PiggyBac transposon system, recombinant vector, gene editing method, recombinant strain and application thereof

Abstract

The invention relates to the technical field of microorganisms and application thereof, and discloses a piggyBac transposon system, a recombinant vector, a gene editing method, a recombinant strain and application of the piggyBac transposon system, the piggyBac transposon system contains piggyBac transposase, an inverted terminal repeat sequence, a target load gene sequence and a selective marker gene, the vector gene is a functional marker gene and / or a reporter gene, and the expression of the transposase is controlled through a promoter PtrpC; wherein the amino acid sequence of the piggyBac transposase is as shown in SEQ ID NO. 1; and the piggyBac transposase system also contains a vector plasmid. The gene editing method of the piggyBac transposon system can be suitable for efficiently and accurately inserting exogenous genes into gibberella zeylanica, traceless and residue-free genome modification is realized, the problem of exogenous gene residue is avoided, and the method has relatively good industrial value.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms and their applications, and in particular to a piggyBac transposon system, a recombinant vector, a gene editing method, a recombinant strain and applications thereof. BACKGROUND

[0002] The smooth progress of high-throughput biological screening programs cannot be achieved without tools that can effectively establish genetic diversity. With the in-depth study of microbial genetics in the field of scientific research, more and more researchers realize that Gibberella fujikuroi, as an important model organism, has attracted high attention due to its ability to synthesize gibberellin (GA3) and its wide application prospects in plant growth regulation, crop growth stimulation, and biopharmaceuticals. In order to fully tap the potential of Gibberella fujikuroi in metabolic regulation, secondary metabolite synthesis, and other industrial applications, and to improve its production efficiency and functional diversity, it is particularly important to establish and optimize genetic tools suitable for this host. Especially in the genome function analysis and modification of Gibberella fujikuroi, the diversity and efficiency of genetic tools become the key to research.

[0003] From the perspective of genome structure, the genome of Gibberella fujikuroi is relatively complex. Its genome consists of a large number of repetitive sequences and special gene structures, which makes it difficult to accurately identify and locate the target gene site when performing gene editing.

[0004] In terms of gene expression regulation, the gene expression regulation network of Gibberella fujikuroi is extremely complex, involving the synergistic action of multiple cis-acting elements and trans-acting factors. This means that even if the exogenous gene is successfully introduced into the bacterial body, due to the complexity of the gene expression regulation mechanism, the exogenous gene may not be able to be normally expressed or the expression level may be unstable, making it difficult to achieve the expected gene editing effect.

[0005] The existing gene editing methods of Gibberella fujikuroi have limitations. First, the transposition site has preference, the vector plasmid has potential risks, and the gene expression regulation is complex. Second, the screening marker gene has limitations, affecting the accuracy of screening and the study of edited strains.

[0006] In addition, the growth environment and physiological characteristics of Gibberella fujikuroi also increase the difficulty of gene editing. It has strict requirements for culture conditions, and its cell state and metabolic pathways will change significantly under different growth stages and environmental conditions, making it difficult to determine the best treatment time and experimental conditions when performing gene editing, further limiting the application of traditional gene editing technology in Gibberella fujikuroi.

[0007] Therefore, it is urgent to develop a piggyBac transposon system suitable for G. moniliformis, and use the piggyBac transposon system to perform efficient and accurate exogenous gene insertion, and realize a gene editing method of scarless and residue-free genome modification. SUMMARY

[0008] The purpose of the present application is to overcome the problems of existing gene editing technology, such as difficulty in accurately identifying and positioning target gene sites in G. moniliformis, inability of introduced exogenous genes to express normally or unstable expression level, and inability to realize scarless and residue-free genome modification. The present application provides a piggyBac transposon system, a recombinant vector, a gene editing method, a recombinant strain and applications thereof. The gene editing method not only enables precise gene insertion in G. moniliformis, but also realizes scarless and residue-free genome modification, avoids the problem of exogenous gene residues that may be caused by traditional genetic tools, and thus improves the accuracy and efficiency of genome editing.

[0009] To achieve the above-mentioned purpose, the first aspect of the present application provides a piggyBac transposon system, which contains a piggyBac transposase, a target payload gene, inverted terminal repeat sequences at both ends of the target payload gene, and a selectable marker gene, wherein the expression of the transposase is controlled by a promoter; wherein the amino acid sequence of the piggyBac transposase is shown in SEQ ID NO. 1.

[0010] The second aspect of the present application provides a recombinant vector, which comprises the piggyBac transposon system according to any one of claims 1 to 4.

[0011] The third aspect of the present application provides a genome editing method, characterized in that the method comprises: transferring the recombinant vector as described above into a starting strain in which the selectable marker gene is deleted.

[0012] The fourth aspect of the present application provides a recombinant strain obtained by the gene editing method as described above.

[0013] The fifth aspect of the present application provides an application of at least one of the piggyBac transposon system as described above, the recombinant vector as described above, the gene editing method as described above, and the recombinant strain obtained by the gene editing method as described above in the fermentation production of gibberellins.

[0014] Through the above technical solutions, this invention provides a piggyBac transposon system, recombinant vector, gene editing method, recombinant strain and its application. This gene editing method can not only introduce precise gene insertion into Fusarium oxysporum, but also achieve traceless and residueless genome modification, avoiding the problem of exogenous gene residue that may be caused by traditional genetic tools, thereby improving the accuracy and efficiency of genome editing and having good prospects for industrial application. Attached Figure Description

[0015] Figure 1 This is the gene map of the pFC332-PtrpC-piggyBac plasmid in Example 1; Figure 2 This is the gene map of the vector pUC-U(5'ITR-VPSS66-3'ITR)RA3 in Example 1; Figure 3 This is the gene map of transposon pFC332-PtrpC-piggyBac-[U-(5'ITR-VPSS66-3'ITR)-RA3] in Example 1; Figure 4 This is a diagram showing the growth of colonies successfully transformed by the transposon system in a basic culture medium lacking uracil in Example 2; Figure 5 This is a comparison chart of the GA3 yield of the original *Gibberella fuciformis* strain in Example 3 and the GA3 yield of the *Gibberella fuciformis* strain successfully transformed by the transposon system. In the chart, A represents the GA3 yield of the original *Gibberella fuciformis* strain, and the rest represent the gibberellin yield of the successfully transformed colonies. Detailed Implementation

[0016] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0017] The first aspect of the present invention provides a piggyBac transposon system, the piggyBac transposon system comprising a piggyBac transposase, a target payload gene, inverted terminal repeat sequences at both ends of the target payload gene, and a selective marker gene, wherein the transposase is expressed under the control of a promoter; wherein the amino acid sequence of the piggyBac transposase is shown in SEQ ID NO. 1.

[0018] In the present application, the piggyBac transposase is the core component of the system, which can recognize the TTAA site of the genome and perform cutting and insertion operations, and is responsible for accurately integrating the target payload gene (exogenous gene) into the target genome; the amino acid sequence is shown as SEQ ID NO. 1, and this specific amino acid sequence enables it to specifically recognize and cut the TTAA site of the genome; the target payload gene sequence is the gene fragment that researchers expect to introduce into the target organism (such as Gibberella fujikuroi) genome, which carries specific genetic information and has specific functions; the inverted terminal repeat sequences at both ends of the target payload gene (including the 5' ITR connected to the 5' end of the target payload gene and the 3' ITR connected to the 3' end of the target payload gene) are the recognition sites of the piggyBac transposase, which can specifically recognize the inverted terminal repeat sequence, then cut the transposon containing the target payload gene sequence and the selectable marker gene, and integrate it into the target genome. The presence of inverted terminal repeat sequences ensures the specificity and accuracy of the transposition process; the selectable marker gene is used to screen cells or strains that have successfully introduced the transposon. During the experimental process of the present application, researchers can distinguish which strains have been effectively transformed by using the selectable marker gene.

[0019] For example, the present application can select the URA3 gene as a marker gene, which is involved in the synthesis of uracil, and uses a medium lacking uracil for screening, only the strains successfully introducing the URA3 gene can grow on this medium, thereby realizing the screening of transformed strains; as a functional marker gene and / or reporter gene, the vector gene can help researchers monitor transposition efficiency, screen successful transformants, and study the function of specific genes; the transposase in the present application is controlled by the promoter PtrpC for expression, and the PtrpC promoter can drive the piggyBac transposase to express at the right time and position, ensuring that the transposase plays a role when gene editing is needed, while avoiding unnecessary expression from causing adverse effects on cells.

[0020] According to the present application, preferably, the nucleotide sequence of the coding gene of the piggBac transposase is shown as SEQ ID NO. 2. The piggyBac transposase encoded by the nucleotide sequence shown in SEQ ID NO. 2 is the core component of the transposon system, and its nucleotide sequence is optimized and designed to ensure that the transposase can specifically recognize the inverted terminal repeat sequence and accurately cut and insert the target payload gene, realizing the site-directed editing of the Gibberella fujikuroi genome.

[0021] Preferably, the nucleotide sequence of the inverted terminal repeat sequence 5'ITR is as shown in SEQ ID NO. 3; preferably, the nucleotide sequence of the inverted terminal repeat sequence 3'ITR is as shown in SEQ ID NO. 4.

[0022] According to the present application, preferably, the promoter is selected from at least one of Pgpd, PtrpC, Ptefl and PenoA. More preferably, the promoter is PtrpC. Further preferably, the nucleotide sequence of the promoter PtrpC is as shown in SEQ ID NO. 5. In this preferred embodiment, the coding gene of the piggyBac transposase as previously described is combined with the promoter PtrpC, which is conducive to further efficient expression of the transposase in the starting strain.

[0023] According to the present application, preferably, the target payload gene is selected from at least one of the coding gene VPSS66 of gamma glutamyltranspeptidase and glutathione hydrolase, the coding gene UGA2 of succinate semialdehyde dehydrogenase and the coding gene PDR of map02010 ABC transporter.

[0024] More preferably, the target payload gene is the coding gene VPSS66 of gamma glutamyltranspeptidase and glutathione hydrolase. Further preferably, the nucleotide sequence of VPSS66 is as shown in SEQ ID NO. 6. The nucleotide sequence as shown in SEQ ID NO. 6 is the VPSS66 coding region sequence optimized for the codon bias of Gibberella fujikuroi, which can significantly improve the transcription and translation efficiency of the gene in the host. The Gibberella fujikuroi edited with this gene performs well in the fermentation production of gibberellins. This not only proves the feasibility of selecting VPSS66 as the target payload gene, but also highlights its advantage in improving the yield of gibberellins, providing a new gene editing scheme for the fermentation production of gibberellins.

[0025] According to the present application, preferably, the selectable marker gene is selected from at least one of antibiotic resistance genes, auxotrophic complementation genes, fluorescent protein genes and enzyme activity genes. More preferably, the selectable marker gene is an auxotrophic complementation gene. Further preferably, the selectable marker gene is a uracil synthesis gene URA3. Still further preferably, the nucleotide sequence of the uracil synthesis gene URA3 is as shown in SEQ ID NO. 7. The present application preferably uses the auxotrophic complementation gene (URA3 gene), which is screened for positive strains through metabolic pathway complementation, without the use of antibiotics, meeting the core advantage of the piggyBac transposon system—achieving "traceless, residue-free" genome modification. The auxotrophic marker gene is only used in the screening stage and can be naturally lost through subculture after editing, avoiding permanent integration of exogenous genes, and is more biologically safe and convenient to operate.

[0026] The second aspect of the present application provides a recombinant vector, wherein the recombinant vector comprises the piggyBac transposon system as described above.

[0027] In the present application, the "vector" used in the recombinant vector can be selected from various vectors known in the art, such as various plasmids, cosmids, bacteriophages and retroviruses available on the market. According to the present application, preferably, the expression vector of the recombinant vector is pFC332 plasmid.

[0028] The third aspect of the present application provides a genome editing method, wherein the method comprises: introducing the recombinant vector as described above into the starting strain with the deletion of the selectable marker gene.

[0029] According to the present application, preferably, the construction process of the recombinant vector comprises: S1, connecting the promoter, the coding gene of piggBac transposase and the expression vector I to obtain the recombinant vector I; adding inverted terminal repeat sequences at both ends of the target payload gene, and then connecting the expression vector II and the selectable marker gene to obtain the recombinant vector II; S2, connecting the recombinant vector I and the recombinant vector II constructed in step S1.

[0030] According to the present application, the starting strain can be a prokaryotic cell or a eukaryotic cell, and preferably, in step S2, the starting strain is Gibberella fujikuroi. Fusarium fujikuroi )。

[0031] The fourth aspect of the present application provides a recombinant strain obtained by the genome editing method as described above.

[0032] The fifth aspect of the present application provides the use of at least one of the piggyBac transposon system as described above, the recombinant vector as described above, the genome editing method as described above and the recombinant strain as described above in the fermentation production of gibberellins.

[0033] For example, the gibberellin yield of the constructed recombinant strain in the fermentation process is significantly higher than that of the original Gibberella fujikuroi, which indicates that the recombinant strain construction method can successfully cultivate a high-yield gibberellin strain, thereby providing a high-quality strain resource for the industrial production of gibberellins.

[0034] The present application will be described in detail through examples below. In the following examples, the pFC332 plasmid and the PUC27-ITR plasmid are purchased from Jin Sui; the Gibberella fujikuroi is purchased from the China Center for Type Culture Collection, and the number is CCTCC AF 200026. The rest of the materials and raw materials are all conventional commercial products. Fusarium fujikuroi

[0035] ​Preparation of uracil-free basic culture medium: The culture medium is specified as a uracil-free basic medium (components: glucose 3g / L, YNB 2g / L, (NH4)2SO4 0.5g / L, 20% agar by weight), with the pH adjusted to 4-5 to suit the growth requirements of *Fujikura fujikura*. It is also stated that "0.01-0.1% by weight of Triton X-100 can be optionally added to inhibit excessive mycelial diffusion." It should be emphasized that this formulation can be extended to liquid screening systems (such as shake flask pre-culture).

[0036] YPD liquid medium: glucose 20 g / L, yeast extract 10 g / L, peptone 20 g / L; YPD solid medium: glucose 20 g / L, yeast extract 10 g / L, peptone 20 g / L, 15% agar by weight.

[0037] The transposon system of this invention is transformed into Fumigia fujikuroi ( Fusarium fujikuroi The colonies of the bacteria were fermented, and the gibberellin content was determined by high-performance liquid chromatography (HPLC). The specific steps for determining the gibberellin content using HPLC include: The fermentation broth of successfully transformed *Fujikura scab* was centrifuged at 13,000 rpm for 5 minutes and filtered through a 0.22 μm filter membrane to collect the supernatant. The concentration of GA3 was quantified by high-performance liquid chromatography (HPLC) using a C18 column equipped with a UV detector (Agilent Infinity Labs, USA). The column and guard column were maintained at 40 °C. 40% (w / w) methanol was dissolved in 1 mL of 0.3 mL of phosphoric acid as the mobile phase. 10 μL of the filtered fermentation broth was injected at a constant flow rate of 1 mL / min, and detection was performed at 210 nm. All data in this study represent SD ± mean. The retention time of GA3 was approximately 8 min.

[0038] Example 1: Construction of the piggyBac transpose system The steps for constructing the piggyBac transposer system are as follows: S1. Construct the piggBac transposase expression vector module pFC332-PtrpC-piggyBac: The piggyBac gene fragment was obtained by gene synthesis, and was specifically commissioned from Jin Siwei Company. A plasmid pUC-fFuCas9-HTBNLS-hph was used as a template, and the plasmid pUC-fFuCas9-HTBNLS-hph was commissioned from Jin Siwei and stored in the laboratory of Professor Huang He of Nanjing Normal University. An upstream primer F-PtrpC (having the nucleotide sequence shown in SEQ ID NO. 8) and a downstream primer R-PtrpC (having the nucleotide sequence shown in SEQ ID NO. 9) target the promoter region, and PtrpC promoter sequence is amplified. The PtrpC promoter fragment obtained by amplification is purified, then ligated with the piggyBac gene fragment, and then the pFC332 plasmid digested with PacI is purified to obtain the pFC332-PtrpC-piggyBac plasmid, wherein the gene map of pFC332-PtrpC-piggyBac is as shown in Figure 1 The reaction conditions are incubation at 50°C for 60 minutes. After assembly, the DH5a competent cells are transformed, the bacterial liquid is spread on an LB plate containing Amp + , and cultured at 37°C for 16 hours. Single colonies are picked for colony PCR verification, piggyBac transposase gene specific primers (having the nucleotide sequence shown in SEQ ID NO. 10), and the positive clones are sequenced to confirm that the piggyBac transposase gene sequence and the connection region are correct and accurate, and the construction of the piggyBac vector is finally completed. The pFC332 plasmid digestion system is shown in Table 1, and the primers used in the construction of pFC332-PtrpC-piggyBac are shown in Table 2; Table 1

[0039] Table 2

[0040] S2, construction of transposon payload gene vector module pUC53-U(5'ITR-VPSS66-3'ITR)RA3: A target payload gene sequence (VPSS66) that may affect gibberellin production is constructed. 5'ITR and 3'ITR sequences are added to both ends of the sequence. Gibson assembly method is used to insert URA3 into vector PUC27-ITR to generate vector U (ITR) RA3. Then the payload gene VPSS66 is inserted to generate vector pUC-U(5'ITR-VPSS66-3'ITR)RA3, wherein the gene map of pUC-U(5'ITR-VPSS66-3'ITR)RA3 is as shown in Figure 2The URA3 gene (synthesized by GenScript) was selected as a marker gene to assist in screening positive strains. The 5'ITR and 3'ITR sequences were added, so they were recognized and cut by piggyBac transposase.

[0041] The construction method comprises the following steps: 1) Adding a URA3 screening marker module: designing a URA3 screening marker: designing primers FURA3-up (having the nucleotide sequence shown in SEQ ID NO. 11), RURA3-up (having the nucleotide sequence shown in SEQ ID NO. 12) to amplify the URA3-up gene, and designing primers FURA3-down (having the nucleotide sequence shown in SEQ ID NO. 13), RURA3-down (having the nucleotide sequence shown in SEQ ID NO. 14) to amplify the URA3-down gene. Homologous sequences of ITR are added at both ends of the gene amplification fragment. Designing primers FITR (having the nucleotide sequence shown in SEQ ID NO. 15), RITR (having the nucleotide sequence shown in SEQ ID NO. 16) to amplify the ITR; 2) Using Gibson Assembly to assemble the obtained fragments to obtain a recombinant vector U (ITR) RA3.

[0042] 3) Using the Gibberella genome as a template, designing primers VPSS66F (having the nucleotide sequence shown in SEQ ID NO. 17) and VPSS66R (having the nucleotide sequence shown in SEQ ID NO. 18) to perform PCR amplification and purification to obtain the VPSS66 sequence (having the nucleotide sequence shown in SEQ ID NO. 6), and then cutting with BamH I and purifying to obtain the U (ITR) RA3 to obtain the fragment U (5'ITR-VPSS66-3'ITR) RA3. The Gibson Assembly assembly system is shown in Table 3; Table 3

[0043] X = (0.02 × number of base pairs of the cloning vector) ng (0.03 pmol) Y = (0.02 × number of base pairs of each fragment) ng (0.03 pmol) S3, introducing the gene editing system described above and the gene editing system obtained according to the construction method described above into the microbial cells. As Figure 1As shown, in step S1, the piggyBac transposase cuts off the VPSS66 sequence flanked by piggyBac ITRs and inserts the entire transposon pFC332-PtrpC-piggyBac-[U-(5'ITR-VPSS66-3'ITR)-RA3] (genomic map as shown) into the G. fujikuroi genome TTAA site. If the transposon integration is successful, an insertional mutant is generated, which can be subsequently selected by screening for growth on media without uracil. Figure 3 As shown, in step S1, the piggyBac transposase cuts off the VPSS66 sequence flanked by piggyBac ITRs and inserts the entire transposon pFC332-PtrpC-piggyBac-[U-(5'ITR-VPSS66-3'ITR)-RA3] (genomic map as shown) into the G. fujikuroi genome TTAA site. If the transposon integration is successful, an insertional mutant is generated, which can be subsequently selected by screening for growth on media without uracil.

[0044] S4, the transformed colonies obtained above are inoculated into basic medium without uracil. At this time, if the piggBac transposase gene is removed from the URA3 gene module, the colonies can grow on the basic medium without uracil, indicating that the URA3 gene transformation is successful.

[0045] Preparation of the transformed strain of Example 2 The transformed strain (prepared in step S4 of Example 1) is recovered and cultured in YPD medium containing uracil for 20 hours (28°C, 180 rpm), and then gradient diluted to 10 3 -10 5 CFU / mL, 100-200 μL of which is uniformly spread on the selection medium plate. Control experiments need to be designed (such as untransformed wild-type strain, empty vector transformed strain inoculation in parallel).

[0046] Cultivation conditions and phenotype observation: the specified cultivation temperature is 28°C, humidity > 70%, and light-free cultivation for 5 days, and the colony growth state is recorded daily. The positive clone determination standard is clearly defined as: forming a single circular colony with a diameter > 1 mm, the edge has no aerial hyphae diffusion, and there is a significant difference from the negative control (no growth) (such as shown). It needs to be stated that "abnormal morphology colonies need to be purified by secondary streaking to exclude chimeras". Figure 4

[0047] Example 3: Detection of GA3 content by high performance liquid chromatography Bacterial culture: the positive transformed strain obtained by screening in step S4 of Example 1 is inoculated into seed culture medium (containing 30 g / L sucrose, 2 mM glutamine, pH 5.8) and cultured at 28°C for 7 days. The culture solution is filtered through a 0.22 μm nylon membrane, and an equal volume of acidified methanol (methanol:0.1 M HCl=7:3, v / v) is added for extraction at 4°C in the dark for 24 hours. Centrifugation (12,000 x g, 10 min) is performed to obtain the supernatant, which is concentrated to 1 / 10 of the original volume by nitrogen blowing, and then passed through a C18 solid phase extraction column (Waters Sep-Pak®). The interfering substances are eluted with 30% (w / w) methanol, and the target component is eluted with 80% methanol. Freeze-drying is used for storage. ​

[0048] Determination of GA3 by high performance liquid chromatography: After 7 days of cultivation, 2 mL of fermentation broth was centrifuged at 13,000 rpm for 5 minutes and filtered through a 0.22 μm filter to collect the supernatant. The concentration of GA3 was quantified by high-performance liquid chromatography (HPLC) using a C18 column equipped with a UV detector (Agilent Infinity Labs, USA). The column and guard column were maintained at 40 °C. 10 μL of filtered fermentation broth was injected as the mobile phase, dissolved in 1 mL of 0.3 mL of phosphoric acid, and detected at a constant flow rate of 1 mL / min using 0.3 mL of filtered phosphoric acid. All data in this study represent SD ± mean. The retention time of GA3 was approximately 8 min.

[0049] Under the same culture conditions, the yield of successfully transformed strains was compared with that of the original *Fusarium oxysporum* GA3 strain (e.g., ...). Figure 5 As shown in the figure, where A represents the GA3 yield of the original *Fujikura fujira*, and the rest represent the gibberellin yield of the successfully transformed colonies. The original *Fujikura fujira* yielded 500 mg / L of GA3, while the transformed *Fujikura fujira* yielded a maximum of 4.5 g / L of GA3. Therefore, it is evident that the piggyBac transposon system and gene editing method provided by this invention can introduce precise gene insertions into *Fujikura fujira*.

[0050] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A piggyBac transposon system, characterized by, The piggyBac transposon system contains a piggyBac transposase, a target payload gene, inverted terminal repeat sequences at both ends of the target payload gene, and a selectable marker gene, and the transposase is controlled to express by a promoter; wherein the amino acid sequence of the piggyBac transposase is shown as SEQ ID NO.

1.

2. The piggyBac transposon system of claim 1, wherein, The nucleotide sequence of the coding gene of the piggBac transposase is shown as SEQ ID NO. 2; Preferably, the nucleotide sequence of the 5' ITR of the inverted terminal repeat sequence is shown as SEQ ID NO. 3; Preferably, the nucleotide sequence of the 3' ITR of the inverted terminal repeat sequence is shown as SEQ ID NO.

4.

3. The piggyBac transposon system according to claim 1 or 2, wherein The promoter is selected from at least one of Pgpd, PtrpC, Ptef1 and PenoA; preferably PtrpC; Preferably, the nucleotide sequence of the promoter PtrpC is shown as SEQ ID NO. 5; Preferably, the target payload gene is selected from at least one of the coding genes of gamma-glutamyl transpeptidase and glutathione hydrolase VPSS66, the coding gene of succinate semialdehyde dehydrogenase UGA2 and the coding gene of map02010 ABC transporter PDR; Preferably, the target payload gene is the coding gene of gamma-glutamyl transpeptidase and glutathione hydrolase VPSS66; Preferably, the nucleotide sequence of the VPSS66 is shown as SEQ ID NO.

6.

4. The piggyBac transposon system according to claim 1 or 2, wherein The selectable marker gene is selected from at least one of antibiotic resistance genes, auxotrophic complementation genes, fluorescent protein genes and enzyme activity genes; Preferably, the selectable marker gene is an auxotrophic complementation gene; Preferably, the selectable marker gene is a uracil synthesis gene URA3; Preferably, the nucleotide sequence of the uracil synthesis gene URA3 is shown as SEQ ID NO.

7.

5. A recombinant vector, characterized in that, The recombinant vector comprises the piggyBac transposon system of any one of claims 1 to 4.

6. The recombinant vector of claim 5, wherein, The expression vector of the recombinant vector is pFC332 plasmid.

7. A method of genome editing, comprising, The method comprises: transferring the recombinant vector of claim 5 or 6 into a starting strain with a deletion of the selectable marker gene.

8. The method of claim 7, wherein, The construction process of the recombinant vector comprises: S1, connecting a promoter, a coding gene of piggBac transposase and an expression vector I to obtain a recombinant vector I; adding inverted terminal repeat sequences at both ends of the target payload gene, and then connecting with an expression vector II and a selectable marker gene to obtain a recombinant vector II; S2, connecting the recombinant vector I and the recombinant vector II constructed in step S1; Preferably, the starting strain is a Magnaporthe grisea strain in which the selective marker gene is deleted Fusarium fujikuroi ).

9. The recombinant strain obtained by the genome editing method of claim 7 or 8.

10. The use of at least one of the piggyBac transposon system of any one of claims 1 to 4, the recombinant vector of claim 5 or 6, the genome editing method of claim 7 or 8, and the recombinant strain of claim 9 in the fermentation production of gibberellins.

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