Fungus broad-spectrum preservative tolerance gene CDC7 and application thereof

Abstract

The invention discloses a fungal broad-spectrum preservative resistance gene CDC7 and application thereof. The nucleotide sequence of the fungal preservative resistance gene CDC7 is at least one of the following (a), (b) and (c): (a) is as shown in SEQ ID NO: 1; (b) a variant sequence which has more than or equal to 95% sequence similarity with SEQ ID NO: 1 and encodes the same functional protein; and (c) a conservative modification sequence which is obtained by carrying out nucleotide substitution, deletion or increase on SEQ ID NO: 1 but does not change the coded amino acid sequence. The broad-spectrum preservative tolerance is endowed to fungi by regulating and controlling a cell signal channel. By constructing a recombinant vector containing the CDC7 gene and transforming the recombinant vector into a sensitive strain, the tolerance of the transgenic strain to preservatives such as potassium sorbate is remarkably enhanced. According to the invention, the sensitivity of the strain to preservatives can be improved, the CDC7 gene can be applied to other ascomycetes across species, and the defects of long period and low efficiency of traditional screening are overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a fungal broad-spectrum preservative tolerance gene CDC7 and its application. Background Art

[0002] With the development of industries such as food, cosmetics, and pharmaceuticals, preservatives play a crucial role in their production processes. However, the use of preservatives has caused varying degrees of inhibition or killing effects on certain microorganisms (such as fungi). However, some fungi have evolved tolerance to preservatives, especially strong tolerance to commonly used preservatives such as potassium sorbate. These preservative-tolerant fungi pose great safety hazards in the production and storage of food, medicine, and other related industries, affecting product quality and safety.

[0003] Therefore, the discovery of new fungal preservative tolerance genes is of great significance for improving the effectiveness of existing preservatives and their application in preservative development. Currently, genetic engineering technology provides an important way to improve and enhance the tolerance of species through transgenic means.

[0004] A large number of practices have proved that the development and utilization of existing fungal preservative tolerance genes is one of the most effective methods for further developing food preservatives. For the cultivation of traditional preservative-tolerant / preservative-sensitive strains, generally, researchers usually first collect a large number of fungal samples and conduct drug resistance screening to identify fungal strains with preservative tolerance. This breeding process is not only cumbersome but also extremely time-consuming. The classical map-based cloning method uses cross-breeding of preservative-tolerant and preservative-sensitive fungal strains, and then sprays preservatives on a large number of segregating offspring, identifies tolerance, conducts genetic mapping, etc. Finally, cloning is carried out based on precise genetic mapping. However, due to its long cycle, time-consuming and laborious nature, and difficulties in separation and allelism detection, it is not suitable for the need of large-scale cloning and screening of preservative tolerance genes. At the same time, due to the unique genetic and variation patterns of ascomycete fungi genes, the application of this method is also greatly limited. The gene mutation rate in ascomycete fungi is one to two orders of magnitude higher than that in ordinary animals and plants, and the nucleotide polymorphism in gene and non-gene regions in ascomycete fungi is also higher (Wang et al., 2020). In this hypermutation process, a large number of sequences with a high Ti / Tv ratio are generated, and this is no exception in the gene region. Utilizing this characteristic can efficiently screen for preservative tolerance genes in ascomycete fungi.

[0005] The CDC7 gene proposed by the present invention is a novel tolerance gene isolated from a strain of Neurospora crassa that exhibits high tolerance to preservatives. This gene belongs to the MAP kinase phosphokinase type gene family. The full name of MAP kinase is "Mitogen-Activated Protein Kinase", which is an important gene family that mainly participates in the cell signal transduction process and regulates various biological processes such as cell proliferation, differentiation, survival, and migration. MAP kinase usually acts as a part of the intracellular signal transduction pathway to respond to external stimulus signals. They participate in the MAPK signal pathway, which is a cascade reaction pathway with MAP kinase as a key component. MAPK has a cascade activation mechanism, and the characteristic of its signal pathway is to form a cascade reaction through the phosphorylation of a series of kinases. Usually, the MAPK pathway includes three layers of kinases: MAPKKK (MAP kinase kinase kinase) can activate the downstream MAPKK; MAPKK (MAP kinase kinase) can activate MAPK; MAPK is the kinase that finally activates the target protein. The functions of MAP kinase in various cell processes include: regulating the cell cycle and proliferation; participating in cell differentiation, migration, and apoptosis; promoting the cell's response to stress (such as oxidative stress, heat stress, etc.). MAP kinase is a key molecule in the cell signal transduction network and regulates a variety of important biological processes. They are not only involved in the normal growth and development of cells but also play an important role in responding to external stress and regulating cell death. By cloning and studying the nucleotide sequence of the CDC7 gene and the amino acid polypeptide sequence encoded by it, the present invention found that this gene exhibits significant tolerance to a variety of preservatives, especially preservatives such as potassium sorbate.

[0006] Currently, there is a lack of a fungal broad-spectrum preservative tolerance gene CDC7 and its application. The innovation of the present invention lies in using this gene to transform Neurospora crassa and evaluating its tolerance to preservatives through experiments, further verifying its application potential in preservative tolerance. This research provides a new perspective for the development of tolerance genes and the in-depth understanding of the preservative tolerance mechanism, and provides a theoretical basis and technical support for the future research and application of preservatives. Summary of the Invention

[0007] The purpose of the present invention is to provide a fungal broad-spectrum preservative tolerance gene CDC7 and its application.

[0008] To solve the problems of the prior art, the present invention provides the following technical solutions: In the first aspect, the present application provides a fungal broad-spectrum preservative tolerance gene CDC7. In the second aspect, the present application provides a protein encoded by the fungal preservative resistance gene CDC7. In the third aspect, the present application provides a promoter for regulating the expression of the fungal preservative resistance gene CDC7. In the fourth aspect, the present application provides a recombinant vector. In the fifth aspect, the present application provides a recombinant strain. In the sixth aspect, the present application provides a gene knockout strain ΔCDC7. In the seventh aspect, the present application provides an application of the fungal preservative resistance gene CDC7 in breeding preservative-tolerant fungal strains. In the eighth aspect, the present application provides a method for preparing a fungal preservative-tolerant strain.

[0009] In the first aspect of the present application, a fungal broad-spectrum preservative tolerance gene CDC7 is provided, and the nucleotide sequence of the fungal preservative resistance gene CDC7 is at least one of the following (a), (b), and (c):

[0010] (a) As shown in SEQ ID NO: 1;

[0011] (b) A variant sequence having a sequence similarity of ≥95% with SEQ ID NO: 1 and encoding the same functional protein;

[0012] (c) A conservative modification sequence obtained by nucleotide substitution, deletion, or addition of SEQ ID NO: 1 without changing the encoded amino acid sequence.

[0013] In the second aspect of the present application, a protein encoded by the fungal preservative resistance gene CDC7 is provided, and the amino acid sequence of the protein encoded by the fungal preservative resistance gene CDC7 is as shown in SEQ ID NO: 2, or a functional variant obtained by substituting, deleting, or adding some amino acids to SEQ ID NO: 2 and retaining the MAP kinase phosphokinase function.

[0014] In the third aspect of the present application, a promoter for regulating the expression of the gene CDC7 is provided, and the nucleotide sequence of the promoter is as shown in SEQ ID NO: 3, or a homologous sequence having the function of regulating the transcription of the CDC7 gene with SEQ ID NO: 3.

[0015] In the fourth aspect of the present application, a recombinant vector is provided, which contains the fungal preservative resistance gene CDC7 and / or the promoter, and the vector is used for genetic transformation of fungal cells.

[0016] In the fifth aspect of the present application, a recombinant strain is provided. By introducing the recombinant vector into a fungal host cell, the strain expresses the CDC7 gene and is tolerant to preservatives; the fungal host is Neurospora crassa.

[0017] The sixth aspect of the present application provides a gene knockout strain ΔCDC7, which is obtained by knocking out the CDC7 gene described in claim 1, and the sensitivity of the strain to preservatives is significantly improved.

[0018] The seventh aspect of the present application provides an application of the fungal preservative resistance gene CDC7 in breeding fungal strains resistant to preservatives, including introducing the gene into strains sensitive to preservatives to improve their preservative tolerance by expressing the CDC7 gene.

[0019] The eighth aspect of the present application provides an application of a protein in the preparation of an enhancer for enhancing fungal preservative tolerance.

[0020] The ninth aspect of the present application provides a method for preparing a fungal strain tolerant to preservatives, including the following steps:

[0021] (a) Construct a recombinant vector containing the fungal preservative resistance gene CDC7;

[0022] (b) Transform the recombinant vector into a target fungal host cell;

[0023] (c) Screen and obtain a transgenic strain expressing the fungal preservative resistance gene CDC7;

[0024] (d) Verify the tolerance of the transgenic strain by adding preservatives.

[0025] Beneficial effects: The present invention not only provides a highly efficient and broad-spectrum fungal preservative tolerance gene and its application scheme, but also solves the problems of long breeding cycle and low efficiency of traditional strains through genetic engineering technology, and has significant industrial application value and scientific significance.

[0026] Compared with the prior art, the present invention has the following advantages: (1) Broad-spectrum resistance advantage: The CDC7 gene belongs to the MAPkinase phosphokinase family, and the signal pathway it regulates has the characteristics of broad-spectrum resistance, and can be tolerant to a variety of chemical preservatives (including but not limited to potassium sorbate), breaking through the limitations of traditional single resistance genes.

[0027] (2) Efficient genetic engineering method: Compared with the traditional methods relying on natural screening or hybridization breeding, the present invention adopts gene cloning, vector construction and transformation technologies, greatly shortening the strain improvement cycle, reducing costs, and the operation is accurate and controllable.

[0028] (4) Cross-species application potential: The CDC7 gene is not only applicable to Neurospora crassa, but can also be transformed into other ascomycetes through genetic engineering technology, providing a general solution for improving the preservative tolerance of different fungal strains in industries such as food, medicine, and cosmetics.

[0029] (5) Mechanism analysis and reverse application: By analyzing the structure of the CDC7 gene and the MAP kinase functional domain of its encoded protein, its resistance mechanism was clarified, laying a foundation for the optimized design of subsequent resistance genes. Meanwhile, knocking out this gene (ΔCDC7 strain) can significantly improve the sensitivity of the strain to preservatives, providing a new strategy for controlling harmful fungal contamination.

[0030] (6) Technical compatibility and scalability: The promoter (SEQ ID NO:3) and recombinant vector provided by the present invention can be combined with other gene editing tools (such as CRISPR / Cas9) or metabolic engineering strategies to achieve multi-gene stacked regulation, further improving the performance of the strain or developing a new preservative tolerance system.

[0031] (7) Promoting the research and development of preservatives: The discovery of the CDC7 gene provides a molecular target for the development of highly effective preservatives or the optimization of existing preservative formulations, helping to design more targeted preservative products and reducing the risk of fungal drug resistance.

[0032] (8) Production safety and economic benefits: By enhancing the preservative tolerance of industrial strains, the dosage or use cost of preservatives in the production process can be reduced, while reducing product losses caused by fungal contamination, improving production efficiency and economic benefits.

[0033] (9) Significantly enhancing the tolerance to preservatives: By introducing the CDC7 gene into preservative-sensitive strains (such as Neurospora crassa), the transgenic strains show significantly enhanced tolerance to common preservatives such as potassium sorbate, effectively reducing the inhibitory effect of preservatives on production strains and ensuring industrial fermentation and product stability. Brief Description of the Drawings

[0034] Figure 1 It is a process schematic diagram of the present invention.

[0035] Figure 2 It is the PCR product of the hygromycin resistance gene of the present invention. Lane 1 is the marker, lanes 2-5 are the PCR amplification products of non-transformants in the ku-70 strain, and lanes 6-8 are the PCR amplification products of knockout transformants in the ku-70 strain.

[0036] Figure 3 It is an identification diagram of the resistance of the transformed strain of the preservative tolerance gene CDC7 in Neurospora crassa of the present invention. Detailed Description of the Invention

[0037] In order to make the technical problems to be solved, technical solutions and beneficial effects of the present application clearer, the following further details the present application with reference to embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0038] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Here, A and B can be singular or plural. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.

[0039] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following items" or similar expressions refer to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can both represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or multiple respectively.

[0040] It should be understood that in various embodiments of this application, the magnitude of the sequence numbers of the above - mentioned processes does not mean the order of execution. Some or all steps can be executed in parallel or successively. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of this application.

[0041] The terms used in the embodiments of this application are only for the purpose of describing specific embodiments, and are not intended to limit this application. The singular forms of "a", "the", and "said" used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0042] The weight of the relevant components mentioned in the specification of the embodiments of this application not only can refer to the specific content of each component, but also can represent the proportional relationship of the weights between the components. Therefore, as long as the content of the relevant components in the specification of the embodiments of this application is scaled up or down proportionally, it is within the scope disclosed in the specification of the embodiments of this application. Specifically, the mass mentioned in the specification of the embodiments of this application can be mass units well - known in the chemical industry such as μg, mg, g, kg, etc.

[0043] The first aspect of the embodiments of this application provides a fungal broad - spectrum preservative tolerance gene CDC7. The nucleotide sequence of the fungal preservative resistance gene CDC7 is at least one of the following (a), (b), and (c):

[0044] (a) As shown in SEQ ID NO:1;

[0045] (b) A variant sequence having a sequence similarity of ≥95% with SEQ ID NO:1 and encoding the same functional protein;

[0046] (c) A conservatively modified sequence of SEQ ID NO:1 with nucleotide substitutions, deletions, or additions that do not change the encoded amino acid sequence.

[0047] In the second aspect of the embodiments of the present application, a protein encoded by the fungal preservative resistance gene CDC7 is provided. The amino acid sequence of the protein encoded by the fungal preservative resistance gene CDC7 is as shown in SEQ ID NO:2, or a functional variant of SEQ ID NO:2 with partial amino acid substitutions, deletions, or additions that retains the MAP kinase phosphokinase function.

[0048] In the third aspect of the embodiments of the present application, a promoter that regulates the expression of the gene CDC7 is provided. The nucleotide sequence of the promoter is as shown in SEQ ID NO:3, or a homologous sequence that contains the function of regulating the transcription of the CDC7 gene and is homologous to SEQ ID NO:3.

[0049] In the fourth aspect of the embodiments of the present application, a recombinant vector is provided, which contains the fungal preservative resistance gene CDC7 and / or the promoter, and the vector is used for genetic transformation of fungal cells.

[0050] In the fifth aspect of the embodiments of the present application, a recombinant strain is provided. By introducing the recombinant vector into a fungal host cell, the strain expresses the CDC7 gene and is tolerant to preservatives; the fungal host is Neurospora crassa.

[0051] In the sixth aspect of the embodiments of the present application, a gene knockout strain ΔCDC7 is provided, which is obtained by knocking out the CDC7 gene described in claim 1, and the sensitivity of the strain to preservatives is significantly increased.

[0052] In the seventh aspect of the embodiments of the present application, an application of the fungal preservative resistance gene CDC7 in breeding fungal strains resistant to preservatives is provided, including introducing the gene into a strain sensitive to preservatives to improve its preservative tolerance by expressing the CDC7 gene.

[0053] In the eighth aspect of the embodiments of the present application, an application of a protein in preparing an enhancer for enhancing fungal preservative tolerance is provided.

[0054] In the ninth aspect of the embodiments of the present application, a method for preparing a fungal strain tolerant to preservatives is provided, including the following steps:

[0055] (a) Construct a recombinant vector containing the fungal preservative resistance gene CDC7;

[0056] (b) Transform the recombinant vector into a target fungal host cell;

[0057] (c) Screen and obtain a transgenic strain that expresses the fungal preservative resistance gene CDC7;

[0058] (d) Verify the tolerance of the transgenic strain by adding preservatives.

[0059] In some embodiments, the preservative is potassium sorbate.

[0060] Determination of the preservative tolerance candidate genes of Neurospora crassa: Based on the preservative-sensitive FGSC2225 strain and the preservative-tolerant ku-70 strain with whole-genome sequencing of Neurospora crassa, hybridization was carried out, and the progeny sexual spores were picked for propagation. Further, the tolerant and sensitive progeny were screened in a medium supplemented with the preservative potassium sorbate. By identifying the parental origin of each gene, we selected 10 candidate preservative-tolerant gene loci. Through processes such as primer design, long-fragment PCR in the Neurospora crassa ku-70 strain, vector construction, transgenesis, and tolerance identification, finally, 1 new preservative-tolerant gene was obtained through systematic identification.

[0061] Unless otherwise specified, the methods used in the following examples are all conventional methods.

[0062] Example 1

[0063] A fungal broad-spectrum preservative tolerance gene CDC7 of the present invention, the nucleotide sequence of the fungal preservative resistance gene CDC7 is at least one of the following (a), (b), and (c):

[0064] (a) As shown in SEQ ID NO:1;

[0065] (b) A variant sequence having ≥95% sequence similarity with SEQ ID NO:1 and encoding the same functional protein;

[0066] (c) A conservative modification sequence obtained by nucleotide substitution, deletion, or addition of SEQ ID NO:1 without changing the encoded amino acid sequence.

[0067] Example 2

[0068] A protein encoded by a fungal preservative resistance gene CDC7 of the present invention, the amino acid sequence of the protein encoded by the fungal preservative resistance gene CDC7 is as shown in SEQ ID NO:2, or a functional variant of SEQ ID NO:2 with partial amino acids substituted, deleted, or added and retaining the MAP kinase phosphokinase function.

[0069] Example 3

[0070] A promoter regulating the expression of gene CDC7 of the present invention, the nucleotide sequence of the promoter is as shown in SEQ ID NO:3, or a homologous sequence having the function of regulating the transcription of gene CDC7 with SEQ ID NO:3.

[0071] Example 4

[0072] A recombinant vector of the present invention comprises a fungal preservative resistance gene CDC7 and / or a promoter, and the vector is used for genetic transformation of fungal cells.

[0073] Example 5

[0074] A recombinant strain of the present invention is obtained by introducing the recombinant vector into a fungal host cell, enabling the strain to express the CDC7 gene and be tolerant to preservatives; the fungal host is Neurospora crassa.

[0075] Example 6

[0076] A gene knockout strain ΔCDC7 of the present invention is obtained by knocking out the CDC7 gene, and the sensitivity of the strain to preservatives is significantly increased.

[0077] Example 7

[0078] An application of a fungal preservative resistance gene CDC7 of the present invention in breeding fungal strains resistant to preservatives includes introducing the gene into a strain sensitive to preservatives to improve its preservative tolerance by expressing the CDC7 gene.

[0079] Example 8

[0080] An application of a protein of the present invention in preparing an enhancer for enhancing fungal preservative tolerance.

[0081] Example 9

[0082] A method for preparing a fungal strain tolerant to fungal preservatives of the present invention includes the following steps:

[0083] (a) Construct a recombinant vector containing a fungal preservative resistance gene CDC7; the preservative is potassium sorbate.

[0084] (b) Transform the recombinant vector into a target fungal host cell;

[0085] (c) Screen to obtain a transgenic strain expressing the fungal preservative resistance gene CDC7;

[0086] (d) Verify the tolerance of the transgenic strain by adding a preservative.

[0087] Example 10

[0088] Cloning, vector construction, identification of sensitive and drug-resistant strains of the preservative tolerance gene CDC7 of Neurospora crassa

[0089] The experimental procedure of this example is as Figure 1 shown.

[0090] 1. Determination of the candidate locus CDC7 gene for preservative tolerance: (1) There is 1 copy of the homologous gene in both FGSC2225 strain and ku-70 strain; (2) There are traces of rapid evolution in its protein region, with a Ti / Tv value as high as 3.9 and a Ka / Ks value as low as 0.1, indicating that it is under strong negative selection.

[0091] 2. Isolation and cloning of the preservative-tolerant gene CDC7 (NCU03071) in Neurospora crassa: Using the newly sequenced FGSC2225 strain and ku-70 strain in our laboratory as reference sequences, primers were designed. The PCR primer sequences are shown in the sequence listing. The CDC7 primer sequence for the ku-70 preservative-tolerant strain is shown in SEQ ID NO:4, and the CDC7 primer sequence for the FGSC2225 preservative-sensitive strain is shown in SEQ ID NO:5.

[0092] The PCR primer sequence list for the ku-70 preservative-tolerant strain of SEQ ID NO:4 is shown in Table 1:

[0093] Table 1

[0094]

[0095] The PCR primer sequence list for the FGSC2225 preservative-sensitive strain of SEQ ID NO:5 is shown in Table 2:

[0096] Table 2

[0097]

[0098] Using the preservative-sensitive FGSC2225 strain, the preservative-tolerant FGSC2489 strain and the ku-70 strain as templates, the candidate gene fragment was amplified by long fragment PCR technology (Long-PCR). The PCR program is as follows: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 60°C for 45 seconds, extension at 68°C for 7 minutes, a total of 35 cycles, then incubation at 72°C for 10 minutes, and finally constant temperature at 10°C. Subsequently, the PCR product was recovered by gel cutting.

[0099] In this invention, the gene knockout mutants are mainly obtained through homologous recombination. The ku-70 strain used is a strain in which the gene mus51 corresponding to the key protein ku70 in the homologous recombination pathway has been mutated. The technique of gene knockout by homologous recombination (HR) has been widely used in fungi. It is a technique that synthesizes new DNA double strands using another homologous DNA as a template at the break point, and only about 1 kb of homologous sequence is required to trigger homologous recombination and perform gene knockout based on the recognition of homologous sequences (Ninomiya et al., 2004). The specific principle is that the resistance gene is constructed into fusion fragments with the 5’ flank and 3’ flank of the target gene ORF respectively and transferred into fungal mycelia. During electroporation, there is a certain probability that the resistance fragment and the target gene ORF will undergo homologous recombination, so that the target gene ORF is replaced by the transferred resistance gene, thus achieving the purpose of gene knockout. After electroporation, the knockout-type and wild-type mycelia and sporophytes coexist, and the purified knockout mutants are gradually obtained through the screening of the resistance gene.

[0100] Plasmid preparation: pCSN44 (containing trpC promoter, hyg, terminator), which can express hygromycin resistance. By transforming into Escherichia coli and performing colony PCR, a fragment that can express hygromycin resistance is obtained. Obtaining of fusion fragments: Connect the ORF 5’ flank or ORF 3’ flank with the hph gene by fusion PCR. Transformation and subculture: Use electroporation to transform the ku-70 knockout mother strain.

[0101] PCR molecular detection: Using the DNA of the transformant as a template, perform PCR reaction on it with the hygromycin resistance gene and its upstream specific primers. Subculture the transformants continuously on the propagation medium containing hygromycin, inoculating as few conidia as possible each time. Generally, after subculturing 20 generations, the homozygous state can be basically achieved.

[0102] Resistance identification of transformants: Select 10 independent transformant offspring strains, extract the genomic DNA of the transformants, and perform PCR identification. The results of resistance identification show that after the candidate gene CDC7 is transferred into the sensitive strain, it shows a certain degree of tolerance to the preservative potassium sorbate, indicating that the candidate gene CDC7 has specific tolerance and has been successfully cloned ( Figure 2 ).

[0103] Analysis of the gene structure and protein structure of the preservative tolerance gene CDC7: The DNA sequence of the CDC7 gene was sequenced using the genome walking method. The DNA length of the CDC7 gene is 4168 bp, containing 2 exons and 1 intron. The protein sequence encoded by the CDC7 gene is shown in SEQ ID NO: 2, encoding a protein polypeptide composed of 1367 amino acid residues. The CDC7 protein belongs to the MAP Kinase protein and contains a typical phosphorylase functional domain.

[0104] The 2 kb sequence before the CDC7 gene sequence was taken as the promoter sequence of this gene, and the TSSPlant software was used to confirm that this sequence contains the typical promoter characteristic sequence fragment - the (TATA-)TSS site.

[0105] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements. The scope of protection required by the present invention is defined by the appended claims, the specification and their equivalents.

Claims

1. A fungal broad-spectrum preservative resistance gene CDC7, characterized in that: The nucleotide sequence of the fungal preservative resistance gene CDC7 is at least one of the following (a), (b), and (c): (a) as shown in SEQ ID NO: 1; (b) a variant sequence having ≥95% sequence similarity to SEQ ID NO: 1 and encoding a protein with the same function; (c) A conservatively modified sequence such as SEQ ID NO: 1 that undergoes nucleotide substitution, deletion or addition but does not change the encoded amino acid sequence.

2. The protein encoded by the fungal preservative resistance gene CDC7 according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the fungal preservative resistance gene CDC7 is shown in SEQ ID NO: 2, or a functional variant of SEQ ID NO: 2 which has been replaced, deleted or added with some amino acids and retains the MAP kinase phosphokinase function.

3. A promoter for regulating the expression of the gene CDC7 according to claim 1, characterized in that: The nucleotide sequence of the promoter is shown in SEQ ID NO: 3, or contains a homologous sequence to SEQ ID NO: 3 that has the function of regulating CDC7 gene transcription.

4. A recombinant vector, characterized in that: The vector comprises the fungal preservative resistance gene CDC7 according to claim 1 and / or the promoter according to claim 3, and is used for genetic transformation of fungal cells.

5. A recombinant strain, characterized in that: By introducing the recombinant vector of claim 4 into a fungal host cell, the strain is made to express the CDC7 gene and have tolerance to preservatives; preferably, the fungal host is Neurospora crassa.

6. A gene knockout strain ΔCDC7, characterized in that: The strain is obtained by knocking out the CDC7 gene described in claim 1, and the sensitivity of the strain to preservatives is significantly improved.

7. Use of the fungal preservative resistance gene CDC7 according to claim 1 in breeding preservative-resistant fungal strains, comprising introducing the gene into a strain sensitive to preservatives, and improving its preservative tolerance by expressing the CDC7 gene.

8. Use of the protein according to claim 2 in the preparation of a fungal preservative tolerance enhancer.

9. A method for preparing a fungal preservative-tolerant strain, characterized in that The steps include: (a) constructing a recombinant vector comprising the fungal preservative resistance gene CDC7 according to claim 1; (b) transforming the recombinant vector into a target fungal host cell; (c) screening and obtaining a transgenic strain expressing the fungal preservative resistance gene CDC7; (d) verifying the tolerance of the transgenic strain by adding preservatives.

10. The preparation method according to claim 9, characterized in that: The preservative is potassium sorbate.

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