An engineered Beauveria bassiana strain Bb-dsMucin expressing dsRNA, its construction method and application

Abstract

The present invention provides a Beauveria bassiana engineering strain Bb-dsMucin expressing dsRNA, its construction method and application. The Beauveria bassiana is the Beauveria bassiana QSE-F1 strain, which can colonize on tomato leaves. The dsRNA can target the key gene BcMucin of Botrytis cinerea and silence the expression of the BcMucin gene by RNAi. The engineering strain is obtained by using the ATMT technique to transfer a vector containing the target gene sequence into the Beauveria bassiana QSE-F1 strain, obtaining the engineering strain Bb-dsMucin capable of synthesizing dsRNA targeting BcMucin. The present invention also provides the application of the engineering strain Bb-dsMucin, and the application is that the engineering strain Bb-dsMucin can interfere with the expression of the target gene BcMucin, thereby effectively controlling tomato gray mold. It is experimentally proven that the provided engineering strain Bb-dsMucin of the present invention can be used to prepare a biological agent for controlling gray mold and has good market application prospects.

Description

Technical Field

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

[0002] RNA interference (RNAi) technology is a relatively advanced gene regulation technology. It can interfere with the expression of target genes through small interfering RNAs (siRNAs) generated by double-stranded RNA (dsRNA), and has potential application value in fields such as 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 the active ingredient, and have the advantages of strong targeting, excellent drug efficacy, high environmental and ecological safety, and low resistance generation. They are considered the "third revolution" in the history of pesticide development. RNA pesticides use biological macromolecules as the active ingredient and are generally considered to fall into the category of biological pesticides, meeting the requirements of green and sustainable agricultural development, and having broad development prospects and application value. At present, there are mainly two ways to apply dsRNA in the agricultural field. One is to obtain transgenic plants expressing dsRNA through plant genetic transformation technology, and use the dsRNA of the pest target gene expressed by the host to interfere with the expression of the target gene, that is, the host-induced gene silencing (HIGS) technology; the other is to directly spray dsRNA in vitro to silence the target gene, that is, the spray-induced gene silencing (SIGS) technology. However, limited by problems such as the lack of a mature genetic transformation system in some crops, poor stability of dsRNA in the environment, low delivery efficiency of dsRNA, and high synthesis and purification costs of dsRNA, these two application ways cannot meet the application and popularization of RNAi technology in the field of plant protection. In order to realize the more effective utilization of RNAi in the prevention and control of plant diseases and pests, new application ways of dsRNA need to be found.

[0003] Synthesizing dsRNA using microorganisms such as Escherichia coli and Saccharomyces cerevisiae can greatly reduce the production cost of dsRNA. However, the dsRNA synthesized by these microorganisms often requires a subsequent purification process, resulting in the loss of dsRNA and increasing the production cost. Using endophytes that are dominant in plant tissues as carriers to construct engineering strains expressing dsRNA and directly using the engineering strains in the form of live microbial agents can continuously release dsRNA to plants while efficiently synthesizing dsRNA by endophytes, effectively solving problems such as high synthesis cost, low delivery efficiency, poor stability, and short application interval of dsRNA, and having 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 in hundreds of insect bodies for pest control and colonize in various plants such as corn and tomatoes, with 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 on its biological characteristics. At the same time, Beauveria bassiana has advantages such as easy cultivation, easy large-scale production, and low production cost, meeting the requirements for constructing an engineering strain expressing dsRNA as a chassis microorganism and having potential application value. Summary of the Invention

[0005] The present invention aims to solve the above problems and provides an engineering strain of Beauveria bassiana Bb-dsMucin expressing dsRNA, its construction method, and application. The engineering strain 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 engineering 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 realized through the following solutions:

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

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

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

[0010] The present invention provides a method for constructing the Beauveria bassiana engineering strain Bb-dsMucin, and the construction method includes the following steps:

[0011] (1) The dsRNA expression framework is composed of arm sequences with the same sequence and opposite directions at both ends and a central sequence that plays a connecting role in the middle, and the dsRNA expression framework is constructed;

[0012] (2) The dsRNA expression framework is ligated 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 framework;

[0013] (3) The successfully constructed plasmid containing the dsRNA expression framework is transferred into the Agrobacterium tumefaciens strain, and then the dsRNA expression framework is transferred into Beauveria bassiana by the ATMT technique, and positive transformants are screened using a resistance plate containing herbicide to obtain the engineering strain Bb-dsMucin strain.

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

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

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

[0017] Furthermore, the preparation method of the spore suspension of the engineering strain Bb-dsMucin is as follows: inoculate the engineering strain Bb-dsMucin into YEPD liquid medium, shake and culture at 25°C and 180 rpm for 4 days, filter the culture solution through three layers of wiped paper to remove mycelia, count the spores in the filtrate, and dilute with clear water to a spore concentration of 10 6 -10 7 cfu / ml before use.

[0018] Furthermore, the engineering strain Bb-dsMucin can effectively inhibit the expression of the gray mold target gene BcMucin, thereby inhibiting the infection of gray mold.

[0019] Furthermore, the engineered strain Bb-dsMucin controls Botrytis cinerea, as evidenced by significantly reduced lesion sizes caused by B. cinerea infection and significantly decreased biomass of B. cinerea on the lesions.

[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 continuously release dsRNA through the colonization of endophytes in plants to exert its effect. The Beauveria bassiana engineered strain Bb-dsMucin provided by the present invention can effectively interfere with the expression of the key target gene BcMucin of B. cinerea, thereby effectively controlling the occurrence of Botrytis cinerea. The Bb-dsMucin strain provided by the present invention can be used for the creation of novel biological pesticides and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of the construction of the expression framework for expressing dsRNA using an engineered bacterium.

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

[0023] Figure 3 It is the PCR detection result of the positive transformant of the engineered strain, where ITS is used as the internal reference gene of the strain.

[0024] Figure 4 It is the detection of dsRNA expressed by the engineered strain through dot blot experiment, and hybridization detection is performed using the dsRNA-specific antibody J2.

[0025] Figure 5 It is the detection of dsRNA expressed by the engineered strain through fluorescence quantitative PCR experiment, where BcActin is used as the internal reference gene.

[0026] Figure 6 It is the detection of the interference effect of the engineered strain Bb-dsGFP on the expression of GFP protein through western blot experiment, and the GFP protein level in the hyphae of B. cinerea is detected by hybridization using the GFP-specific antibody.

[0027] Figure 7 It is the control effect of the biocontrol engineered strain Bb-dsMucin on tomato Botrytis cinerea, with the Bb-dsGFP strain as the control.

[0028] Figure 8 It is that the biocontrol engineered strain Bb-dsMucin significantly reduces the biomass of B. cinerea, with the Bb-dsGFP strain as the control.

[0029] Figure 9The biocontrol engineering strain Bb-dsMucin significantly reduces the mRNA level of the target gene BcMucin, with the Bb-dsGFP strain as the control. Detailed implementation manners

[0030] The technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. Unless otherwise specified, the experimental methods in the following embodiments are all conventional methods and will not be elaborated; for unconventional experimental operations, the specific steps are described in detail later. 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 three repeated experiments and the average value is taken.

[0031] Example 1

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

[0033] On October 2, 2022, 5 tomatoes were collected from the greenhouse in Qingdao. About 1 cm of stem was cut from the main stem of the plant, disinfected with 0.1% sodium hypochlorite for 3 min, 70% ethanol for 1 min, and washed three times with sterile water; then placed in a sterile mortar and ground into juice, and the juice was serially diluted with sterile water; 100 μl of the tissue suspensions diluted 100-fold and 1000-fold were respectively taken, and evenly spread on PDA plates (200 g of potatoes were cut into pieces and boiled in boiling water for 15 min, the supernatant was taken after filtering with three layers of gauze, 20 g of glucose, 15 g of agar powder, added water to 1 L, autoclaved at 121 °C for 20 min), and cultured in an incubator at 28 °C for 24-48 h. Single colonies grown on the PDA plates were picked and inoculated onto new PDA plates to obtain single colonies.

[0034] The purified strain was inoculated into YEPD liquid medium (10 g of yeast powder, 20 g of peptone, 20 g of glucose, added water to 1 L, autoclaved at 121 °C for 20 min), and cultured with shaking at 25 °C and 180 rpm for 4 days. The culture solution was centrifuged at 12000 rpm, and the supernatant was further filtered through a 0.45 μm sterile filter to obtain a cell-free fermentation broth. The fermentation broth was mixed with PDA medium and poured into plates, and then inoculated with Botrytis cinerea cakes, and continued to be cultured in an incubator at 25 °C for 2-3 d. The inhibition of Botrytis cinerea mycelial growth was observed, and a strain with a strong inhibitory effect on Botrytis cinerea mycelial growth was obtained and named QSE-F1.

[0035] II. Classification and identification of QSE-F1 strain

[0036] 1. Strain morphological identification: The QSE-F1 strain was inoculated onto PDA medium and cultured at 28 °C. The formed colonies were flat, villous, thick in the middle and thin at the edges; the surface of the colonies was initially milky white 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 powdery milky white; Aerial hyphae of QSE-F1 were picked and observed under a microscope. The hyphae were colorless, transparent, septate, and about 1.73 ± 0.27 μm in diameter; The conidia were mostly spherical and oval, with sizes of about 1.52 ± 0.17 μm - 3.52 ± 0.23 μm.

[0037] 2. Molecular identification: Genomic DNA of the 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; ddH2O 34 μl; Total 50 μl. The PCR reaction program was as follows: Pre-denaturation at 95 °C for 5 min; Denaturation at 95 °C for 30 s; Annealing at 58 °C for 30 s; Extension at 72 °C for 40 s; Final extension at 72 °C for 5 min; 34 cycles; Hold at 4 °C.

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

[0039] The Beauveria bassiana QSE-F1 screened in the present invention was preserved. The preservation unit: China Center for Type Culture Collection; Address: Wuhan University, Wuhan, China; Preservation date: July 22, 2024; The preservation 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 a plasmid containing a dsRNA expression cassette

[0042] The dsRNA expression framework is composed of arm sequences with the same sequence and opposite directions at both ends, and a central sequence that plays a connecting role in the middle. The arm sequences are partial coding sequences of the BcMucin gene or partial coding sequences 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 respectively connected forward and backward to both ends of the central sequence to construct the expression framework of the target gene dsRNA, and the specific structure is as Figure 1 shown. The sequence of the dsRNA expression framework (BcMucin-dsRNA) expressing BcMucin is shown in SEQ ID No.1, and the sequence of the dsRNA expression framework (GFP-dsRNA) expressing GFP is shown in SEQ ID No.5.

[0043] The expression frameworks BcMucin-dsRNA and GFP-dsRNA are 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 as Figure 2 shown, and the vector sequence is shown in SEQ ID No. 6. The pDHt-Bar vector contains a herbicide resistance gene (Bar), which can be used as a screening marker to screen transformants.

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

[0045] The plasmids containing the GFP-dsRNA and BcMucin-dsRNA expression frameworks that were successfully constructed were respectively transferred into the Agrobacterium tumefaciens AGL1 strain, and then the dsRNA expression framework was transferred into the Beauveria bassiana QSE-F1 strain using the ATMT technique. Positive transformants were screened using a resistance plate containing herbicide (Basta). The transformants were verified for the target gene by PCR, and the results are as Figure 3 shown. Bands of the same size as those on the plasmid were detected in different transformants, while no bands were detected in the wild-type QSE-F1 strain, indicating that the dsRNA expression framework was successfully transferred into the chassis strain. The chassis strains containing the GFP-dsRNA and BcMucin-dsRNA expression frameworks were respectively named engineering strains Bb-dsGFP and Bb-dsMucin strains.

[0046] Example 3: Detection of dsRNA expression in engineering strains

[0047] 1. Detection of dsRNA expression in engineering strains

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

[0049] The 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 shown. Compared with the QSE-F1 strain, the Cq values of GFP amplification in the Bb-dsGFP strain and BcMucin amplification in the Bb-dsMucin strain were significantly reduced, indicating that the total RNA of the two engineered strains contained the RNA of the target genes.

[0050] 2. Detection of the effect of the engineered strains on silencing the expression of target genes

[0051] Conidia of Botrytis cinerea (B05.10-GFP) labeled with GFP were collected and inoculated into 3 bottles of YEPD liquid medium in equal amounts and cultured with shaking. Conidia of the wild-type strain QSE-F1 of Beauveria bassiana and the engineered strain Bb-dsGFP collected were added to two of the bottles respectively, and the total amount of added conidia was the same. After culturing with shaking for 2 days, the hyphae of Botrytis cinerea were collected, and the total protein of the hyphae was extracted. The level of GFP in the total protein was detected by western blot, with histone H3 as the internal reference. The results were as follows Figure 6 shown. Compared with the group without adding Beauveria bassiana and the group adding conidia of the QSE-F1 strain of Beauveria bassiana, the level of GFP in the total protein of Botrytis cinerea added with conidia of the engineered strain Bb-dsGFP decreased significantly, indicating that the engineered strain Bb-dsGFP could effectively interfere with the expression of the GFP gene in B05.10-GFP.

[0052] Example 4: Application of the biocontrol engineered strain Bb-dsMucin in controlling Botrytis cinerea

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

[0054] The results are as Figure 7 shown. The lesion area on the leaves treated with the spore suspension of the engineered strain Bb - dsMucin is significantly smaller than that on the leaves treated with the spore suspension of the Bb - dsGFP strain. Take leaves of the same size containing lesions, extract total DNA, and detect the biomass of Botrytis cinerea in the leaves by fluorescence quantitative PCR. The results are as Figure 8 shown. The biomass of Botrytis cinerea in the lesions on the leaves treated with the spore suspension of the engineered strain Bb - dsMucin is significantly lower than that on the 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] Collect Botrytis cinerea hyphae from the lesions, extract total RNA, and reverse - transcribe to obtain cDNA, and detect the mRNA level of the target gene BcMucin by fluorescence quantitative PCR. The results are as Figure 9 shown. After treatment with the engineered strain Bb - dsMucin, the mRNA level of the target gene BcMucin of Botrytis cinerea in the leaf lesions is 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 cassette into the chassis bacterium QSE - F1 can successfully express dsRNA and effectively interfere with the expression of the target gene. The engineered strain Bb - dsMucin expressing 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 Botrytis cinerea. It is a new biocontrol engineering strain and can be developed into a biological agent for effective prevention and control of tomato Botrytis cinerea.

[0057] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions required to be protected by the present invention.

Claims

1. A strain of Beauveria bassiana expressing dsRNA ( Beauveria bassiana ) The engineered strain Bb-dsMucin, 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 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) constructing a plasmid containing a dsRNA expression frame by connecting the dsRNA expression frame to a pDHt-Bar vector having a vector sequence of SEQ ID NO: 6 and containing a constitutive promoter oliC suitable for Beauveria bassiana; (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.

4. The construction method according to claim 3, 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.

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

6. The use according to claim 5, characterized in that: The engineered strain Bb-dsMucin is prepared into a spore suspension and evenly sprayed on the entire plant.

7. The use according to claim 6, 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.

8. The use according to claim 7, 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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