chloramphenicol) and incubated at 26℃for about 5 days until resistant colonies appeared.
6) A second selection was performed and resistant colonies were picked to 1/4SDAY medium (containing 100. Mu.g/mL glufosinate, 25. Mu.g/mL chloramphenicol) and incubated at 26℃for about 5d, the results of which are shown in FIG. 6.
7) And (3) picking a normal-growth resistant colony, extracting genome DNA, performing PCR verification by using a primer Bbsp-F/VRF1-R, and preliminarily determining that the colony is a positive transformant if the size of the band is correct.
The PCR verification electrophoresis detection result is shown in FIG. 7, wherein the size of the PCR product band of 8 positive transformants is 1485bp, the size of the PCR product band is consistent with the size of a target band, and the pPK2-Bar-gBVft is preliminarily determined to be transferred into beauveria bassiana, so that the positive transformants (called beauveria bassiana positive transformants for short) of the beauveria bassiana containing the gene sequences for expressing the parasitic wasp venom proteins are obtained.
3. Detection of venom protein MmVRF1 of red side cocoon bee expressed by beauveria bassiana positive transformant
Mycelium with a wet weight of about 0.1g was scraped off on 1/4SDAY medium corresponding to the beauveria bassiana positive transformant. The mycelium was added to SDB liquid medium and cultured at constant temperature of 26℃and 200rpm for 3 days. The culture supernatant was precipitated with an equal volume of pre-chilled acetone at-20℃for 4h. Centrifuging at 12000rpm for 5min, and collecting appropriate amount of ddH 2 Western-blot is carried out after O is dissolved and precipitated, mmVRF1 is detected, and the result is shown in a figure 8, wherein lanes 1-8 are MmVRF1 bands secreted into a culture medium in the liquid culture process of 8 beauveria bassiana positive transformants respectively.
Finally, 8 recombinant beauveria bassiana engineering bacteria capable of expressing MmVRF1 are obtained, 1 st, 2 nd and 7 th strains with high MmVRF1 expression in a Western-blot result are selected, and further growth characteristic indexes are measured.
4. Determination of growth characteristic index of recombinant beauveria bassiana engineering bacteria for expressing MmVRF1
4.1 measuring the spore yield of recombinant beauveria bassiana engineering bacteria expressing MmVRF1
Preparing 1X 10 conidium of recombinant beauveria bassiana engineering bacteria expressing MmVRF1 7 Mu.l of spore suspension of each spore/mL was placed in the center of the PDA solid medium plate and incubated in an inverted incubator at 26℃for 14d. Colonies were scraped with a punch of 5mm diameter from the center to 1/2 of the edge of the spore-producing portion of the colonies, placed in 5mL of sterile water containing 0.05% (v/v) Tween-20, vortexed for 2min, and the spore production was measured and calculated with a hemocytometer. The quantity of the obtained spore yield is converted into the unit area (cm) of the culture medium 2 ) Spore yield. The 1 st, 2 nd and 7 th strains with high VRF1 expression in the Western-blot result are measured and named as Bb-VRF1-1, bb-VRF1-2 and Bb-VRF1-7, 3 PDA plates are selected for each strain to detect the spore yield, and wild beauveria bassiana (Beauveria bassiana ARSEF 2860, abbreviated as Bb 2860) is used as a control for comparison, and the result is shown in figure 9, the spore yield of only Bb-VRF1-2 is obviously lower than that of Bb2860, and the spore yields of Bb-VRF1-1 and Bb-VRF1-7 are not obviously different from that of Bb 2860.
4.2 determination of spore germination Rate
After the recombinant beauveria bassiana engineering bacteria expressing MmVRF1 produce spores, selecting a proper amount of spores, inoculating the spores into an SDB liquid culture medium, performing shake culture at 26 ℃ at 200rpm/min for 24 hours, and performing microscopic examination. 3 shaking tubes were taken for each strain, and each tube was subjected to microscopic examination in 3 fields using a blood cell counting plate, and germination rates were calculated. As shown in FIG. 10, only Bb-VRF1-2 had a spore germination rate significantly lower than that of Bb2860, and Bb-VRF1-1 and Bb-VRF1-7 had no significant difference from that of Bb 2860.
4.3 determination of colony growth Rate
3 single colonies of recombinant beauveria bassiana engineering bacteria expressing MmVRF1 are selected and inoculated into a PDA culture medium, and the beauveria bassiana (Beauveria bassiana ARSEF 2860) is used as a control group for comparison. The colony diameter was measured once daily with a vernier caliper for a total of 15d. Colony growth rate (mm/d) =colony diameter/15. As a result, as shown in FIG. 11, colony growth rates of Bb-VRF1-1, bb-VRF1-2 and Bb-VRF1-7 were not significantly different from that of Bb 2860.
4.4 colony morphology observations
3 solid media were prepared: (1) CDM Czapek-Dox Medium (Coolabier, MM1010-250 g); (2) 1/4SDAY, SDAY Medium (Hopebio, HB 0235-6) containing only 1/4 of the components; (3) PDA potato dextrose medium, potato Dextrose Agar Medium (Coolaber, PM0520-250 g). Mu.l of spore suspensions of Bb2860 and Bb-VRF1 were dropped in the center of the plate and incubated at 26℃for 20 days, and the morphology of colonies on different media was observed. As shown in FIG. 12, the colony morphology of Bb2860 and Bb-VRF1 was consistent on 3 solid media, indicating that the colony morphology of Bb-VRF1 was not affected by the transfer of parasitic wasp venom protein.
The expression quantity, spore yield, germination rate, colony growth speed and colony morphology of the parasitic wasp venom are comprehensively considered to finally obtain a recombinant beauveria bassiana Bb-VRF1-1 (BbVRF 01 for short) which stably grows and expresses MmVRF1, and the BbVRF01 strain is preserved in 2021 at 7 months and 14 days, wherein the preservation number is CGMCC NO.23043 and is preserved in China general biological center (address: north Chen West road No.1 of the Korean area of Beijing city).
EXAMPLE 2 insecticidal efficacy determination of recombinant beauveria bassiana expressing MmVRF1
The insecticidal efficiency is measured by using an insect dipping method, and the specific method is as follows: preparing a control group Bb2860 wild beauveria bassiana (Beauveria bassiana ARSEF 2860, abbreviated as Bb 2860) and a treatment group BbVRF01 recombinant beauveria bassiana (BbVRF 01) conidium suspension, and adjusting the suspension concentration to 1 multiplied by 10 8 spores/mL. And (3) immersing the test insects in the suspension for 20s, taking out the test insects, and absorbing excessive water by using water absorbing paper. Each treatment was performed in 3 replicates, 24 replicates each for test insects. The test insects are placed in an artificial climate box with the temperature of 26 ℃ and the relative humidity of 80% and the photoperiod L: D=14h:10h, the insect-raising box is cleaned regularly, and the feed is replaced timely. Observing every 24h, removing dead insect corpses, and placing into culture plateAnd (5) subsequent observation, statistics and production of a survival curve. An estimate of time in death was calculated from the data processed differently.
The insecticidal efficiency is measured by using an injection method, and the specific method is as follows: preparing a control group Bb2860 wild beauveria bassiana (Beauveria bassiana ARSEF 2860, abbreviated as Bb 2860) and a treatment group BbVRF01 recombinant beauveria bassiana (BbVRF 01) conidium suspension, and adjusting the suspension concentration to a proper concentration. Mu.l of spore suspension was aspirated using a microinjector and the injection was slowly performed at the base of the second pair of tarsopoda of the test insects, which were anesthetized on ice in advance. Each treatment group was repeated 3 times, 24 test insects per treatment. The insects for testing after injection are placed in a climatic chamber with the temperature of 26 ℃ and the relative humidity of 50% and the photoperiod L: D=14h:10h, the insect-culturing box is cleaned regularly, and the feed is replaced timely. Every 24 hours, removing dead insect corpses, placing the insect corpses in a culture plate independently for subsequent observation, counting and making a survival curve. An estimate of time in death was calculated from the data processed differently.
Determination of the insecticidal efficacy against Heliothis armigera Helicoverpa armigera
The method comprises the steps of infecting cotton bollworms on day 1 of 3 years by using an insect dipping method, carrying out insecticidal efficiency measurement by taking cotton bollworms infected by wild beauveria bassiana (Beauveria bassiana ARSEF 2860, abbreviated as Bb 2860) as a control group Bb2860 and cotton bollworms infected by recombinant beauveria bassiana (BbVRF 01) as a treatment group BbVRF01, and regulating spore suspension to 1 multiplied by 10 8 Spores/ml, each test insect is taken out after being soaked for 20s, and is normally fed and is observed later. As shown in FIG. 13, the survival curve of the cotton bollworms in the treated group BbVRF01 was steeper and the mortality was higher than that in the control group Bb2860, indicating that secretion of the parasitic wasp venom protein enhanced the killing capacity of beauveria bassiana to cotton bollworms.
Infection of 1 st day of 5 th age with injection method, measurement of insecticidal efficiency with cotton bollworm infected with wild beauveria bassiana (Beauveria bassiana ARSEF 2860, abbreviated as Bb 2860) as control Bb2860, and cotton bollworm infected with recombinant beauveria bassiana (BbVRF 01) as treatment group BbVRF01, and adjustment of spore suspension to 5×10 6 Spores/ml, per microinjectorThe first test insects were injected with 5 μl of suspension for infection, fed normally and observed subsequently. The survival curves are shown in fig. 14 and the pathology is shown in fig. 17 a, the survival curves of cotton bollworms in the treatment group BbVRF01 are steeper and the mortality rate is higher compared to the control group Bb2860, indicating that secretion of parasitic wasp venom proteins enhances the killing capacity of beauveria bassiana on cotton bollworms.
Second, determination of insecticidal efficiency against Spodoptera frugiperda Spodoptera frugiperda
The injection method is used for infecting spodoptera frugiperda at day 1 of 6 years, spodoptera frugiperda of wild beauveria bassiana (Beauveria bassiana ARSEF 2860, abbreviated as Bb 2860) is used as a control group Bb2860, spodoptera frugiperda infected by recombinant beauveria bassiana (BbVRF 01) is used as a treatment group BbVRF01 for measuring the insecticidal efficiency, and the spore suspension concentration is adjusted to 2 multiplied by 10 6 Spores/ml, 5 μl of suspension was injected with each test insect with a microinjector for infection, normal feeding and subsequent observation. The survival curve is shown in fig. 15, and the pathology is shown in fig. 17B, and the survival curve of spodoptera frugiperda in the treatment group BbVRF01 is steeper and the mortality is higher than that of the control group Bb2860, indicating that secretion of parasitic wasp venom protein can enhance the lethality of beauveria bassiana to spodoptera frugiperda.
Third, the insecticidal efficiency of the plutella xylostella Plutella xylostella is measured
The method comprises the steps of (1) infecting plutella xylostella on day 3 by using an insect dipping method, taking plutella xylostella infected by wild beauveria bassiana (Beauveria bassiana ARSEF 2860, abbreviated as Bb 2860) as a control group Bb2860, taking plutella xylostella infected by recombinant beauveria bassiana (BbVRF 01) as a treatment group BbVRF01, measuring insecticidal efficiency, and regulating spore suspension to 1 multiplied by 10 8 Spores/ml, each test insect is taken out after being soaked for 20s, and is normally fed and is observed later. The survival curves are shown in FIG. 16 and the symptoms are shown in FIG. 17C, and the survival curves of the plutella xylostella in the control group Bb2860 are steeper and the mortality is higher than that of the treatment group BbVRF01, which indicates that the killing capacity of beauveria bassiana to plutella xylostella is not enhanced by secreting parasitic wasp venom protein due to the fact that the plutella xylostella has a relationship more distant than that of spodoptera frugiperda, and the enhancement of biocontrol by using the parasitic wasp venom is proved from the sideThe bacterial virulence is not easy to miss targets, and beneficial insects in the application environment can not be damaged.
Fourthly, the cotton bollworm Helicoverpa armigera LD is treated by wild beauveria bassiana (Beauveria bassiana ARSEF 2860, abbreviated as Bb 2860) and recombinant beauveria bassiana engineering bacteria (BbVRF 01) 50 And LT 50 Is (are) determined by
Supplemental determination of Bb2860 and BbVRF01 lethal ability to 5-year-old bollworms at 3 concentration gradients using injection and their LD based on the Probit regression method based on the measured data 50 And LT 50 Calculations were performed (using software SPSS ver.21).
Table 3 shows LD against 5-year-old bollworm injection infection using Bb2860 50 The experimental data obtained were measured. Table 4 shows LD against 5-year-old bollworm injection infection using BbVRF01 50 The experimental data obtained were measured. Calculated based on Probit regression method, bb2860 LD for 5-year-old cotton bollworm 50 Is 2.15X10 3 LD of individual spores, bbVRF01, against 5-year-old cotton bollworm 50 1.29×10 3 And (3) spores. LD of BbVRF01 50 Reduced by 1.67 times compared with Bb-2860.
Table 5 LT for 5-year-old bollworm infection by Bb2860 50 The experimental data obtained were measured. Table 6 LT for 5-year-old bollworm infection with BbVRF01 50 The experimental data obtained were measured. Calculated based on the Probit regression method, bb2860 is used for LT of 5-year-old cotton bollworms 50 LT of BbVRF01 against 5-year-old cotton bollworm for 5.327 days 50 3.432. LT of BbVRF01 50 The time is shortened by 1.55 times compared with Bb-2860.
Table 3Bb2860 vs. bollworm LD 50 Determination of experimental data
TABLE 4BbVRF01 vs. Helicoverpa armigera LD 50 Determination of experimental data
Table 5Bb2860 vs. Helicoverpa armigera LT 50 Determination of experimental data
TABLE 6BbVRF01 vs. Helicoverpa armigera LT 50 Determination of experimental data
It should be understood that the foregoing examples of the present invention are provided merely for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention, and that various other changes and modifications may be made therein by one skilled in the art without departing from the spirit and scope of the present invention as defined by the appended claims.
Claims (10)
1. A recombinant beauveria bassiana, characterized in that the recombinant beauveria bassiana is capable of expressing parasitic wasp venom proteins.
2. The recombinant beauveria bassiana according to claim 1, wherein the recombinant beauveria bassiana is integrated with an exogenous gene encoding a parasitic wasp venom protein, the gene encoding the parasitic wasp venom protein being any one of the following nucleotide sequences:
a1 Nucleotide sequence of SEQ ID NO.1 at positions 67-1449;
b1 Nucleotide sequence of SEQ ID NO.2 at positions 67-1449;
c1 A) the complement of a 1) or b 1) above;
d1 A polynucleotide derived from the nucleotide sequence of SEQ ID NO.1 or SEQ ID NO.2 due to the degeneracy of the genetic code.
3. The recombinant beauveria bassiana according to claim 2, wherein the recombinant beauveria bassiana has integrated therein an endogenous gene encoding a beauveria bassiana chitinase chit1 signal peptide before the exogenous gene encoding a parasitic bee venom protein, the gene encoding the beauveria bassiana chitinase chit1 signal peptide having a nucleotide sequence as set forth in any one of the following:
a2 Nucleotide sequence of SEQ ID NO. 3;
b2 A) the complement of a 2) above;
c2 A polynucleotide derived from the nucleotide sequence of SEQ ID NO.3 due to the degeneracy of the genetic code.
4. The recombinant beauveria bassiana is characterized in that the preservation number of the recombinant beauveria bassiana is CGMCC NO.23043.
5. An insecticide comprising the recombinant beauveria bassiana of any one of claims 1-4, or a progeny thereof, or a conidium produced thereby, or a mycelium produced thereby, or any combination thereof; preferably, an agropharmaceutically acceptable carrier is also included.
6. The insecticide according to claim 5, wherein the insecticide is used for controlling cotton bollworms and/or spodoptera frugiperda.
7. Use of the recombinant beauveria bassiana of any one of claims 1-4, or a progeny of said recombinant beauveria bassiana, or a conidium produced thereby, or a mycelium produced thereby, or any combination thereof, in the preparation of a pesticide.
8. The preparation method of the recombinant beauveria bassiana is characterized by comprising the following steps: the gene encoding the beauveria bassiana chitinase chip 1 signal peptide and the gene encoding the parasitic wasp venom protein are operably introduced into beauveria bassiana.
9. The method of claim 8, wherein the introducing is by agrobacterium-mediated.
10. A method of killing insects comprising the step of administering the recombinant beauveria bassiana of any one of claims 1-4, or a progeny of the recombinant beauveria bassiana, or a conidium produced thereby, or a mycelium produced thereby, or any combination thereof, or the insecticide of any one of claims 5 or 6, or the recombinant beauveria bassiana produced by the method of claim 8 or 9.
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