Genetic transformation method of PEG / CaCl2 mediated Penicillium citrinum
A highly efficient genetic transformation system for Penicillium finger of citrus was successfully constructed using a PEG/CaCl2-mediated method, solving the problems of low transformation efficiency and dependence on Agrobacterium in existing technologies, and achieving rapid and stable gene introduction and expression.
Patent Information
- Application Number
- CN202512007675.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-29
- Publication Date
- 2026-05-15
AI Technical Summary
Existing genetic transformation methods are difficult to efficiently construct stable transformation systems for Penicillium finger of citrus, and they rely on Agrobacterium strains, which pose risks of gene contamination and immune responses.
Using a PEG/CaCl2-mediated method, the target gene fragment was added to a suspension of Penicillium finger protoplasts, followed by an ice bath reaction, and then PTC buffer and TB3 liquid medium were added for resuscitation. Stable transformants were then prepared by dark culture and screening.
It achieves efficient and rapid genetic transformation, avoids dependence on Agrobacterium, reduces gene contamination and immune response, is applicable to different fungal and plant species, and has high transformation efficiency and is easy to operate.
Smart Images
Figure CN122038434A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of microbial genetic transformation technology, and more specifically, it relates to a method for genetic transformation of Penicillium finger of citrus mediated by PEG / CaCl2. Background Technology
[0002] Citrus fruits are beloved by consumers for their delicious taste and rich nutrients, and they hold a significant position in my country's national economy. However, citrus fruits are susceptible to fungal infections during post-harvest storage, primarily caused by Penicillium finger-like mold. Penicillium digitatum ), Italian penicillin ( P. italicum ) and citrus acid rot fungus ( Geotrichum citri-aurantii This causes significant economic losses. It has been reported that approximately 50% of postharvest losses can be attributed to various postharvest pathogens occurring at different stages of storage, with green mold caused by *Penicillium digitatum* being the most severe. *Penicillium digitatum* (… P.digitatum *Penicillium digitatum* is the most important pathogen causing citrus rot during storage. Typically, 20-30% of citrus fruits are lost due to rot after harvest, and over 90% of this is caused by *Penicillium digitatum* or *Penicillium italicum*. The pathogen mainly infects through three routes: (1) wounds caused by mechanical damage or insect infestation; (2) natural openings such as stomata; and (3) contact between diseased and healthy fruits or airborne transmission. Its biological characteristics are: optimal growth temperature 25-28°C (it can still grow slowly under refrigerated conditions at 4-10°C); suitable relative humidity >90%; preference for a slightly acidic environment (pH 4–6), highly compatible with the pH value (3–4) of citrus fruits. It is worth noting that highly mature fruits and epidermal damage caused during harvesting and transportation significantly increase the risk of infection.
[0003] Genetic transformation technology has been widely applied in the genetic transformation systems of filamentous fungi. Fungal mycelia, spores, and protoplasts can all serve as transformation recipients, enabling the target gene to be stably inherited and expressed within the recipient. Existing fungal genetic transformation methods include electroporation-mediated transformation, lithium acetate-mediated transformation, restriction endonuclease-mediated transformation, CaCl2-PEG-mediated transformation, Agrobacterium-mediated transformation, gene gun-mediated transformation, and transposon-mediated transformation. Among these, CaCl2-PEG-mediated transformation is simple to operate, low in cost, and does not require complex equipment or techniques. It is particularly suitable for the transformation and isolation of filamentous fungi with thick cell walls that do not readily produce spores, and is therefore widely used.
[0004] Currently, research on *Penicillium digitatum* is limited, and our understanding of its pathogenesis and occurrence patterns is insufficient. Establishing a genetic transformation system is a fundamental prerequisite for studying the pathogenic mechanisms of pathogenic fungi. Constructing an efficient and stable genetic transformation system will help provide technical evidence for subsequent research on the pathogenic functional genes of the fungus. To effectively control the losses caused by *Penicillium digitatum* to citrus and other fruits, it is necessary to conduct in-depth research at the molecular level on the pathogenic mechanisms, growth and development patterns, and effective targets of fungicides. An effective genetic transformation system is a prerequisite for conducting molecular biological research on *Penicillium digitatum*. The establishment of a *Penicillium digitatum* genetic transformation system not only lays the foundation for molecular biological research on *Penicillium digitatum* but also provides a reference for the molecular biology and genetic engineering of other species in the *Penicillium* genus. Currently, there are genetic transformation systems for *Penicillium digitatum* in citrus using *Agrobacterium tumefaciens*, but no system has been developed using the CaCl2-PEG mediated method for transformation of *Penicillium digitatum* in citrus. Summary of the Invention
[0005] The purpose of this invention is to provide a method for genetic transformation of Penicillium finger of citrus mediated by PEG / CaCl2, which has the advantages of higher transformation efficiency, independence from Agrobacterium strains, and faster transformation speed.
[0006] To achieve the above objectives, this invention provides a method for genetic transformation of *Penicillium fingering* citrus mediated by PEG / CaCl2, comprising the following steps: S1. Add the target gene fragment to the protoplast suspension of Penicillium finger of citrus and perform an ice bath reaction; the target gene fragment is obtained by PCR amplification using pJET-hph-02944 plasmid as template and hygromycin B resistance gene primer pair with gene sequences as shown in SEQ ID No. 1 and SEQ ID No. 2; S2. Add PTC buffer to the mixture, mix well and let stand; S3. Continue to add TB3 liquid medium containing ampicillin for resuscitation culture; S4. Mix the revived protoplasts with melted TB3 solid medium, pour the plates, and perform the first dark culture. Then cover with TB3 solid medium containing hygromycin B and ampicillin, and perform a second dark culture to obtain transformants. Transfer the transformants to PDA plates containing hygromycin B for screening.
[0007] Furthermore, it also includes the following steps: S5. After multiple subcultures of the selected transformants, stable transformants are obtained by screening with hygromycin B.
[0008] Furthermore, the citrus penicillium finger protoplast suspension was prepared using the following method: The citrus penicillium strain was inoculated onto PDA plates and cultured. Then, mycelia were scraped and suspended in PDB liquid medium for shaking culture. After cultivation, collect the mycelia and rinse them with KC buffer. Enzymatic hydrolysate was then added to the mycelium for enzymatic hydrolysis. After hydrolysis, the solution was filtered to obtain a filtrate containing protoplasts. The filtrate was centrifuged to remove the supernatant and obtain a protoplast precipitate. The protoplast precipitate was washed with KC buffer and then with STC solution. Finally, it was resuspended with STC solution.
[0009] Furthermore, the shaking culture time is 24-72 h.
[0010] Furthermore, the enzymatic hydrolysate contains at least one of the following: a breakdown enzyme, a snail enzyme, and a wall-lysing enzyme.
[0011] Furthermore, the enzymatic hydrolysate contains 2% catalytic enzyme and 2% snail enzyme by mass concentration.
[0012] Furthermore, the enzymatic hydrolysis reaction takes 1-5 hours.
[0013] Furthermore, the shaking culture time is 48 h, and the enzymatic hydrolysis reaction time is 3 h.
[0014] Furthermore, the concentration of protoplasts in the *Penicillium finger* protoplast suspension is 1.0 × 10⁻⁶. 7 The target gene fragment was added at a concentration of CFU / mL: 15 μg of the target gene fragment was added to every 300 μL of the *Penicillium finger* protoplast suspension.
[0015] Furthermore, the ice bath reaction time is 20-30 min.
[0016] Compared with the prior art, the present invention has the following technical effects: The PEG / CaCl2-mediated genetic transformation method of Penicillium fingering of citrus of the present invention uses CaCl2-PEG to mediate the transformation of Penicillium fingering of citrus, which has higher transformation efficiency, faster transformation speed and wide applicability; CaCl2-PEG-mediated transformation does not depend on other microorganisms, is not limited by the compatibility between the host and Agrobacterium, and can avoid gene contamination and reduce immune response. Attached Figure Description
[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0018] Figure 1 The spectrum of the pJET-hph-02944 plasmid provided in the embodiments of the present invention; Figure 2 Gel electrophoresis image of the target gene fragment provided in the embodiments of the present invention; Figure 3 Micro-electron microscopy images of protoplasts under different oscillation culture times provided in embodiments of the present invention; Figure 4 These are electron micrographs of protoplasts under the action of different enzymatic hydrolysates provided in the embodiments of the present invention. Figure 5 Micro-electron micrographs of protoplasts under different enzymatic hydrolysis solutions and different enzymatic hydrolysis times provided in the embodiments of the present invention; Figure 6 The embodiment of the present invention provides a solution containing 160 μg·mL -1 Transformation subgraphs grown on Hyg B PDAs; Figure 7 The embodiment of the present invention provides a solution containing 160 μg·mL -1 Comparison of wild-type P44 and transformant colonies grown on Hyg B PDA; Figure 8 The image shows an electrophoresis diagram of the transformants provided in an embodiment of the present invention; in the diagram, 1 is a positive control (plasmid), 2 is a negative control (water), 3 is a negative control (wild bacteria), and 4, 5, 6, 7, and 8 are transformant bands; Figure 9 The transformants provided in this embodiment of the invention, after three generations of culture, contained 160 μg·mL⁻¹ -1 Growth diagram of Hyg B on a PDA. Detailed Implementation
[0019] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.
[0020] The terminology used in the embodiments of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. The singular forms “a,” “the,” and “the” as used in the embodiments of this invention and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.
[0021] The culture media and reagents used in the embodiments of this invention are as follows: PDA medium: Boil and filter 200 g potatoes, add distilled water to 1 L, add 2% glucose by weight, add 1.5%–2% agar, sterilize at 121℃ for 15 min, and use for strain activation and culture.
[0022] PDB medium: Boil and filter 200 g potatoes, add distilled water to 1 L, add 2% glucose by weight, sterilize at 121℃ for 15 min, and use for strain propagation.
[0023] Enzyme system: Driselase, Lysing Enzyme, and Snailase were purchased from Sigma-Aldrich and Beijing Dingguo Changsheng Biotechnology Co., Ltd., respectively.
[0024] KC buffer: 0.64 mol / L KCl + 0.2 mol / L CaCl2, sterilized by 0.22 μm filter membrane.
[0025] TB3 liquid medium: 3 g yeast extract + 3 g acid-hydrolyzed casein + 200 g sucrose, bring to a final volume of 1 L, and sterilize at 121℃ for 20 min.
[0026] TB3 solid medium: 3 g yeast extract + 3 g acid-hydrolyzed casein + 200 g sucrose, bring to a final volume of 1 L, add 1.5%–2% agar, stir well, and sterilize at 121℃ for 20 min.
[0027] STC buffer: 200g sucrose + 7.88 g Tris-HCl + 5.55 g CaCl2, bring to a final volume of 1 L, and adjust pH to 7.5; PTC buffer: Dissolve 6 g PEG 4000 in 4 mL STC buffer and sterilize with a 0.22 μm filter membrane.
[0028] Screening antibiotics: Hygromycin B (Hyg B).
[0029] Ampicillin (AMP).
[0030] This invention provides a method for genetic transformation of Penicillium finger of citrus mediated by PEG / CaCl2, comprising the following steps: S1. Add the target gene fragment to the protoplast suspension of Penicillium finger of citrus and perform an ice bath reaction for 20-30 min. The target gene fragment is obtained by PCR amplification using pJET-hph-02944 plasmid as template and primers with gene sequences as shown in SEQ ID No. 1 and SEQ ID No. 2 for hygromycin B resistance gene. S2. Add PTC buffer to the mixture, mix well and let stand; S3. Continue to add TB3 liquid medium containing ampicillin for resuscitation culture; S4. Mix the revived protoplasts with melted TB3 solid medium, pour the plates, and perform the first dark culture. Then cover with TB3 solid medium containing hygromycin B and ampicillin, and perform a second dark culture to obtain transformants. Transfer the transformants to PDA plates containing hygromycin B for screening.
[0031] In step S1 above, the preparation of the Penicillium finger protoplast suspension includes the following steps: (1) Pick out citrus penicillium colonies, inoculate the citrus penicillium strain onto PDA plates for culture, then scrape off the hyphae and suspend them in PDB liquid medium for shaking culture. The shaking culture time can be 24-72 h, preferably 48 h. (2) After the culture is completed, filter the bacterial solution with sterilized gauze to collect the mycelium, and rinse with KC buffer. (3) Then add the enzymatic hydrolysate to the hyphae for enzymatic hydrolysis. After enzymatic hydrolysis, filter to obtain a filtrate containing protoplasts. Centrifuge the filtrate to remove the supernatant and obtain protoplast precipitate. Wash the protoplast precipitate with KC buffer and then with STC solution. Finally, resuspend it with STC solution.
[0032] In this embodiment, the enzymatic hydrolysate contains at least one of a breakdown enzyme, a snail enzyme, and a wall-lysing enzyme. Preferably, the enzymatic hydrolysate contains 2% breakdown enzyme and 2% snail enzyme by mass concentration. The enzymatic hydrolysis reaction time can be 1-5 hours, preferably 3 hours.
[0033] To obtain a more stable transformant, the transformation method of this embodiment of the invention further includes the following steps: S5. After multiple subcultures of the selected transformants, stable transformants are obtained by screening with hygromycin B.
[0034] The following specific embodiment illustrates a method for genetic transformation of Penicillium finger of citrus mediated by PEG / CaCl2 according to an embodiment of the present invention.
[0035] I. Protoplast Preparation and Transformation 1. Mycelial Culture: Select *Penicillium fingertips* colony P44, inoculate the P44 strain onto PDA plates, and incubate at 28 ℃ for 4 days. Scrape the mycelia and suspend them in PDB liquid medium, incubating at 28 ℃ and 200 r·min. -1 Shaking culture for 48 h.
[0036] 2. Protoplast preparation: Collect 0.4 g of mycelium and wash 2-3 times with KC buffer. Add 10 mL of enzymatic digestion solution (2% snailase + 2% catabolase, prepared with KC buffer), and incubate at 28 ℃ and 110 r·min. -1 Enzymatic hydrolysis for 4 h (50 mL centrifuge tubes placed horizontally). Filter the hydrolysate through three layers of sterile lens paper, discard the precipitate, and incubate at 4 °C and 2500 r·min. -1 Centrifuge for 5 min, discard the supernatant, take the protoplast precipitate, wash with KC buffer and then with STC solution, and finally resuspend with STC for later use. Count and adjust the concentration to obtain the protoplast suspension.
[0037] II. PEG / CaCl2-mediated conversion 1. Obtaining the target gene fragment (hygromycin gene fragment): using the pJET-hph-02944 plasmid (plasmid pattern shown in figure) Figure 1 Using the template shown in SEQ ID No. 1 (HYG-F) and SEQ ID No. 2 (HYG-R) as the gene sequence, PCR amplification was performed using primer pairs for the hygromycin B resistance gene. The amplified products were subjected to agarose gel electrophoresis and gel imaging (as shown in SEQ ID No. 1 (HYG-F) and SEQ ID No. 2 (HYG-R)). Figure 2 As shown in the figure, gene fragments in the gel were recovered using a DNA kit.
[0038] SEQ ID No.1: 5′-ACTGTGAGAATAATTCCTGAAAAAAAAAGG-3′; SEQ ID No. 2: 5′-AGCTCGGTACCTCGCGAATGCATCTAGATC-3′.
[0039] 2. Take 300 μL of protoplast suspension (concentration of 1.0 × 10⁻⁶). 7 Add 15 μg of the target gene fragment (CFU / mL) to a 50 mL centrifuge tube and incubate on ice for 25 min.
[0040] 3. Add 1 mL of PTC buffer and mix well. Let stand at room temperature for 25 min.
[0041] 4. Add 8 mL of TB3 (with 50 μg / mL AMP) to the previous step and incubate overnight in the dark (28 °C, 110 rpm).
[0042] 5. Mix the revived protoplasts with TB3 solid medium melted at 45-50 ℃, pour the plates, and incubate in the dark for the first time for 48 h. Then cover with TB3 solid medium containing 130 μg / ml Hyg B and 50 μg / ml AMP and continue to incubate in the dark for a second time for 3-4 days. After one week, pick the transformants.
[0043] III. Transformer Validation 1. Resistance screening: Transformants were inoculated with 160 μg / mL solution. -1 Hyg B was cultured on a PDA plate at 28 °C for 7 days, and the growth was observed.
[0044] 2. PCR identification: DNA was extracted using a fungal genomic DNA extraction kit and amplified by PCR using Hyg B gene-specific primers (SEQ ID No.3 (HYG-F-1): 5′-CTTCTGCGGGCGATTTGTG-3′; SEQ ID No.4 (HYG-R-1): 5′-CGGAGGCTATGGATGCGAT-3′). The target band was detected by agarose gel electrophoresis.
[0045] 3. Genetic stability test: Transformants were passaged three times consecutively to test the stability of their resistance.
[0046] IV. Experimental Results This invention investigated the protoplast preparation method of Penicillium digitatum strain P44 and determined the following optimal conditions: 1. Optimal mycelial age: The highest protoplast yield, reaching 8.2 × 10⁻⁶, was achieved when the mycelium was cultured in PDB liquid medium for 48 h. 7 Cells / mL. Both too short (24 h) and too long (72 h) culture times will result in a significant decrease in yield. Figure 3 Micro-electron microscopy images of protoplasts cultured for different times are shown.
[0047] 2. Optimal enzyme hydrolysate system: The enzymes used in the experiment were: lysis enzyme (D), snail enzyme (S), and lysozyme (L).
[0048] The highest protoplast yield (1.14 × 10⁻⁶) was achieved when using a mixed enzymatic hydrolysate of 2% cytokinase (D) + 2% snailase (S). 7 The preparation efficiency of any single enzyme or other enzyme combination was significantly lower than that of 2% collapse enzyme (D) + 2% snail enzyme (S), as shown in Table 1 below. Figure 4 As shown.
[0049] Table 1
[0050] 3. Optimal enzymatic hydrolysis time Protoplast production peaks after 3 hours of enzymatic hydrolysis. Insufficient hydrolysis (1 hour) results in incomplete hydrolysis, while excessive hydrolysis (5 hours) leads to the rupture of released protoplasts and a decrease in yield (e.g., ...). Figure 5 (As shown).
[0051] V. Screening and Resistance Identification of Transformants This invention successfully obtained multiple hygromycin B-resistant transformants (e.g., using a PEG / CaCl2-mediated transformation method) through a PEG / CaCl2-mediated transformation method. Figure 6 (As shown).
[0052] On PDA plates containing 160 μg / mL Hyg B, the transformants grew normally, while the wild-type strain (P44) was completely inhibited and could not grow (e.g., Figure 7 (As shown).
[0053] The transformants showed significant differences in colony morphology compared to the wild type: some transformedants had slower growth rates, some had faster growth rates, some had thinner mycelial growth, some had reduced sporulation, some had increased sporulation, some had mycelia that adhered more closely to the culture medium surface, some had fewer mycelia, some had more mycelia, some had increased pathogenicity, and some had decreased pathogenicity.
[0054] VI. Molecular identification of transformants (PCR verification) PCR amplification of the genomic DNA of transformants was performed using primers specific to the hygromycin B resistance gene. Electrophoresis results showed that all tested transformants amplified a specific band of 592 bp, exactly as expected. Figure 8 The wild-type strain and the negative control using ddH2O as a template showed no amplification bands. This indicates that the exogenous Hyg B resistance gene has been successfully integrated into the transformant's genome.
[0055] VII. Genetic stability testing After three consecutive subcultures of positive transformants on antibiotic-free PDA plates, they were then inoculated again onto plates containing hygromycin B. The transformants continued to grow normally. This indicates that the exogenous gene (Hyg B) has been stably integrated into the chromosome and can be stably inherited and expressed. Figure 9 ).
[0056] In this embodiment of the invention, the plasmid contains a hygromycin B resistance gene. Hygromycin B is added to the culture medium to create a selective medium. On this medium, Penicillium digitatum transformed by transgene is selectively cultured. The transformed Penicillium digitatum can grow normally and form colonies because it contains the hygromycin B resistance gene, while the untransformed Penicillium digitatum does not have this resistance gene and is extremely sensitive to hygromycin B, and therefore cannot grow.
[0057] The embodiments of the present invention have successfully established Penicillium digitatum The highly efficient protoplast genetic transformation system of strain P44 enables the target gene to be stably inherited and expressed in the recipient. This marker strain provides a powerful tool for subsequent real-time and intuitive studies of the infection process, colonization dynamics, and pathogenic mechanisms of this pathogen. The transformation method of this invention is simple and yields stable transformants, thus laying the foundation for molecular operations such as gene expression and functional analysis of *Penicillium digitatum*.
[0058] Compared with existing Agrobacterium-mediated genetic transformation methods, the method of this invention has the following advantages: 1. Higher transformation efficiency: CaCl2-PEG-mediated protoplast transformation usually has a high transformation efficiency under certain conditions because it acts directly on the protoplasts of fungal cells without the obstruction of the cell wall, making it easier for exogenous DNA to enter the cell.
[0059] 2. Wide applicability: The CaCl2-PEG-mediated transformation method is widely applicable to different fungal or plant species.
[0060] 3. Independent on Agrobacterium strains: CaCl2-PEG does not depend on any microorganisms, and therefore is not limited by the compatibility between the host and Agrobacterium.
[0061] 4. Fast conversion speed: The CaCl2-PEG method is relatively simple and fast, and the conversion can be completed in a short time.
[0062] 5. Avoid gene contamination: Agrobacterium transformation involves the insertion of exogenous genes into Agrobacterium T-DNA, which carries the risk that some non-target genes may be integrated into the host genome. The CaCl2-PEG method reduces the possibility of such gene contamination by directly introducing DNA.
[0063] 6. Reduced immune response: The CaCl2-PEG method is a physicochemical approach that is less likely to induce an immune response in the host.
[0064] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims.
Claims
1. A method for genetic transformation of *Penicillium fingering* citrus mediated by PEG / CaCl2, characterized in that, Includes the following steps: S1. Add the target gene fragment to the protoplast suspension of Penicillium finger of citrus and perform an ice bath reaction; the target gene fragment is obtained by PCR amplification using pJET-hph-02944 plasmid as template and hygromycin B resistance gene primer pair with gene sequences as shown in SEQ ID No. 1 and SEQ ID No. 2; S2. Add PTC buffer to the mixture, mix well and let stand; S3. Continue to add TB3 liquid medium containing ampicillin for resuscitation culture; S4. Mix the revived protoplasts with melted TB3 solid medium, pour the plates, and perform the first dark culture. Then cover with TB3 solid medium containing hygromycin B and ampicillin, and perform a second dark culture to obtain transformants. Transfer the transformants to PDA plates containing hygromycin B for screening.
2. The method for genetic transformation of *Penicillium fingering* citrus mediated by PEG / CaCl2 as described in claim 1, characterized in that, It also includes the following steps: S5. After multiple subcultures of the selected transformants, stable transformants are obtained by screening with hygromycin B.
3. The method for genetic transformation of *Penicillium fingering* citrus mediated by PEG / CaCl2 as described in claim 1, characterized in that, The citrus penicillium finger protoplast suspension was prepared using the following method: The citrus penicillium strain was inoculated onto PDA plates and cultured. Then, mycelia were scraped and suspended in PDB liquid medium for shaking culture. After cultivation, mycelia were collected and washed with KC buffer. Then, an enzymatic hydrolysate was added to the mycelia for enzymatic hydrolysis. After hydrolysis, the mycelium was filtered to obtain a filtrate containing protoplasts. The filtrate was centrifuged to remove the supernatant and obtain a protoplast precipitate. The protoplast precipitate was washed with KC buffer and then with STC solution. Finally, it was resuspended in STC solution.
4. The method for genetic transformation of *Penicillium fingering* citrus mediated by PEG / CaCl2 as described in claim 3, characterized in that, The shaking culture time is 24-72 h.
5. The method for genetic transformation of *Penicillium fingering* citrus mediated by PEG / CaCl2 as described in claim 3, characterized in that, The enzymatic hydrolysate contains at least one of the following: a breakdown enzyme, a snail enzyme, and a wall-breaking enzyme.
6. The method for genetic transformation of *Penicillium fingering* citrus mediated by PEG / CaCl2 as described in claim 5, characterized in that, The enzymatic hydrolysate contains 2% breakdown enzyme and 2% snail enzyme by mass concentration.
7. The method for genetic transformation of *Penicillium fingering* citrus mediated by PEG / CaCl2 as described in claim 3, characterized in that, The enzymatic hydrolysis reaction takes 1-5 hours.
8. The method for genetic transformation of *Penicillium fingering* citrus mediated by PEG / CaCl2 as described in claim 3, characterized in that, The shaking culture time was 48 h, and the enzymatic hydrolysis reaction time was 3 h.
9. A method for genetic transformation of *Penicillium fingering* citrus mediated by PEG / CaCl2 as described in any one of claims 1-8, characterized in that, The concentration of protoplasts in the *Penicillium finger* protoplast suspension was 1.0 × 10⁻⁶. 7 The concentration of CFU / mL and the amount of the target gene fragment added are: 15 μg of the target gene fragment added to every 300 μL of the *Penicillium finger* protoplast suspension.
10. A method for genetic transformation of *Penicillium fingering* citrus mediated by PEG / CaCl2 as described in any one of claims 1-8, characterized in that, The ice bath reaction time is 20-30 min.