Beauveria bassiana engineering strain Bb-dsMucin for expressing dsRNA as well as construction method and application of Beauveria bassiana engineering strain Bb-dsMucin

Abstract

The invention provides a beauveria bassiana engineering strain Bb-dsMucin for expressing dsRNA (double-stranded ribonucleic acid) as well as a construction method and application of the beauveria bassiana engineering strain Bb-dsMucin. The beauveria bassiana is a beauveria bassiana QSE-F1 strain, and the strain can be colonized at the tomato leaves. The dsRNA can target a botrytis cinerea key gene BcMucin, and the expression of the BcMucin gene is silenced in an RNAi (Ribonucleic Acid Interference) mode. According to the engineering strain, a carrier containing a target gene sequence is transferred into a beauveria bassiana QSE-F1 strain by utilizing an ATMT technology, and the engineering strain Bb-dsMucin capable of synthesizing dsRNA of targeted BcMucin is obtained. The invention also provides an application of the engineering strain Bb-dsMucin, and the application is that the engineering strain Bb-dsMucin can interfere with the expression of a target gene BcMucin, so that the botrytis cinerea can be effectively prevented and treated. Experiments prove that the provided engineering strain Bb-dsMucin can be used for preparing biological agents for preventing and treating gray mold and has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of crop disease prevention and control genes, and specifically relates to an engineering strain of Beauveria bassiana Bb-dsMucin expressing dsRNA, and a construction method and application thereof. Background Art

[0002] RNA interference (RNAi) technology is a cutting-edge gene regulation technology that can interfere with the expression of target genes through small interfering RNA (siRNA) produced by double-stranded RNA (dsRNA), and has potential application value in the fields of medicine and agriculture. RNA pesticides developed based on RNAi technology use dsRNA that specifically targets and interferes with the expression of key genes of pests as an active ingredient. They have the advantages of strong targeting, excellent efficacy, high environmental and ecological safety, and low resistance to pesticides. They are considered to be the "third revolution" in the history of pesticide development. RNA pesticides use biological macromolecules as active ingredients and are generally considered to be classified as biological pesticides. They meet the needs of green and sustainable agricultural development and have broad development prospects and application value. At present, there are two main approaches to the application of dsRNA in the agricultural field. One is to obtain transgenic plants expressing dsRNA through plant genetic transformation technology, and use dsRNA of the target gene of the pest expressed by the host to interfere with the expression of the target gene, that is, host-induced gene silencing (HIGS) technology; the other is to silence the target gene by directly spraying dsRNA in vitro, that is, exogenous spray-induced gene silencing (SIGS) technology. However, due to the limitations of the lack of mature genetic transformation systems for some crops, poor stability of dsRNA in the environment, low dsRNA delivery efficiency, and high cost of dsRNA synthesis and purification, these two application approaches cannot meet the application and promotion of RNAi technology in the field of plant protection. In order to achieve more effective use of RNAi in plant disease and insect pest control, it is necessary to find new dsRNA application approaches.

[0003] The use of microorganisms such as Escherichia coli and Saccharomyces cerevisiae to synthesize dsRNA can greatly reduce the production cost of dsRNA. However, dsRNA synthesized by these microorganisms often requires a later purification process, resulting in dsRNA loss and increased production costs. Using endophytes that are dominantly colonized in plant tissues as carriers, engineering strains expressing dsRNA are constructed, and the engineering strains are directly used in the form of live bacterial agents. While using endophytes to efficiently synthesize dsRNA, dsRNA is continuously released to plants, which can effectively solve the problems of high dsRNA synthesis cost, low delivery efficiency, poor stability, and short application intervals. It has great research and application value in the development and field application of RNA pesticides.

[0004] Beauveria bassiana is a dual biocontrol fungus that can parasitize hundreds of insect species and be used to control pests; it can also colonize in a variety of plants such as corn and tomatoes, and has multiple functions such as promoting plant growth and improving plant disease resistance. Beauveria bassiana is a widely used biocontrol fungus with a good research foundation for its biological characteristics. At the same time, Beauveria bassiana has the advantages of being easy to culture, easy to scale up, and having low production costs. It meets the requirements of being a chassis microorganism to construct an engineered strain expressing dsRNA and has potential application value. Summary of the invention

[0005] The present invention aims to solve the above problems and provides an engineered strain of Beauveria bassiana Bb-dsMucin expressing dsRNA and a construction method and application thereof. The engineered strain Bb-dsMucin utilizes the Beauveria bassiana QSE-F1 strain to express dsRNA of the target gene BcMucin gene of Botrytis cinerea. The engineered strain can effectively interfere with the expression of the target gene BcMucin and has a significant control effect on tomato gray mold.

[0006] To achieve the above invention objectives, the present invention is implemented through the following scheme:

[0007] The invention provides an engineered strain of Beauveria bassiana Bb-dsMucin expressing dsRNA. The engineered strain of Beauveria bassiana Bb-dsMucin is obtained by transferring a dsRNA expression framework capable of interfering with the expression of a target gene BcMucin into Beauveria bassiana. The sequence of the dsRNA expression framework is shown in SEQ ID No. 1.

[0008] Furthermore, the Beauveria bassiana is a QSE-F1 strain, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241661.

[0009] Furthermore, the dsRNA can target BcMucin, a key target gene of Botrytis cinerea.

[0010] The present invention provides a method for constructing the Beauveria bassiana engineered strain Bb-dsMucin, which comprises the following steps:

[0011] (1) The dsRNA expression framework is composed of arm sequences with the same sequence at both ends and opposite directions and a central sequence in the middle that acts as a connector to construct the dsRNA expression framework;

[0012] (2) Connecting the dsRNA expression frame to the multiple cloning site of the pDHt-Bar vector containing the constitutive promoter oliC suitable for Beauveria bassiana to construct a plasmid containing the dsRNA expression frame;

[0013] (3) The successfully constructed plasmid containing the dsRNA expression framework was transferred into the Agrobacterium tumefaciens strain, and then the dsRNA expression framework was transferred into Beauveria bassiana using the ATMT technology. Positive transformants were screened using a resistance plate containing herbicide to obtain the engineered bacteria Bb-dsMucin strain.

[0014] Furthermore, the arm sequence in step (1) is a partial coding sequence of the BcMucin gene, the sequence is shown in SEQ ID No. 2, and the central sequence is shown in SEQ ID No. 3.

[0015] The invention also provides application of the engineering strain Bb-dsMucin in preventing and controlling tomato gray mold.

[0016] Furthermore, during application, the spore suspension of the engineered strain Bb-dsMucin is evenly sprayed on the entire tomato plant.

[0017] Furthermore, the spore suspension of the engineered strain Bb-dsMucin is prepared by inoculating the engineered strain Bb-dsMucin into a YEPD liquid culture medium, shaking and culturing at 25°C and 180 rpm for 4 days, filtering the culture solution through 3 layers of clean paper to remove mycelium, counting the spores in the filtrate, and diluting the spore suspension with clean water to a spore concentration of 10 6 -10 7 cfu / ml before use.

[0018] Furthermore, the engineered strain Bb-dsMucin can effectively inhibit the expression of the target gene BcMucin of Botrytis cinerea, thereby inhibiting the infection of Botrytis cinerea.

[0019] Furthermore, the engineered strain Bb-dsMucin controls gray mold, which is reflected in that the lesions produced by gray mold infection are significantly reduced, and the biomass of gray mold on the lesions is significantly reduced.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects: the present invention utilizes plant endophytes to express dsRNA of key target genes of pathogens, which can not only produce dsRNA at low cost, but also utilize the colonization of endophytes in plants to continuously release dsRNA to exert its effects. The engineered strain of Beauveria bassiana Bb-dsMucin provided by the present invention can effectively interfere with the expression of BcMucin, a key target gene of Botrytis cinerea, thereby effectively preventing and controlling the occurrence of gray mold. The Bb-dsMucin strain provided by the present invention can be used for the creation of new biopesticides, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of constructing an expression framework for expressing dsRNA using engineered bacteria.

[0022] Figure 2 is a map of the pDHt-Bar vector.

[0023] Figure 3 It is the PCR test result of the positive transformant of the engineering strain, in which ITS is used as the internal reference gene of the strain.

[0024] Figure 4 The dsRNA expressed by the engineering strain was detected by dot blot experiment, and the dsRNA-specific antibody J2 was used for hybridization detection.

[0025] Figure 5 The dsRNA expressed by the engineered strain was detected by fluorescence quantitative PCR experiment, in which BcActin was used as an internal reference gene.

[0026] Figure 6 The interference effect of the engineered strain Bb-dsGFP on GFP protein expression was detected by western blot experiments, and the GFP protein level in Botrytis cinerea hyphae was detected by hybridization using GFP-specific antibodies.

[0027] Figure 7 This is the control effect of the biocontrol engineering strain Bb-dsMucin on tomato gray mold, with the Bb-dsGFP strain as a control.

[0028] Figure 8 The biocontrol engineering strain Bb-dsMucin significantly reduced the biomass of Botrytis cinerea, and the Bb-dsGFP strain was used as a control.

[0029] Fig. 9The biocontrol engineering strain Bb-dsMucin significantly reduced the mRNA level of the target gene BcMucin, and the Bb-dsGFP strain was used as a control. DETAILED DESCRIPTION

[0030] The technical scheme of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. The experimental methods in the following embodiments are conventional methods unless otherwise specified and are not described in detail; for non-conventional experimental operations, the specific steps are described in detail as follows. The experimental materials, drugs, instruments, etc. used in the following embodiments are all commercially available products unless otherwise specified. The quantitative statistics in the following embodiments are all repeated experiments three times and the average value is taken.

[0031] Example 1

[0032] 1. Isolation and screening of QSE-F1 strain

[0033] On October 2, 2022, 5 tomato samples were collected in a greenhouse in Qingdao. A stem of about 1 cm was cut from the main stem of the plant, disinfected with 0.1% sodium hypochlorite for 3 minutes, 70% ethanol for 1 minute, and washed three times with sterile water; then placed in a sterile mortar to grind out the juice, and the juice was diluted with sterile water; 100 μl of the 100-fold and 1000-fold diluted tissue suspensions were taken respectively, and evenly spread on the PDA plate with a spreader (200 g of potato was cut into pieces and boiled in boiling water for 15 minutes, filtered through three layers of gauze, the supernatant was taken, 20 g of glucose, 15 g of agar powder, water was added to 1 L, and autoclaved at 121°C for 20 minutes), and placed in a 28°C incubator for 24-48 hours. Pick a single colony grown on the PDA plate and inoculate it on a new PDA plate to obtain a single colony.

[0034] The purified strain was inoculated into YEPD liquid medium (10g yeast powder, 20g peptone, 20g glucose, add water to 1L, and sterilize at 121℃ for 20min), and cultured at 25℃, 180rpm for 4 days. The culture solution was centrifuged at 12000rpm, and the supernatant was filtered through a 0.45μm sterile filter to obtain a fermentation solution without bacteria. The fermentation solution was mixed with PDA medium and poured onto a plate, and then inoculated with Botrytis cinerea cake, and continued to be cultured in a 25℃ incubator for 2-3 days. The inhibition of Botrytis cinerea hyphae growth was observed, and a strain with a strong inhibitory effect of the fermentation solution on the growth of Botrytis cinerea hyphae was obtained, named QSE-F1.

[0035] 2. Classification and Identification of QSE-F1 Strain

[0036] 1. Morphological identification of strains: The QSE-F1 strain was inoculated on PDA medium and cultured at 28°C. The colonies formed were flat, villi-like, thick in the middle and thin at the edges. The surface of the colonies was milky white at first, and gradually turned light yellow with the increase of culture time. Spores began to be produced after 5-7 days, and the surface of the colonies was milky white powder. QSE-F1 aerial hyphae were picked and observed under a microscope. The hyphae were colorless, transparent and had septa, with a diameter of about 1.73±0.27 μm. Conidia were mostly spherical and oval, with a size of about 1.52±0.17μm - 3.52±0.23 μm.

[0037] 2. Molecular identification: The genomic DNA of QSE-F1 strain was extracted and used as a template for PCR amplification; the PCR reaction system was as follows: 5×TransStart R FastPfu Buffer 10 μl; 10 mM dNTP mix 1 μl; template 2 μl; primer-F (10 μM) 1 μl; primer-R (10 μM) 1 μl; TransStart R FastPfu DNA polymerase 1 μl; ddH2 O 34 μl; Total 50 μl. The PCR reaction procedure was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; 58℃ annealing for 30 s; 72℃ extension for 40 s; 72℃ extension for 5 min; 34 cycles; 4℃ hold.

[0038] After the PCR product was run on the gel, the gel was cut and recovered, and the recovered product was sent to a sequencing company for sequencing. The sequences obtained by sequencing were compared with the sequences in the NCBI database. The sequence of the strain to be tested had the highest homology with that of Beauveria bassiana. Based on the sequence alignment of the gene fragments, a phylogenetic tree was constructed. Through comprehensive analysis, strain QSE-F1 was determined to be Beauveria bassiana.

[0039] The Beauveria bassiana QSE-F1 screened by the present invention is deposited in China Center for Type Culture Collection; address: Wuhan University, Wuhan, China; deposit date: July 22, 2024; the deposit number of Beauveria bassiana QSE-F1 is CCTCC NO: M 20241661.

[0040] Example 2: Preparation of an engineered strain of Beauveria bassiana expressing dsRNA

[0041] 1. Construction of plasmid containing dsRNA expression framework

[0042] The dsRNA expression framework is composed of arm sequences with the same sequence at both ends and opposite directions, and a central sequence in the middle that acts as a connector. The arm sequence is a partial coding sequence of the BcMucin gene or a partial coding sequence of GFP, and the sequences are shown in SEQ ID No.2 and SEQ ID No.4, respectively. The central sequence is the first intron of the endogenous gene BcTubulin (Bcin01g08040) of Botrytis cinerea, and the sequence is shown in SEQ ID No.3. The arm sequences are connected to the two segments of the central sequence in the forward and reverse directions to construct the expression framework of the target gene dsRNA. The specific structure is shown in Figure 1 The sequence of the dsRNA expression frame for expressing BcMucin (BcMucin-dsRNA) is shown in SEQ ID No.1, and the sequence of the dsRNA expression frame for expressing GFP (GFP-dsRNA) is shown in SEQ ID No.5.

[0043] The expression frames BcMucin-dsRNA and GFP-dsRNA were respectively connected to the multiple cloning site (MCS) of the pDHt-Bar vector containing the constitutive promoter oliC suitable for Beauveria bassiana. The map of the pDHt-Bar vector is shown in Figure 2 The vector sequence is shown in SEQ ID No. 6. The pDHt-Bar vector contains a herbicide resistance gene (Bar) and can be used as a selection marker to select transformants.

[0044] 2. Transformation of Beauveria bassiana QSE-F1 strain with plasmid containing dsRNA expression framework

[0045] The plasmids containing GFP-dsRNA and BcMucin-dsRNA expression frameworks were transformed into Agrobacterium tumefaciens AGL1 strain, and then the dsRNA expression framework was transformed into Beauveria bassiana QSE-F1 strain using ATMT technology. Positive transformants were screened using resistance plates containing herbicide (Basta). The transformants were verified by PCR for the target gene, and the results were as follows: Figure 3 As shown, bands of the same size as those on the plasmid were detected in different transformants, but no bands were detected in the wild-type QSE-F1 strain, indicating that the dsRNA expression frame was successfully transferred into the chassis strain. The chassis strains containing the GFP-dsRNA and BcMucin-dsRNA expression frames were named engineered bacteria Bb-dsGFP and Bb-dsMucin strains, respectively.

[0046] Example 3: Detection of dsRNA expressed by engineered strains

[0047] 1. Detection of dsRNA expressed by engineered strains

[0048] Total RNA from the wild-type QSE-F1 strain, Bb-dsGFP strain, and Bb-dsMucin strain of Beauveria bassiana was extracted using Trizol reagent, and dsRNA in the total RNA was detected using a dsRNA antibody (J2) by dot blot experiment. The results are shown in Figure 4 As shown, dsRNA could be detected in the Bb-dsGFP and Bb-dsMucin strains, but not in the QSE-F1 strain, indicating that both strains could successfully express dsRNA.

[0049] Total RNA was reverse transcribed to obtain cDNA, and the target genes GFP and BcMucin were detected by fluorescence quantitative PCR. The results were as follows Figure 5 As shown, the Cq values ​​for amplifying GFP in the Bb-dsGFP strain and BcMucin in the Bb-dsMucin strain were significantly reduced compared with those in the QSE-F1 strain, indicating that the total RNA of the two engineered strains contained RNA of the target gene.

[0050] 2. Detection of the effect of silencing target gene expression in engineered strains

[0051] Conidia of GFP-labeled Botrytis cinerea (B05.10-GFP) were collected and inoculated into three bottles of YEPD liquid culture medium in equal amounts for shaking culture; two of the bottles were added with the collected conidia of the wild-type strain QSE-F1 of Beauveria bassiana and the engineered strain Bb-dsGFP, and the total amount of conidia added was the same. After shaking culture for 2 days, the hyphae of Botrytis cinerea were collected, the total protein of the hyphae was extracted, and the level of GFP in the total protein was detected by western blot, with histone H3 as the internal reference. The results are shown in Figure 6 As shown, compared with the conidia without Beauveria bassiana and the conidia of Beauveria bassiana QSE-F1 strain, the GFP level in the total protein of Botrytis cinerea with the conidia of the engineered strain Bb-dsGFP was significantly decreased, indicating that the engineered strain Bb-dsGFP can effectively interfere with the expression of the GFP gene in B05.10-GFP.

[0052] Example 4: Application of the biocontrol engineering strain Bb-dsMucin in controlling gray mold

[0053] The engineered strains Bb-dsGFP and Bb-dsMucin were inoculated into YEPD liquid medium and cultured at 25°C and 180 rpm for 4 days. The culture solution was filtered through 3 layers of clean paper to remove mycelium, and the spores in the filtrate were counted and diluted with clean water to a spore concentration of 10 6 -10 7cfu / ml to make a spore suspension of the engineered strain. The fermentation stock solution needs to be diluted about 2-4 times, that is, the concentration of the fermentation solution is about 25-50%. Select 4-5 week old tomato seedlings with consistent growth, take 20 leaves with basically consistent growth, randomly divide them into 2 groups, 10 tomato leaves in each group, and place them on moist filter paper. Use a small spray bottle to evenly spray the above-mentioned Bb-dsGFP and Bb-dsMucin spore suspensions respectively. Collect conidia from the PDA plate with gray mold, count and dilute to a conidia concentration of 10 6 After the liquid on the surface of the above-treated leaves dried naturally, 6ul of gray mold spore liquid was inoculated on the leaves. Keep moist for 60-96 hours, observe the gray mold infection, measure the diameter of the lesions by the cross method, and perform statistical analysis on the lesion areas on the two groups of leaves.

[0054] The results are as follows Figure 7 As shown in the figure, the lesions on the leaves treated with the spore suspension of the engineered strain Bb-dsMucin were significantly smaller than those on the leaves treated with the spore suspension of the Bb-dsGFP strain. Leaves with lesions of the same size were taken, total DNA was extracted, and the biomass of Botrytis cinerea in the leaves was detected by fluorescent quantitative PCR. The results are shown in the figure. Figure 8 As shown, the biomass of Botrytis cinerea in the lesions on leaves treated with the spore suspension of the engineered strain Bb-dsMucin was significantly lower than that on leaves treated with the spore suspension of the Bb-dsGFP strain. The above results indicate that the engineered strain Bb-dsMucin has a significant control effect on Botrytis cinerea.

[0055] Mycelium of Botrytis cinerea was collected from the lesions, total RNA was extracted, and cDNA was obtained by reverse transcription. The mRNA level of the target gene BcMucin was detected by fluorescence quantitative PCR. Fig. 9 As shown, after treatment with the engineered strain Bb-dsMucin, the mRNA level of BcMucin, the target gene of Botrytis cinerea, in the leaf lesions was significantly lower than that in the leaves treated with the spore suspension of the Bb-dsGFP strain, indicating that the engineered strain Bb-dsMucin can interfere with the expression of BcMucin.

[0056] Based on all the above results, the engineered strain constructed by transferring the dsRNA expression frame into the chassis fungus QSE-F1 can successfully express dsRNA and effectively interfere with the expression of the target gene. The engineered strain Bb-dsMucin, which expresses dsRNA targeting the key gene BcMucin of Botrytis cinerea, can effectively inhibit the expression of the target gene BcMucin, and thus has a significant control effect on tomato gray mold. It is a new type of biocontrol engineering strain, which can be developed into a biological agent for the effective prevention and control of tomato gray mold.

[0057] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. An engineered strain of Beauveria bassiana Bb-dsMucin expressing dsRNA, characterized in that: The engineered strain of Beauveria bassiana Bb-dsMucin is obtained by transferring a dsRNA expression framework capable of interfering with the expression of the target gene BcMucin into Beauveria bassiana. The sequence of the dsRNA expression framework is shown in SEQ ID No.

1.

2. The engineered strain of Beauveria bassiana Bb-dsMucin according to claim 1, characterized in that: The Beauveria bassiana is a QSE-F1 strain, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241661.

3. The engineered strain of Beauveria bassiana Bb-dsMucin according to claim 1, characterized in that: The dsRNA expression framework contains a partial coding sequence of the target gene BcMucin as an arm sequence, and the arm sequence is shown in SEQ ID No.

2.

4. The method for constructing the Beauveria bassiana engineered strain Bb-dsMucin according to claim 1, characterized in that: The construction method comprises the following steps: (1) The dsRNA expression framework is composed of arm sequences with the same sequence at both ends and opposite directions and a central sequence in the middle that acts as a connector, and a dsRNA expression framework containing the target gene BcMucin is constructed; (2) connecting the dsRNA expression framework to a plasmid containing a gene suitable for transformation into Beauveria bassiana to construct a plasmid containing the dsRNA expression framework; (3) The successfully constructed plasmid containing the dsRNA expression framework was transferred into the Agrobacterium tumefaciens strain, and then the dsRNA expression framework was transferred into Beauveria bassiana using the ATMT method. Positive transformants were screened using a resistance plate containing herbicide to obtain the engineered strain Bb-dsMucin.

5. The construction method according to claim 4, characterized in that: The arm sequence in step (1) is a partial coding sequence of the target gene BcMucin, the sequence is shown in SEQ ID No. 2, and the central sequence is shown in SEQ ID No.

3.

6. The construction method according to claim 4, characterized in that: The Beauveria bassiana in step (2) is a QSE-F1 strain, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20241661.

7. Use of the engineered strain of Beauveria bassiana Bb-dsMucin according to claim 1 in preventing and treating gray mold.

8. The use according to claim 7, characterized in that: The engineered strain Bb-dsMucin is prepared into a spore suspension and evenly sprayed on the entire plant.

9. The use according to claim 8, characterized in that: The preparation method comprises the following steps: inoculating the engineered strain into a YEPD liquid culture medium for shaking culture, filtering the culture medium through filter paper to remove hyphae, and obtaining a spore suspension containing spores.

10. The use according to claim 9, characterized in that: The spore concentration of the engineered strain Bb-dsMucin in the spore suspension containing spores is 10 6 -10 7 cfu / ml, the engineered strain Bb-dsMucin can inhibit the expression of the target gene BcMucin of Botrytis cinerea and effectively inhibit the occurrence of tomato gray mold.

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