Application of genetically engineered bacteria in enhancing ultraviolet radiation resistance of beauveria bassiana
By highly expressing the Bbrad1 gene in Beauveria bassiana and enhancing its photorepair ability, the problem of Beauveria bassiana being sensitive to UVB radiation was solved, and efficient application under strong light conditions was achieved.
Patent Information
- Application Number
- CN202510846803.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-24
- Publication Date
- 2025-09-23
AI Technical Summary
After application in the field, the conidia of Beauveria bassiana are sensitive to UVB ultraviolet radiation from sunlight, resulting in inactivation and failure, which limits its effectiveness in all-weather application in summer.
By over-expressing the Bbrad1 gene in Beauveria bassiana, its photorepair ability is enhanced, a genetically engineered strain is constructed, and the upper limit of tolerance to UVB radiation is increased.
It significantly improved the anti-ultraviolet radiation ability of Beauveria bassiana, enhanced the photoreactivation rate of conidia under different lighting conditions, and prolonged its effectiveness under strong sunlight.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of genetic engineering, and particularly relates to the application of genetically engineered bacteria in enhancing the ultraviolet radiation resistance of Beauveria bassiana. Background Art
[0002] With increasing awareness of the hazards of overuse of chemical pesticides and a growing demand for pollution-free, green food, environmental awareness has intensified. Green pest control technologies for agriculture and forestry have emerged and are rapidly developing. Biological pest control based on entomopathogenic fungi is considered a highly effective and environmentally friendly approach. Beauveria bassiana is the entomopathogenic fungus with the broadest host range known to date and is widely used to control insect pests and mites in agriculture, forestry, and livestock. Conidia are the vectors that B. bassiana infects its host. Conidia first attach to the insect's body wall and, under appropriate conditions, germinate to form germ tubes. These conidia extend into hyphae that penetrate the body wall and enter the host's hemocoel, where they transform into blastospores. These blastospores rapidly reproduce in a manner similar to yeast budding, utilizing nutrients from the host's body, leading to the host's ossification and death.
[0003] After field application, formulated conidia of Beauveria bassiana are inevitably exposed to environmental stresses, particularly intense ultraviolet (UV) radiation from summer sunlight, which can inactivate them and become a major limitation to the round-the-clock application of fungal insecticides (acaricides) in the summer. Fungi have evolved two mechanisms to resist UV radiation: rapid photorepair mediated by photolyases under visible light, and nucleotide excision repair (NER) under dark conditions, also known as dark repair, involving numerous UV-resistant BBRAD (anti-UV radiation) family proteins. The processes of restoring UV-damaged cells through photorepair and dark repair are known as photoreactivation and dark reactivation, respectively.
[0004] Direct exposure to solar radiation for several hours can inactivate most fungal conidia. Sublethal doses of UV radiation can reduce the germination rate and virulence of conidia. In recent years, the depletion of the ozone layer has led to increased UVB radiation at the Earth's surface. The negative effects of increased UV radiation doses are particularly severe during the late germination phase of conidia, when fungi are most sensitive to UV radiation. Therefore, studying the effects and mechanisms of UV radiation resistance in Beauveria bassiana and using these as targets for strain improvement to enhance UV radiation resistance is of great significance for improving pest control effectiveness and the widespread application of biocontrol strains. Summary of the Invention
[0005] In response to the above problems, the purpose of the present invention is to provide a technical solution for an ultraviolet radiation-resistant strain of Beauveria bassiana based on high expression of the endogenous gene Bbrad1, as well as a construction method and use thereof. This invention largely overcomes the sensitivity of conidia, which are the active ingredients of fungal insecticides, to sunlight UVB radiation after field application, and can significantly enhance the ability of fungal insecticides to resist ultraviolet radiation, thus having important application prospects.
[0006] To achieve the above object, the present invention provides the following technical solutions:
[0007] In the first aspect, the present invention provides the use of genetically engineered bacteria in enhancing the ultraviolet radiation resistance of Beauveria bassiana. The genetically engineered bacteria highly express the Bbrad1 gene, which can specifically increase the upper limit of the tolerance of Beauveria bassiana to UVB radiation under photorepair conditions, thereby enhancing its ultraviolet radiation resistance.
[0008] Furthermore, the accession number of the Bbrad1 gene in the NCBI library is BBA_07749.
[0009] Furthermore, under the illumination condition of L:D 5:19, the recombinant engineered strain has a conidia light resurrection rate of ≥15% after being irradiated with a lethal dose of UVB, and the dark resurrection rate of the genetically engineered strain after being irradiated with a lethal dose of UVB is not significantly improved.
[0010] In the present application, by overexpressing the Bbrad1 gene in Beauveria bassiana, the ultraviolet radiation resistance of Beauveria bassiana was significantly improved. Under different lighting conditions, the photoreactivation rate of conidia was increased after exposure to a lethal dose of UVB radiation, while the dark reactivation rate did not change significantly, indicating that the Bbrad1 gene mediates the photorepair process in Beauveria bassiana.
[0011] In a second aspect, the present invention provides a method for enhancing the ultraviolet radiation resistance of Beauveria bassiana, wherein the Bbrad1 gene is highly expressed in Beauveria bassiana to enhance its ultraviolet radiation resistance.
[0012] Furthermore, the specific steps for over-expressing the Bbrad1 gene in Beauveria bassiana are as follows:
[0013] (1) Using the cDNA genome of Beauveria bassiana as a template, a cDNA fragment of the Bbrad1 gene was cloned;
[0014] (2) Connecting the cDNA fragment obtained in step (1) to an expression vector by homologous recombination to construct a high expression vector;
[0015] (3) The high expression vector was transferred into the wild strain of Beauveria bassiana using the Agrobacterium-mediated method to obtain a Beauveria bassiana strain that highly expressed the Bbrad1 gene.
[0016] Furthermore, in step (1), the primers used to amplify the cDNA fragment of the Bbrad1 gene are shown in SEQ ID NO. 1-2.
[0017] Furthermore, in step (2), the expression vector is the plasmid p0380-Ptef1-sur linearized by XmaI / BamHI enzyme digestion.
[0018] Furthermore, in step (3), the Agrobacterium is Agrobacterium tumefaciens AGL1.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] The present invention provides the use of genetically engineered bacteria in enhancing the ultraviolet radiation resistance of Beauveria bassiana. Specifically, the strong endogenous promoter Ptef of Beauveria bassiana is used to drive the high expression of the Bbrad1 gene in wild strains of Beauveria bassiana. The transcriptional expression level of Bbrad1 in the screened engineered strain is increased by 18-24 times compared with the wild strain, thereby increasing the upper limit of the tolerance of conidia produced by the engineered strain to UVB radiation under light repair conditions, while the dark resurrection rate after exposure to a lethal dose of UVB radiation is not significantly improved. This indicates that high expression of the Bbrad1 gene in Beauveria bassiana can enhance ultraviolet radiation resistance, and strong sunlight ultraviolet radiation is the main factor limiting the all-weather application of fungal insecticides (mite) in summer. Therefore, the Beauveria bassiana with high expression of the Bbrad1 gene obtained by the present invention has important practical application value. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 Schematic diagram of the construction and identification of a strain with high expression of the Bbrad1 gene of Beauveria bassiana; A is a schematic diagram of the construction of the high expression vector; B is a schematic diagram of the PCR identification of the high expression transformant; and C is a schematic diagram of the qPCR analysis of the high expression strain.
[0022] Figure 2 Schematic diagram of the dark resurrection rate trend of conidia of the Bbrad1 gene highly expressed strain of Beauveria bassiana when cultured in the dark for 12 (A) or 24 h (B) after irradiation with gradient UVB doses as the radiation dose changes.
[0023] Figure 3 The UVB dose LD corresponding to 75% and 50% dark resuscitation of the strain with high expression of Bbrad1 gene of Beauveria bassiana in dark culture treatment 12h (A) or 24h (B) after UVB irradiation 25 and LD 50 Schematic diagram.
[0024] Figure 4 Schematic diagram of the trend of photoreactivation rate of conidia of the Bbrad1 gene highly expressed strain of Beauveria bassiana after UVB irradiation at L:D 3:9 (A) and L:D 5:7 (B).
[0025] Figure 5 The trend of photoreactivation rate of conidia of the Bbrad1 gene highly expressed strain of Beauveria bassiana after UVB irradiation at L:D 3:21 (A) and L:D 5:19 (B) treatments as the UVB dose changes.
[0026] Figure 6 The UVB doses LD corresponding to 75% and 50% photoreactivation of the Bbrad1 gene highly expressed strain of Beauveria bassiana under L:D 3:9 (A), L:D 5:7 (B), L:D 3:21 (C) and L:D 5:19 (D) treatments after UVB irradiation are shown in Figure 2. 25 and LD 50 Schematic diagram. DETAILED DESCRIPTION
[0027] In order to make those skilled in the art better understand the present invention, the technical solution of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and is only a part of the embodiments of the present invention, rather than all embodiments.
[0028] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work should fall within the scope of protection of the present invention.
[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.
[0030] In the following examples, the Beauveria bassiana strain used was B. bassiana ARSEF 2860, which was sourced from the ARSEF strain bank of the United States Department of Agriculture; and the Agrobacterium used was Agrobacterium tumefaciens AGL1.
[0031] In the following examples, the accession number of the Bbrad1 gene in the NCBI database is BBA_07749.
[0032] Example 1 Construction of a strain with high expression of the Bbrad1 gene
[0033] (1) PCR amplification of Bbrad1 gene cDNA.
[0034] The wild strain contained 10 7 100 mL of a suspension containing 100 conidia / mL was evenly spread on a glassine-covered SDAY medium plate and incubated for 3 days at 25°C under optimal conditions of a 12:12 photoperiod. The culture was then ground into a powder using liquid nitrogen. Total RNA was extracted from the ground material using RNAiso™ Plus Reagent (TaKaRa) and reverse-transcribed into cDNA using the PrimeScript RT reagent kit (TaKaRa).
[0035] PCR amplification was performed using the cDNA described above as a template and the sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 as primers using KODFX (TOYOBO). The PCR reaction system consisted of 25 μL of 2× reaction buffer, 2 μL of each primer, 4 μL of Beauveria bassiana cDNA template, 10 μL of dNTPs, and ddH2O to a final volume of 50 μL. The reaction procedure was: 94°C / 2 min; 98°C / 10 s, 58°C / 30 s, 68°C / 2 min, 35 cycles; and 68°C / 5 min. A cDNA fragment of the Bbrad1 gene was amplified.
[0036] (2) Construction of Bbrad1 gene high expression vector.
[0037] The cDNA fragment of the Bbrad1 gene obtained in (1) was inserted into the plasmid p0380-Ptef1-sur linearized by XmaI / BamHI enzyme digestion using the homologous recombination enzyme ClonExpress II OneStep Cloning Kit (Novozyme Biotechnology Co., Ltd.) to obtain the high expression vector p0380-Ptef-Bbrad1-sur, in which Ptef1 is an endogenous strong promoter (such as Figure 1 The recommended reaction system was as shown in Figure A. The following reaction was performed using the following: 2 μL 5× reaction buffer, 1 μL vector recovered after enzyme digestion, 3 μL fusion fragment, 1 μL ligase, and 10 μL ddH2O. Ligation was performed in a PCR instrument at 37°C for 30 minutes, and E. coli was transformed. The correctness of the recombinant vector, p0380-Ptef-Bbrad1-sur, was confirmed by plasmid extraction, enzyme digestion, and sequencing.
[0038]
[0039] (3) Construction of transgenic strains with high expression of Bbrad1
[0040] After sequencing verification, the plasmid was transformed into the wild-type strain using Agrobacterium-mediated fungal transformation. The culture medium and specific transformation method used are as follows:
[0041] Transformation media: IM liquid medium: 40% 2.5 mm saline solution, 5‰ glycerol, 10 mM glucose, 40 mM morpholineethanesulfonic acid (MES), 200 μM acetosyringone (AS), adjusted to pH 5.3. Note that MES and AS must be filtered and added after the sterilized medium has cooled to 55°C. IM solid medium: 10 mM glucose, 1.5% agar powder. Other components are the same as for liquid medium. M-100 screening medium: 1% glucose, 0.3% potassium nitrate, 6.25% M-100 saline solution, 1.5% agar powder.
[0042] Specific transformation method: A single colony of Agrobacterium tumefaciens transformed with the correct plasmid vector was inoculated into 3 mL of YEB liquid medium (containing 50 μg / mL carbenicillin and 50 μg / mL kanamycin), and cultured overnight at 28°C and 220 rpm (16-20 h); the next day, the Agrobacterium cells were collected by centrifugation at 10,000 rpm for 1 min, and the cells were resuspended in 1 mL of IM liquid medium and the concentration was adjusted to OD660 of 0.15, and then cultured at 28°C and 200 rpm for 6 h. The conidia suspension of the cultured Agrobacterium and the wild strain of Beauveria bassiana (concentration of 1×10 7 Mix equal volumes of 500 μg / mL of the bacterial solution and evenly spread 150 μL of the solution onto IM solid culture medium. Blow dry in a clean hood and incubate in a dark incubator at 25°C for 2 days. After 2 days, transfer the bacterial solution to M-100 screening medium (containing 500 μg / mL cefotaxime sodium and 10 μg / mL chlorimuron-ethyl) for continued screening. Incubate at 25°C for approximately 3-4 days until resistant colonies appear.
[0043] (4) Screening of positive transformants
[0044] Use a sterile toothpick to pick a single colony and inoculate it onto a 48-well SDAY plate with the same resistance. Incubate at 25°C for about 2-3 days. Use a sterile toothpick to pick the colony growing in the 48-well plate again. Use mycelial DNA as a template and the sequences shown in SEQ ID NO.3 and SEQ ID NO.4 as primers to perform PCR amplification using KOD FX to verify potential positive transformants. The PCR amplification system is as described above. The results are shown in Figure 2. Figure 1 As shown in B. The positive transformants were transferred and cultured and used as the experimental materials in the next step.
[0045] (5) Screening the expression of OEBbrad1 in highly expressed strains using quantitative PCR
[0046] After the positive transformants were cultured on SDAY plates for 3 days, total RNA was extracted and reverse transcribed into cDNA according to the method described in (1). Using this cDNA as a template, the β-actin gene as an internal reference, and the sequences shown in SEQ ID NO.5 and SEQ ID NO.6 as primers, fluorescence real-time quantitative PCR analysis was performed to detect the expression level of the target gene Bbrad1 in each transformant relative to the wild strain. Two strains of Bbrad1 gene high expression colonies were screened and named OEBbrad1-1 / 2. The expression levels in three repeated samples were increased by 24 times and 18 times respectively compared with the wild strain (as shown in Figure 2). Figure 1 C), an average increase of 21 times.
[0047] Example 2 Determination of light resurrection rate and dark resurrection rate of high expression strains
[0048] (1) Determination of dark resurrection rate
[0049] 100 μL of the highly expressed OEBbrad1-1 / 2 strain was added to 7 The suspension of 100 conidia / mL was evenly spread on GM germination plates with a diameter of 7 cm, dried aseptically for 10 minutes, and placed on Bio-Sun ++ The sample tray of the UV radiation box was irradiated with UVB at a weighted wavelength of 312 nm at 0.1 to 0.8 J / cm 2 The gradient UVB dose irradiation (with the unirradiated plate as the control) was repeated three times. The error of each radiation dose was controlled by a microprocessor embedded in the radiation box within 1μJ / cm 2 (10 -6 ), the processor automatically adjusts the UVB intensity and wavelength 4 times per second (manufacturer's guidelines), and the irradiation time for each dose does not exceed 5 minutes. The irradiated plates are immediately covered and incubated in the dark at 25°C for 12 or 24 hours. The number of germinated and non-germinated conidia in three fields of view of each plate is observed and counted under a microscope. The ratio of the germination rate of conidia after irradiation to the germination rate of conidia in the control group is defined as the conidia survival index Is (0≤Is≤1). The trend of Is that varies with the UVB radiation dose (d) is fitted with the modified logistic equation Is=1 / [1+exp(a+rd)], where a and r are two parameters to be fitted. When Is is 0.75 and 0.5 respectively, the solution of the fitting equation can calculate the dark resurrection of conidia 75% (LD 25 ), 50% (LD 50 ) was used as an indicator to evaluate the dark resurrection characteristics of conidia.
[0050] The trend of dark resurrection rate of conidia of high-expression strain OEBbrad1-1 / 2 and wild strain after gradient UVB dose irradiation at 12 and 24 h in dark culture with UVB dose, as shown in Figure 2. Figure 2 A and B show the LD of wild-type strains. 25 and LD 50 In the 12 h dark treatment, the values were 0.058 and 0.105 J / cm, respectively. 2 (like Figure 3 A), and increased to 0.153 and 0.217 J / cm, respectively, in the dark for 24 h. 2 (like Figure 3 B), compared with the wild-type strain, the LD of the high-expressing strain in the above treatments 25 and LD 50 All of them have been improved to varying degrees. 50 For example, the overexpression strain OEBbrad1-1 / 2 increased by about 20% and 27% respectively compared with the wild-type strain in 12h dark treatment, and increased by about 27% in 24h dark treatment. However, the dark resuscitation rate of the overexpression strain after high-dose UVB radiation did not increase significantly. 2 After irradiation with a high lethal dose and cultured in the dark for 24 hours, the dark resurrection rate of the OEBbrad1 strain was only about 4% higher than that of the wild strain.
[0051] (2) Determination of photoreactivation rate
[0052] The conidia of the highly expressed strain OEBbrad1-1 / 2 and the wild strain were isolated at 0.1-0.8 J / cm 2 After irradiation with graded UVB doses, the conidia were cultured in the light for 3 or 5 h and then in the dark for 9 or 7 h (L:D 3:9 or 5:7, simulating field conditions) and 21 or 19 h (L:D 3:21 or 5:19). Other steps were the same as (1) to evaluate the photoreactivation characteristics of the conidia of each strain.
[0053] The results showed that the photoreactivation rate of the strain with high expression of Bbrad1 gene was significantly higher than that of the wild type strain, especially after high dose of UVB radiation. Figure 4 A) and L:D 5:7 (as Figure 4 B) treatment, 0.45 J / cm 2 The conidia of the wild strain irradiated with UVB doses did not show photoreactivation (germination), while the OEBbrad1 strain still retained about 7.7% and 8.5% photoreactivation rates. Figure 5 A) and L:D 5:19( Figure 5 B) During treatment, the upper limit of UVB tolerance of wild strains was 0.7 J / cm 2, under this radiation dose, the OEBbrad1 strain still retained about 15% and 26% photoreactivation rates.
[0054] The model fitting analysis showed that the photoreactivation rate of OEBbrad1 strain changed with the UVB dose in the above L:D treatments. Compared with the wild-type conidia, the OEBbrad1 strain had a higher photoreactivation rate in the L:D 3:9 treatment (such as Figure 6 A), L:D 5:7 treatment (as shown Figure 6 B), L:D 3:21 treatment (as shown Figure 6 C), L:D 5:19 treatment (as shown Figure 6 D) corresponds to the LD 25 and LD 50 There are significant improvements. 50 For example, the high-expression strain OEBbrad1-1 increased by about 13%, 13%, 20% and 14% after the above light treatments compared with the wild-type strain, and OEBbrad1-2 increased by about 18%, 15%, 21% and 17%.
[0055] The results showed that high expression of the Bbrad1 gene significantly improved the photoreactivation activity of Beauveria bassiana, especially increasing the upper limit of the fungus's tolerance to UVB radiation, and extending the dark culture time after light treatment would enhance the photoreactivation effect.
Claims
1. The use of genetically engineered bacteria in enhancing the ultraviolet radiation resistance of Beauveria bassiana, characterized in that: The Bbrad1 gene is highly expressed in the genetically engineered bacteria, which specifically increases the upper limit of the tolerance of Beauveria bassiana to UVB radiation under photorepair conditions, thereby enhancing the ultraviolet radiation resistance of Beauveria bassiana.
2. The use according to claim 1, characterized in that The accession number of the Bbrad1 gene in the NCBI database is BBA_07749.
3. The use according to claim 1, characterized in that The genetically engineered bacteria has a conidia photoreactivation rate of ≥15% after being irradiated with a lethal dose of UVB under the illumination condition of L:D 5:
19.
4. A method for enhancing the ultraviolet radiation resistance of Beauveria bassiana, characterized in that: High expression of the Bbrad1 gene in Beauveria bassiana enhances its ability to resist ultraviolet radiation.
5. The method according to claim 4, characterized in that The specific steps for overexpressing the Bbrad1 gene in Beauveria bassiana are as follows: (1) Using the cDNA genome of Beauveria bassiana as a template, a cDNA fragment of the Bbrad1 gene was cloned; (2) Connecting the cDNA fragment obtained in step (1) to an expression vector by homologous recombination to construct a high expression vector; (3) The high expression vector was transferred into the wild strain of Beauveria bassiana by using the Agrobacterium-mediated method, and the strain was screened by PCR and RT-qPCR to obtain a Beauveria bassiana strain that highly expressed the Bbrad1 gene.
6. The method according to claim 4, characterized in that In step (1), the primers used to amplify the cDNA fragment of the Bbrad1 gene are shown in SEQ ID NO. 1-2.
7. The method according to claim 4, characterized in that In step (2), the expression vector is the plasmid p0380-Ptef1-sur linearized by XmaI / BamHI enzyme digestion.
8. The method according to claim 4, characterized in that In step (3), the Agrobacterium is Agrobacterium tumefaciens AGL1.