System and method for recycling selection marker of gibberellic gibberellic acid based on inducible recombinase

Abstract

The invention provides a gibberellic gibberellic selection marker recovery system and method based on inducible recombinase. According to the system, recombinase expression is guided through an inducible promoter, recombinase recognizes a specific recombination site to carry out recombination recovery on a selection marker, and it is guaranteed that no resistance gene residues exist on a genome. Experiments prove that the screening marker can be recycled and reused, the problems that the variety of resistance genes in gibberellin is few and the reusability is poor are solved, and a feasible genetic engineering strategy is used.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to a screening label recovery method, and more specifically to a screening label recovery system and method for Fumigranobacter fumonis based on inducible recombinase. Background Technology

[0002] Gibberellins (GAs) belong to the tetracyclic diterpenoid family and are a group of ubiquitous plant hormones essential for regulating plant growth. Their wide application in crops, brewing, and horticulture has made them well-established commercial products. Since the first isolation of GAs in 1917, 136 structurally similar gibberellins have been identified from plants, bacteria, and fungi. GA1, GA3, GA4, and GA7 are the main active substances, with GA3 being the most prominent commercial product.

[0003] The industrial production of gibberellins primarily relies on the liquid fermentation of *Fusarium fujikuroi*, a filamentous fungus originally identified as a plant pathogen causing rice bakanae disease. To date, GA3, GA4, and GA7 are mature commercial products produced by *Fusarium fujikuroi* fermentation. To achieve efficient industrial-scale production, constructing genetically stable, high-yielding gibberellin-producing engineered strains is crucial. Over the past decade, the gibberellin biosynthesis pathway of *Fusarium fujikuroi* has been fully elucidated, and genetic engineering tools have been developed, becoming a new means of obtaining high-yielding strains. However, genetic engineering research relies on the introduction of selection tags, and multiple iterations of genetic modification require the efficient recovery of used selection tags to release genomic manipulation sites and maintain genetic stability. Currently, a universal technical system for the precise and efficient removal of selection tags from *Fusarium fujikuroi* is still lacking. Summary of the Invention

[0004] To address the lack of existing methods for the recovery of screening markers for Fusarium oxysporum, this invention provides a Fusarium oxysporum screening marker recovery system and method based on inducible recombinase, which can recover screening markers by recombination at specific sites through induced expression of recombinase.

[0005] The technical solution adopted in this invention is: a Fujikura gibberellic acid (Fujikura) screening label recovery system based on inducible recombinase, comprising an inducible promoter, a site-specific recombinase regulated by the inducible promoter, a screening label, and specific recombination sites located on both sides of the screening label; the site-specific recombinase recognizes the specific recombination sites and cleaves the screening label; the specific recombinase is an Flp recombinase or a Cre recombinase; the specific recombination site is FRT or loxP; the Flp recombinase recognizes the specific recombination site FRT; the Cre recombinase recognizes the specific recombination site loxP.

[0006] This invention is based on the recognition and recombination of specific recombination sites by recombinases. Recombinases can recognize specific sites on the DNA sequence, such as FLP recognizing the FRT site and CRE recognizing the loxP site. These specific sites typically consist of two palindromic sequences and an intermediate spacer sequence. The spacer sequence between the palindromic sequences is asymmetrical, thus defining the orientation of the specific site. The recombinase recognizes the orientation of the specific site and performs cleavage and exchange on the spacer sequence, achieving the purpose of recombination recovery and selection of tags. (Reference) Figure 1 After gene editing of *Fujikura scab* with the aforementioned selection tag recovery system, an inducible promoter is used to guide recombinase expression. The recombinase recognizes specific recombination sites located flanking the selection tag and recovers the tag through recombination. For example, a tetracycline-induced Tet-on system is used to express the recombinase FLP, recognizing the FRT sites flanking hygromycin resistance, recovering hygromycin resistance through recombination for use in the next gene editing. By recovering the selection tag, the engineered strain loses antibiotic resistance or reforms auxotrophic traits, making it usable in the next gene editing.

[0007] Preferably, the inducible promoter is a chemically induced promoter, which includes the nitrogen-starved promoter glnA, the ethanol-responsive promoter alcA, the tetracycline-responsive promoter Tet-on, or the Tet-off.

[0008] Preferably, the screening markers include antibiotic resistance genes and auxotrophic complement genes. The antibiotic resistance genes include the hygromycin resistance gene *hph*, the herbicides resistance gene *nptII*, the northerly styramine resistance gene *nat1*, and the bleomycin resistance gene *bleo*. The auxotrophic complement genes include the lysine auxotrophic complement gene *ppt1*, the uracil auxotrophic complement gene *pyr4*, and the nitrate auxotrophic complement gene *niaD*.

[0009] This invention also provides a method for screening, labeling, and recovering Fusarium oxysporum based on inducible recombinase, comprising the following steps:

[0010] S1. Construct a plasmid containing the target gene expression cassette and the described Fujikura gibberellin selection marker recovery system;

[0011] S2. The plasmid constructed in step S1 was transformed into Gibberella fuciformis protoplasts using the protoplast chemical transformation method to obtain transformants;

[0012] S3. Induce the expression of site-specific recombinase in transformants to obtain engineered Fumigranorhizium without selection markers.

[0013] Preferably, the method includes the following steps:

[0014] S1. Construct a plasmid containing a target gene expression cassette and the system described above; the system includes a tetracycline-responsive promoter Tet-on, an Flp recombinase regulated by the tetracycline-responsive promoter Tet-on, a hygromycin resistance gene hph, and specific recombination sites FRT located on both sides of the hygromycin resistance gene hph.

[0015] S2. The plasmid constructed in step S1 was transformed into Gibberella fuciformis protoplasts using the protoplast chemical transformation method to obtain transformants;

[0016] S3. Add tetracycline to induce transformants to express Flp recombinase and obtain engineered Fuchsia fucoidan strain that does not contain the hygromycin resistance gene hph.

[0017] The beneficial effects of this invention are:

[0018] This invention provides a system and method for recovering selection markers from *Fujikura fusarium* using inducible recombinase. The system guides recombinase expression via an inducible promoter, and the recombinase recognizes specific recombination sites to recover the selection markers, ensuring no residual resistance genes remain on the genome. Experiments have shown that this method allows for the recovery and reuse of selection markers, solving the problems of limited types of resistance genes and poor reusability in *Fujikura fusarium*, and represents a feasible genetic engineering strategy. Attached Figure Description

[0019] Figure 1 This is the technical approach of the present invention.

[0020] Figure 2 This is a gel image of colony PCR verification in the protoplast preparation method of Example 1 of the present invention; lanes a, b, c, and d are PCR products of transformant colonies, of which lanes a and d are positive.

[0021] Figure 3 This is a comparison of bacterial colonies between resistant plates with successfully recovered resistance labels and plates without resistance labels in Example 1 of the present invention.

[0022] Figure 4 This is a gel image of colony PCR verification during the screening marker recovery verification in Example 1 of the present invention; lanes a, b, and c are colony PCR products, of which lanes b and c are negative. Detailed Implementation

[0023] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. Unless otherwise specified, the methods used in the embodiments of the present invention are conventional methods, and the reagents used are commercially available.

[0024] Example 1

[0025] I. Protoplast Preparation Method

[0026] Using a sterile inoculation spatula, a 1 cm² cell block was scooped from the slant culture medium in an eggplant flask and transferred to 30 mL of YEPD medium (YEPD medium: 30 g / L yeast extract, 10 g / L peptone, 20 g / L glucose, solvent: water, sterilized at 115°C for 30 min), and incubated at 250 rpm and 28°C for 2 days. The cells were then collected by Miracloth filtration and washed with 0.8 M NaCl solution. The cells were resuspended in 10 mL of enzyme solution (containing 150 mg of enzyme, snail enzyme, and yatalase 1:2, dissolved in 10 mL of 0.8 M NaCl solution; the mixture was sterilized using a microporous membrane) to enzymatically hydrolyze the cell walls. This mixture was incubated in a water bath at 28°C and 150 rpm for 3 hours, manually inverted every 30 minutes. Finally, the hydrolysis product was filtered through a Miracloth to collect the protoplasts. The protoplast supernatant was then centrifuged at 900×g for 5 minutes to remove insoluble particles. The particles containing protoplasts were collected and washed twice with STC solution. Finally, the protoplasts were resuspended in STC solution to a concentration of 1×10⁻⁶. 7 Cells / mL, stored at 4°C for later use.

[0027] II. Overexpression of key gibberellin synthesis genes using protoplast chemical transformation method

[0028] 1. A plasmid was constructed with pUC19 as the backbone, containing a gpdA structural strong promoter-guided expression cassette of the endogenous gene ggs2 from Fumigranobacter fumonis, a Tet-on-guided tetracycline-inducible FLP recombinase expression cassette, and a hygromycin expression cassette flanked by FRT-specific recognition sites. The sequence is shown in SEQ ID NO. 1.

[0029] 2. Add the system according to Table 1 below.

[0030] Table 1. Protoplast Transformation System

[0031] Group protoplast plasmids PEG 6000 STC experimental group 160 oz 100 oz 60 oz / Positive control group 160 oz / 60 oz 100 oz negative control group 160 oz / 60 oz 100 oz

[0032] STC solution: Weigh 14.56 g of sorbitol, 0.6 g of Tris-base, and 0.5548 g of CaCl2, dissolve in 80 mL of ultrapure water, adjust the pH to 7.5 with HCl, and bring the volume to 100 mL. Autoclave at 121°C for 20 min.

[0033] 3. Place the 2 mL EP tubes containing the mixture on ice, invert them every 10 min to mix, and after 30 min, add 1.5 mL of 60% PEG6000 solution to each tube, mix thoroughly, and let stand at room temperature for 25 min.

[0034] 4. Add 6 mL of MYG soft agar medium, 3 mL of STC buffer, and antibiotic stock solution to a 15 mL centrifuge tube (do not add antibiotic stock solution to the medium used for the positive control group). Then, add the mixture from the 2 mL EP tube to the 15 mL centrifuge tube and mix well. Divide the mixture from the 15 mL centrifuge tube into two portions and pour them onto two MYG medium plates (MYG solid medium: yeast extract 5 g / L, maltose monohydrate 5 g / L, sucrose 171 g / L, glucose 10 g / L, agar 20 g / L; soft agar medium: agar 10 g / L, natural pH, autoclaved at 115℃ for 30 min). Gently rotate the plates to spread the soft agar evenly. Incubate at 28℃ for 3-7 days.

[0035] 5. Pick transformants and transfer them to PDA solid medium (PDA solid medium: potato 200 g / L, glucose 20 g / L, agar powder 20 g / L, solvent: water, sterilized at 115℃ for 30 min) and continue culturing for 2-4 days. Colony PCR is then used to verify successful transformation. Figure 2 ).

[0036] 6. Preparation of seed culture for engineered strains: Select a single colony that has grown on the plate in step 5 and has been verified, and inoculate it into seed culture medium. Incubate at 28℃ and 250 rpm for 48 h to obtain seed culture. Seed culture medium: corn starch 20 g / L, sucrose 15 g / L, peanut powder 15 g / L, soybean powder 3 g / L, KH2PO4 1 g / L, MgSO4 1 g / L, water as solvent, sterilize at 121℃ for 20 min.

[0037] 7. Fermentation culture of engineered strains: 40 mL of fermentation medium was placed in a 250 mL shake flask. Seed culture was inoculated at a concentration of 6% (v / v) and fermented at 28℃ and 250 rpm for 7 days. The fermentation medium consisted of: corn starch 75 g / L, rice flour 87.5 g / L, soybean flour 5 g / L, peanut flour 5 g / L, KH₂PO₄ 0.5 g / L, K₂SO₄ 5 g / L, MgSO₄•7H₂O 0.11 g / L, with water as the solvent. The medium was sterilized at 121℃ for 20 min. After 7 days, the supernatant was collected, appropriately diluted, and GA was determined by high-performance liquid chromatography (HPLC). 4+7 Production.

[0038] III. Tetracycline-induced Tet-on expression recombinase recovery and screening markers

[0039] 1. Tetracycline-induced Tet-on expression of FLP recombinase recovery and screening markers: The engineered strain with the highest gibberellin yield in Example 2 was selected and inoculated into YEPD medium. Tetracycline was added to a final concentration of 1 μg / mL, and the culture was carried out at 28℃ and 250 rpm for 48 h to induce the expression of recombinase FLP. The cultured bacterial cells were prepared into protoplasts according to the above method, and after appropriate dilution, they were revived on MYG medium.

[0040] 2. Screening marker recovery verification: The revived single colonies from the previous step were picked and placed onto PDA solid medium and PDA solid medium supplemented with hygromycin resistance, respectively. For example... Figure 3 As shown, after tetracycline-induced Tet-on expression and FLP recombinase recovery and screening, the engineered strain could not grow on hygromycin-resistant PDA solid medium. Colonies that could grow on non-resistant PDA solid medium but not on resistant PDA solid medium were selected for further colony PCR verification of the loss of the hygromycin resistance gene. Figure 4 Experiments have shown that this method can recover screening tags and reuse them in subsequent gene editing, making it a practical and feasible gene editing tool.

[0041] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A screening, labeling, and recovery system for *Fujikura scab* based on inducible recombinase, characterized in that, This includes inducible promoters, site-specific recombinases regulated by inducible promoters, selection markers, and specific recombination sites located on either side of the selection markers; The site-specific recombinase recognizes the specific recombination site and cleaves the selection marker; The specific recombinase is either Flp recombinase or Cre recombinase; The specific recombination site is FRT or loxP; The Flp recombinase recognizes a specific recombination site, FRT. The Cre recombinase recognizes a specific recombination site loxP.

2. The system as described in claim 1, characterized in that, The inducible promoter is a chemically induced promoter.

3. The system as described in claim 2, characterized in that, The chemically induced promoters include the nitrogen-starved promoter glnA, the ethanol-responsive promoter alcA, and the tetracycline-responsive promoters Tet-on or Tet-off.

4. The system as described in claim 1, characterized in that, The screening markers include antibiotic resistance genes and auxotrophic complement genes.

5. The system as described in claim 4, characterized in that, The antibiotic resistance gene includes the hygromycin resistance gene. hph Genetic mycotoxin resistance gene nptII Norscin resistance gene nat1 Bleomycin resistance gene bleo .

6. The system as described in claim 4, characterized in that, The auxotrophic complement gene includes the lysine auxotrophic complement gene. ppt1 Uracil auxotrophic complement gene pyr4 Nitrate auxotrophic gene complementation niaD .

7. A method for screening, labeling, and recovering *Fujikura scab* based on inducible recombinase, characterized in that, The steps include: S1. Construct a plasmid containing a target gene expression cassette and the system described in any one of claims 1 to 6; S2. The plasmid constructed in step S1 was transformed into Gibberella fuciformis protoplasts using the protoplast chemical transformation method to obtain transformants; S3. Induce the expression of site-specific recombinase in transformants to obtain engineered Fumigranorhizium without selection markers.

8. The method as described in claim 7, characterized in that, The steps include: S1. Construct a plasmid containing the target gene expression cassette and the system described above; the system includes a tetracycline-responsive promoter Tet-on, an Flp recombinase regulated by the tetracycline-responsive promoter Tet-on, and a hygromycin resistance gene. hph Located in the hygromycin resistance gene hph FRT, the specific recombination sites on both sides; S2. The plasmid constructed in step S1 was transformed into Gibberella fuciformis protoplasts using the protoplast chemical transformation method to obtain transformants; S3. Add tetracycline to induce transformants to express Flp recombinase, obtaining transformants without the hygromycin resistance gene. hph The engineered fungus *Fujikura fusarium*.

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