LU504051B1 - Transgenic method for improving heat stress resistance of beauveria bassiana - Google Patents
Transgenic method for improving heat stress resistance of beauveria bassiana Download PDFInfo
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- LU504051B1 LU504051B1 LU504051A LU504051A LU504051B1 LU 504051 B1 LU504051 B1 LU 504051B1 LU 504051 A LU504051 A LU 504051A LU 504051 A LU504051 A LU 504051A LU 504051 B1 LU504051 B1 LU 504051B1
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- beauveria bassiana
- vvmapk
- gene
- insdseq
- volvariella volvacea
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Abstract
The invention relates to the field of biotechnology, in particular to a transgenic method for improving heat stress resistance of Beauveria bassiana. In the invention, the Vvmapk gene of Volvariella volvacea is overexpressed in Beauveria bassiana to obtain a Beauveria bassiana strain overexpressing Vvmapk. The Beauveria bassiana strain overexpressing Vvmapk is driven by the promoter of the glyceraldehyde 3- phosphate dehydrogenase gene of Volvariella volvacea to overexpress the Vvmapk gene, and the experimental verification shows that the molecular spores of this strain have good heat resistance, and the germination rate of conidia suspension is still about 76.3% after heat treatment at 45℃ for 60 min, which is superior to that of wild-type conidia powder of Beauveria bassiana, which is of great significance for prolonging the shelf life and improving the practical application value.
Description
DESCRIPTION LU504051
TRANSGENIC METHOD FOR IMPROVING HEAT STRESS RESISTANCE OF
BEAUVERIA BASSIANA
The invention relates to the field of biotechnology, in particular to a transgenic method for improving heat stress resistance of Beauveria bassiana.
Beauveria bassiana is a pathogenic fungus widely used in the prevention and control of agricultural and forestry pests. It mainly reproduces asexually with conidia, which germinates and grows hyphae. The hyphae differentiate into conidiophore under the stimulation of external environment such as light and air, and then further differentiate into conidia, which spreads with the wind and animal movement. When conidia are attached to the insect body wall, they germinate to form attached cells and infected nails, and secrete hydrolase to penetrate the insect body wall, forming insect bodies in the host body to escape the immune response of the host. When the nutrients in the host are exhausted, Beauveria bassiana passes through the body wall in the form of mycelium and continues to differentiate into conidia. Therefore, conidia are the starting point of development and are very important to the virulence of the host.
However, in the process of diffusion and practical application, conidia of Beauveria bassiana will encounter external environment such as temperature, humidity and ultraviolet rays, which limits its application scope, and the stress resistance of conidia becomes an important limiting factor of its insecticidal toxicity.
MAPK pathway is involved in the transformation of fungal dimorphism, the formation of chlamydospore, the synthesis of white pigment and cell wall and the change of virulence. The entomogenous fungus MAPK cascade pathway receives external signals,
and regulates the growth and differentiation, cell wall integrity and hyperosmotic stred$J504051 response of bacteria through Mapk1, SIt2 and Hog1 respectively. Hog1 pathway of
Beauveria bassiana regulates various stress responses. The cascade pathways of
Metarhizium anisopliae Fus3-MAPK, Hog1-MAPK and SIt2-MAPK can regulate the development of conidia. After the corresponding components in the cascade pathway are deleted, the conidiophore of the strain is damaged or the development is delayed, resulting in the decrease of conidia yield. Moreover, the phosphorylation level of
Hog1-MAPK is regulated by SIt2-MAPK. When hog1 is knocked out, the transcription levels of the pathway factors of conidia development center, such as Mr-AbaA, Mr-WetA, and the genes related to pigment synthesis are significantly reduced. Protein phosphorylation may also be involved in cell signal transduction of Saccharomyces cerevisiae to cold stress, involving gene transcription, protein folding and degradation, cell cycle regulation and morphogenesis. It was also found in Aspergillus fumigatus that this pathway participated in various stress reactions such as cold and heat. However, there is no report on whether the phosphorylation pathway of Volvariella volvacea participates in cold and heat stress and its mechanism.
The purpose of the present invention is to provide a transgenic method for improving heat stress resistance of Beauveria bassiana, so as to solve the problems existing in the prior art. By over-expressing the Vvmapk gene of Volvariella volvacea in
Beauveria bassiana under the drive of the glyceraldehyde 3-phosphate dehydrogenase gene promoter, the constructed transgenic strain significantly improves the heat stress resistance compared with wild Beauveria bassiana.
In order to achieve the above objectives, the present invention provides the following scheme:
The invention provides a transgenic method for improving heat stress resistance of
Beauveria bassiana, comprising: overexpressing Volvariella volvacea gene Vvmapk in
Beauveria bassiana to obtain Beauveria bassiana strains overexpressing Vvmapk.
Optionally, the transgenic method comprises: LU504051 (1) respectively cloning the Volvariella volvacea Vvmapk gene and the Volvariella volvacea glyceraldehyde 3-phosphate dehydrogenase gene promoter which drives the expression of the gene, and then carrying out gene fusion; (2) introducing the fusion fragment obtained in (1) into an expression vector to construct a recombinant vector; (3) transferring the recombinant vector into Agrobacterium for genetic transformation, screening out resistant Agrobacterium, then mixing the resistant
Agrobacterium with Beauveria bassiana in equal volume, and culturing and screening to obtain Beauveria bassiana strain overexpressing Vvmapk.
Optionally, in (1), the primer used for cloning the Volvariella volvacea gene Vvmapk is shown in SEQ ID NO: 1-2, and the primer used for cloning the glyceraldehyde 3-phosphate dehydrogenase gene promoter of Volvariella volvacea is shown in SEQ ID
NO: 3-4.
Optionally, in (2), the expression vector comprises pk2(bar).
Optionally, in (3), the recombinant vector is transformed into Agrobacterium by electric shock, and the conditions of electric shock are: 2200 V, 4-5 ms.
Optionally, in (3), IM solid medium and CZA medium added with cefotaxime sodium antibiotic and herbicide PPT are used for culture screening in sequence, wherein the IM solid culture medium comprises: 7.8 g MES, 5 mL 1M glucose, 0.3 g NaCl, 0.3 g K2HPO4, 0.3 g MgSO4+7H20, 5 mL glycerol, 15 g agar, 100 uL 200 mM acetosyringone, and ddHzO to make up to 1000 mL.
The invention provides application of Volvariella volvacea gene Vvmapk in improving the heat stress resistance of Beauveria bassiana, the heat stress resistance of
Beauveria bassiana is improved by overexpressing Volvariella volvacea gene Vvmapk in
Beauveria bassiana.
The invention provides application of a recombinant vector containing the Volvariella volvacea gene Vvmapk in improving the heat stress resistance of Beauveria bassiana, the heat stress resistance of Beauveria bassiana is improved by overexpressing the recombinant vector containing the Volvariella volvacea gene Vvmapk in BeauveriäJ504051 bassiana.
The invention provides application of host bacteria containing the recombinant vector according to claim 8 in improving the heat stress resistance of Beauveria bassiana.
The invention discloses the following technical effects.
According to the invention, it is discovered for the first time and verified by experiments that the Volvariella volvacea Vvmapk gene is overexpressed in Beauveria bassiana, so that the heat-resistant treatment ability of Beauveria bassiana conidia can be improved. When the conidia suspension of Beauveria bassiana with over-expression of Vvmapk gene is heat-treated at 45°C for 60 min, the conidia germination rate showed a downward trend, but compared with WT (wild type), the conidia germination rate of the over-expression strain was significantly higher than WT(P<0.01). It shows that the heat stress resistance of Volvariella volvacea Vvmapk gene in Beauveria bassiana can be significantly improved, and the heat resistance is an important factor to prolong the shelf life and improve the practical application value of Beauveria bassiana conidia powder, so the Beauveria bassiana overexpressing Vvmapk gene obtained by the invention has practical application value.
In order to explain the embodiments of the present invention or the technical scheme in the prior art more clearly, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention, and other drawings can be obtained according to these drawings without creative work for ordinary people in the field.
Fig. 1 is a schematic diagram of the construction of pK2(bar)-Vvgpd:mapk vector,
Fig. 2 shows the construction of overexpression vector and PCR verification of transformant; A: amplification of Vvgpd gene promoter; B: pk2 (bar)-OE Vvmapk vector is verified by EcoRI digestion; C: PCR verification of Agrobacterium transformation with recombinant vector, using Vvgpd-F/PtrpC as primer; D: PCR verification of Beauveria)504051 bassiana OEVvmapk transformant, with Vvmapk-F/PtrpC as primer; Marker stands for standard DNA molecule; 1, 2 and 3 represent different strains;
Fig. 3 shows the mRNA level of Vvmapk of OEVvmapk strain; WT stands for wild strains system; #1, #2, #3 and #4 respectively represent different strains;
Fig. 4 shows the response of conidia of OE Vvmapk strain to heat stress; **. P<0.01.
A number of exemplary embodiments of the present invention will now be described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention.
It should be understood that the terminology described in the present invention is only for describing specific embodiments and is not used to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Intermediate values within any stated value or stated range, as well as each smaller range between any other stated value or intermediate values within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.
Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. Although the present invention only describes the preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and/or materials related to the documents. In case of conflict with any incorporated document, the contents of this specification shall prevail.
It is obvious to those skilled in the art that many improvements and changes can bé/504051 made to the specific embodiments of the present invention without departing from the scope or spirit of the present invention. Other embodiments will be apparent to the skilled person from the description of the invention. The description and example of that present invention are exemplary only.
The terms "comprising", "including", "having" and "containing" used in this article are all open terms, which means including but not limited to.
Example 1 1. Volvariella volvacea Vvmapk is overexpressed in Beauveria bassiana. 1.1 fusion of Vvmapk gene of Volvariella volvacea and Vvgpd promoter. (1) preparation of Volvariella volvacea DNA template
The mycelium of Volvariella volvacea grown on PDA medium was picked with a sterile toothpick, immersed in 5 pL of 0.3 mol/L NaOH solution, heated at 95°C for 3 min in a PCR instrument for mycelium lysis, and then added with 106 pL of neutralization solution to mix evenly, and the mixed solution was the crude DNA extract. Formula of neutralization solution: 5 mL 1M Tris-HCI (pH=8.0), 20 mL 0.3 M HCI, and the volume is adjusted to 400 mL with ddH2O. (2) PCR amplification of Vvgpd gene promoter of Volvariella volvacea.
The PCR reaction system is: 10 uL of 2xKeyPo Master Mix (Dye Plus) reaction mixture, 1 uL of Vvgpd-F/R primers (see Table 1) and 8 uL of DNA template, with a total volume of 20 JL.
The reaction procedures are: 98°C/3 min, 98°C/10 s, 55°C/5 s, 72/5 s, 72/5 min, in which 30 cycles are carried out in the second to fourth steps.
Table 1 Primers used in the test LU504051
Name Sequence (5-3) Serial numbe r
Vvgpd-F CAGCTATGACCATGATTACGAATTCGGAATACTAACCCAGCA (SEQ
ATC ID
NO :1)
Vvgpd-R GCTTGAGCGTGCTCAGGCATGATGACTTAGGTGCGTAGGA (SEQ
ID
NO :2)
Vvmapk-F TCCTACGCACCTAAGTCATCATGCCTGAGCACGCTCAAGC (SEQ
ID
NO :3)
Vvmapk-R CTTCTGTCGACACTAGTGAATTCCTACAAGTTGATGGT (SEQ
ID
NO :4) qVvmapk-Fl GAGGAGGCATTAGCTCACCC//GATGGTGGGTTGGAACGTC (SEQ
R A ID
NO :5) qactin-F/R GTCAAGTCATCACCATTGGC//GAGGAGCAATGATCTTGAC (SEQ
C ID
NO :6)
PtrpC-R CATTGTTGACCTCCACTAGC (SEQ
ID
NO :7) (3) PCR amplification of Vvmapk gene of Volvariella volvacea.
RNA was extracted from the mycelium of Volvariella volvacea, cDNA was obtained by reverse transcription, and PCR amplification was carried out using the cDNA as a template. The PCR amplification system is: 10 uL of 2xKeyPo Master Mix (Dye Plus) reaction mixture, 1 pL of Vvmapk-F/R primers (see Table 1), 1 uL of cDNA template, and ddH20 to make the total reaction volume 20uL. The reaction procedures were: 98°C/3/504051 min, 98°C/10 s, 55°C/5 s, 72/10 s, 72/5 min, in which 30 cycles were carried out in the second to fourth steps to obtain the Vvmapk gene of Volvariella volvacea (GenBank:
FJ906769.1). (4) The promoter of Vvmapk gene and Vvgpd gene (GenBank: KF528323.1) (1200 bp before the start codon ATG) was fused by PCR. Using Vvgpd-F/Vvmapk-R as primers, 1 UL of fragments recovered from steps (2) and (3) were used as templates for PCR amplification. The reaction procedure is the same as step (2). 1.2 Construction of recombinant vector
The specific method is as follows: the fusion fragment is connected to pk2(bar) vector by homologous recombination, and the reaction system is carried out according to the system recommended by the instruction of CloneExpress MultiS One Step Cloning
Kit (Nanjing Novezan Biotechnology Co., Ltd.): 2 uL of 5xreaction buffer, 1 pL of vector recovered after EcoRI digestion, 3 pL of fusion fragment, 1uL ligase, make up 10 pL ddH20, and connect at 37°C/30 min in PCR instrument to transform Escherichia coli. The correctness of the recombinant vector pK2(bar)-OE Vvmapk was confirmed by plasmid extraction, restriction enzyme digestion and sequencing. 1.3 Agrobacterium transformation
The recombinant vector pk2(bar)-OE Vvmapk was transformed into Agrobacterium
AGL-1 by electric shock, and the electric shock condition was 2200 V for 4-5 ms, and it was screened on YEB medium with kanamycin (working concentration was 50 pg/mL). 1.4 culture of single colony
The Agrobacterium selected above was cultured in YEB liquid medium with kanamycin (working concentration: 50 pg/mL) until OD600=0.5, centrifuged at 12000 r/min for 1 min to collect the precipitate, and then the supernatant was discarded, and then the concentration was adjusted to ODeoo=0.15 with IM liquid medium, and the culture was continued for 6 h at 28°C and 200 r/min. The cultured conidia suspension of
Agrobacterium and Beauveria bassiana (concentration: 1x10° spores /mL) was mixed in equal volume, vortexed, and the mixed bacteria liquid was coated on IM solid medium with microporous filter membrane in advance, and cultured at 26°C for 48 h in the dark.
Then transferring the filter membrane to CZA medium with cefotaxime sodium antibioti¢J504051 (working concentration is 500 ug/mL) and herbicide PPT [18%(wt%) glufosinate solution diluted 4 times with sterile water, and then added according to the ratio of 1:1000] for 5-7 days, until a single colony appears.
Among them, the formula of IM liquid culture medium in the above test steps is: 7.8 g MES, 10 mL 1M glucose, 0.3 g NaCl, 0.3 g KHPO4, 0.3 g MgS04+7H20, 5 mL glycerol, 100 uL 200 mM AS (Acetosyringone), adding ddHzO to a constant volume of 1000 mL, and adjusting pH to 5.3. Note that glucose and AS need to be filtered and then added after the culture medium is cooled to 55 °C Formula of IM solid medium: 5 mL 1M glucose, 15 g agar powder, and other components are the same as liquid medium. 1.5 Screening positive transformants
Single colonies were selected with toothpicks and inoculated on 48-well plates with the same resistance for screening. Once again, the colonies growing in 48-well plates were picked out with sterile toothpicks, and the potential positive transformants were verified by PCR amplification with mycelium DNA as template. The positive transformants were transferred and amplified to be used as the next experimental materials. 1.6 Screening the expression of over-expressed strain OEVvmapk by quantitative
PCR.
Real-time quantitative PCR was performed using iCycler iQ multicolor real-time
PCR detection system with SYBR Green (Bio-Rad). 5 uL of reaction mixture, 0.5 pL of upstream and downstream primers (see qVvmapk-F/R and gactin-F/R in Table 1, with a concentration of 10 umol/L), 3 uL of cDNA template diluted 20 times, 1 uLof ddH20, with a total volume of 10 pL, and mixed evenly by centrifugation for a short time; pre-denaturation at 95°C/5 min; then according to 95°C/15 s; 60°C/15 s; 39 cycles of amplification were performed at 72°C/30 s. The relative expression level of the gene was determined with the expression level of actin as a reference. 1.7 Determination of conidia germination rate
The conidia of the over-expressed strain were treated by thermal shock at 45°C/60 min. Adjust the concentration of conidia suspension to 1x108 /mL, heat shock the conidia suspension at 45°C for 60 min in a metal bath, then coat it on CZA culture medium, and)504051 cultivate it for 24-76 h at 26°C. Cut the culture medium blocks with the size of 1 cmx1 cm with a sharp blade every 12 h, and observe and take photos with an inverted microscope to calculate the germination rate of conidia. 2. Results and analysis (1) Construction of recombinant vector
The schematic diagram of the construction of recombinant vector is shown in Fig. 1.
By fusing the ORF frame of Vvmapk gene of Volvariella volvacea with Vvgpd promoter, adding EcoRl restriction sites at both ends of the fusion fragment by designing primers, and connecting with pK2(bar) vector by homologous recombination, the overexpression vector pK2(bar)-OE Vvmapk was constructed. (2) Construction of overexpression vector and PCR verification of transformant.
Using Volvariella volvacea DNA as template and Vvgpd-F/R as primer, the Vvgpd gene promoter was amplified, and the amplified fragment size was about 1200 bp (A in
Figure 2).
Using Volvariella volvacea cDNA as template and Vvmapk-F/R as primer to amplify
Vvmapk gene, the obtained Vvmapk fragment (1150 bp) and Vvgpd gene promoter were fused by PCR, and then cloned into pK2(bar) vector. After enzyme digestion, the fragment with the size of 2350 bp (B in Figure 2) can be cut, which is consistent with the expected fragment size.
Further sequencing confirmed the correctness of the cloned fragment. The obtained pK2(bar)-OEVvmapk recombinant vector was transformed into Agrobacterium tumefaciens, and with Vvgpd-F/PtrpC-R as primer, a fragment of about 2350 bp (C in
Figure 2) could be amplified, indicating that the recombinant vector was transformed into
Agrobacterium. The recombinant vector pK2(bar)-OEVvmapk was transformed into
Beauveria bassiana by Agrobacterium-mediated method, and a fragment of about 1200 bp could be amplified with Vvmapk-F/PtrpC-R (see Table 1) as primer, indicating that the recombinant vector has been transformed into Beauveria bassiana (D in Figure 2). (3) Different strains overexpressing Vvmapk are verified by quantitative PCR.
Four different over-expressed strains were selected for quantitative PCR verificatid/504051
Compared with WT, the expression levels of Vvmapk in the four strains were all increased in different degrees, among which the gene expression levels of strains 2#, 3# and 4# were 2.2-2.7 times that of WT (Figure 3), and these three strains were used as follow-up experimental materials. (4) Detecting the germination rate
When the conidia suspension was treated with heat shock at 45°C for 60 min, the germination of conidia was not observed after 24 h of culture. After 48 h of culture, the germination rate was observed, and it was found that the average germination rate of over-expressed strains was 76.3%, which was significantly higher than that of wild-type conidia (see Figure 4).
The above-mentioned embodiments only describe the preferred mode of the invention, and do not limit the scope of the invention. Under the premise of not departing from the design spirit of the invention, various modifications and improvements made by ordinary technicians in the field to the technical scheme of the invention shall fall within the protection scope determined by the claims of the invention.
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Claims (9)
1. A transgenic method for improving heat stress resistance of Beauveria bassiana, comprising: overexpressing Vvmapk gene of Volvariella volvacea in Beauveria bassiana to obtain Beauveria bassiana strains overexpressing Vvmapk.
2. The transgenic method according to claim 1, wherein the transgenic method comprises: (1) respectively cloning the Vvmapk gene of Volvariella volvacea and the Volvariella volvacea glyceraldehyde 3-phosphate dehydrogenase gene promoter which drives the expression of the gene, and then carrying out gene fusion; (2) introducing the fusion fragment obtained in (1) into an expression vector to construct a recombinant vector; (3) transferring the recombinant vector into Agrobacterium for genetic transformation, screening out resistant Agrobacterium, then mixing the resistant Agrobacterium with Beauveria bassiana in equal volume, and culturing and screening to obtain Beauveria bassiana strain overexpressing Vvmapk.
3. The transgenic method according to claim 2, wherein in (1), the primer used for cloning the Vvmapk gene of Volvariella volvacea is shown in SEQ ID NO: 1-2, and the primer used for cloning the glyceraldehyde 3-phosphate dehydrogenase gene promoter of Volvariella volvacea is shown in SEQ ID NO: 3-4.
4. The transgenic method according to claim 2, wherein in (2), the expression vector comprises pk2(bar).
5. The transgenic method according to claim 2, wherein in (3), the recombinant vector is transformed into Agrobacterium by electric shock, and the conditions of electric shock are: 2200 V, 4-5 ms.
6. The transgenic method according to claim 2, wherein in (3), IM solid medium and/504051 CZA medium which is added with cefotaxime sodium antibiotic and herbicide PPT are used for culture screening in sequence, wherein the IM solid medium comprises: 7.8 g MES, 5 mL 1M glucose, 0.3 g NaCl, 0.3 g K:HPO4, 0.3 g MgSO4+7H20, 5 mL glycerol, 15 g agar, 100 uL 200 mM acetosyringone, and ddHzO to make up to 1000 mL.
7. Application of Vvmapk gene of Volvariella volvacea in improving the heat stress resistance of Beauveria bassiana, characterized in that the heat stress resistance of Beauveria bassiana is improved by overexpressing Vvmapk gene of Volvariella volvacea in Beauveria bassiana.
8. Application of a recombinant vector containing the Vvmapk gene of Volvariella volvacea in improving the heat stress resistance of Beauveria bassiana, characterized in that the heat stress resistance of Beauveria bassiana is improved by overexpressing the recombinant vector containing the Vvmapk gene of Volvariella volvacea in Beauveria bassiana.
9. Application of host bacteria containing the recombinant vector according to claim 8 in improving the heat stress resistance of Beauveria bassiana.
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