Agrobacterium-mediated transformation method of panax ginseng alternaria alternata gene
Patent Information
- Application Number
- CN202610949821.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-06-29
- Publication Date
- 2026-08-28
AI Technical Summary
然而，针对该类真菌的遗传转化长期存在显著的技术难点：
[0031] (1) Breaking through the conventional "double-layer screening method" design, unexpected decontamination and high positive rate effects were achieved: Conventional transformation screening often uses direct application of co-cultures onto single-layer screening plates containing antibiotics, which easily leads to excessive proliferation of Agrobacterium and false positives in Alternaria ginseng transformation. This invention innovatively adopts a double-layer screening strategy of "after a thin layer of fungi (5-8 Alternaria ginseng colonies, with few hyphae and hyphal area accounting for 30%-60% of the total area of IM medium) and a few Agrobacterium (no more than 10), pouring in a layer of PDA solid medium containing hygromycin to cover it". This innovative design, which breaks away from conventional physical isolation and single-concentration screening, utilizes hygromycin to slowly diffuse from the upper PDA solid medium to the lower IM medium, creating a gradient distribution of antibiotic concentration from high to low between the two media. This unexpectedly and completely blocks the excessive proliferation of Agrobacterium. At the same time, the slow penetration of hygromycin provides sufficient recovery time for the transformed Alternaria ginseng, increasing the true positive rate of the screened clones to over 90% (compared to less than 10% in conventional methods), and greatly reducing false positive interference.
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Abstract
Description
Technical Field
[0001] This invention relates to the fields of bioengineering and fungal genetic transformation technology, and in particular to an Agrobacterium-mediated transformation method for Alternaria ginseng genes. Background Technology
[0002] In fungal gene function research, establishing a stable and efficient genetic transformation system is a crucial prerequisite. *Alternaria panax*, a major pathogenic fungus causing ginseng black spot disease, requires efficient genetic transformation tools for genome analysis and pathogenic mechanism research. However, significant technical challenges have long existed in the genetic transformation of this type of fungus:
[0003] First, the cell wall of Alternaria ginseng is relatively thick, and the sporulation rate is easily limited under in vitro culture conditions, resulting in extremely low infection efficiency of conventional Agrobacterium.
[0004] Second, this bacterium exhibits a certain degree of background tolerance or "escape" from selective antibiotics such as hygromycin. In conventional Agrobacterium transformation methods (such as direct monolayer coating screening), Agrobacterium overgrowth (i.e., Agrobacterium contamination) is highly likely to occur after co-culturing Agrobacterium and fungi. This not only inhibits the recovery growth of Alternaria ginseng but also leads to an extremely high number of "false positive" transformed clones, resulting in a huge workload and a very low success rate for subsequent screening.
[0005] Third, improper selection of parameters such as spore concentration, Agrobacterium concentration (OD value), and induction conditions during co-culture can easily lead to fungal death or the inability of exogenous genes to be effectively integrated.
[0006] As can be seen from the above, the traditional Agrobacterium-mediated transformation method often has extremely low transformation efficiency in Alternaria ginseng, making it difficult to obtain stable transgenic lines. Currently, there is an urgent need for a transformation method that can overcome these shortcomings, effectively inhibit the excessive growth of Agrobacterium, and significantly improve the true positive rate. Summary of the Invention
[0007] The technical problem to be solved by the present invention is to provide an Agrobacterium-mediated transformation method for Alternaria ginseng gene, which can effectively inhibit the excessive growth of Agrobacterium and significantly improve the true positive rate.
[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:
[0009] An Agrobacterium-mediated transformation method for Alternaria ginseng gene, comprising:
[0010] (1) Co-cultivation:
[0011] The recombinant Agrobacterium containing the target gene, after activation and induction culture, was co-cultured with a suspension of Alternaria ginseng mycelium on IM medium.
[0012] (2) Screening:
[0013] After co-culturing, when 5-8 Alternaria ginseng colonies are observed, the mycelial area of the colonies accounts for 30%-60% of the total area of the IM medium, and the number of Agrobacterium plaques does not exceed 10, pour a layer of PDA solid medium containing screening antibiotics onto the IM medium for double-layer screening.
[0014] (3) Verification:
[0015] Once the ginseng Alternaria colonies have grown again, their hyphae will be selected for transformation verification.
[0016] As a further improvement of the present invention, in (1), the recombinant Agrobacterium containing the target gene after induction culture, OD 600 =0.5-0.6; the number of ginseng Alternaria spores in the mycelial suspension is 1×10⁻⁶. 5 spores / mL - 1×10 7 spores / mL.
[0017] Furthermore, in (1), the number of Alternaria ginseng spores in the mycelial suspension is 1 × 10⁻⁶. 6 spores / mL.
[0018] Further, in step (1), the induction culture includes: mixing the activated recombinant Agrobacterium bacterial suspension with IM induction liquid medium at a ratio of 1:9 to make 20 mL, adding 20 μL of 50 mg / mL kanamycin and 20 μL of 0.2 M acetylsyl syringone, and inducing in the dark at 28 ℃ to OD. 600 =0.5-0.6.
[0019] Further, in (1), the preparation method of the ginseng Alternaria mycelial suspension is as follows: sterile, enzyme-free water is placed on a PDA solid culture medium covered with ginseng Alternaria, and mycelia are scraped from the surface of the culture medium using a spreader, so that the number of ginseng Alternaria spores in the mycelial suspension is 1×10⁻⁶. 6 spores / mL.
[0020] Furthermore, in (2), the screening antibiotic is hygromycin B, and the screening concentration is 25 μg / mL.
[0021] Furthermore, in (1), the target gene is labeled with GFP;
[0022] In step (3), the transformation verification includes PCR primer amplification and fluorescence microscopy observation.
[0023] Furthermore, in (3), the PCR primers include:
[0024] GFP forward primer 5'-CTTCAAAGCAAGTGGATTGATG-3';
[0025] GFP reverse primer 5'-CGTCGCCGTCCAGCTCGACCAG-3';
[0026] Hygromycin B forward primer 5'-CTGTCGAGAAGTTTCTGATCG-3';
[0027] Hygromycin B reverse primer 5'-CTGATAGAGTTGGTCAAGACC-3'.
[0028] Furthermore, the gene transformation method is a gene overexpression transformation method.
[0029] Furthermore, the recombinant Agrobacterium containing the target gene is EHA105 containing pCAMBIA1300-35S-GFP-ApX, wherein ApX is the target gene of Alternaria ginseng.
[0030] By adopting the above technical solution, the present invention has at least the following beneficial effects:
[0031] (1) Breaking through the conventional "double-layer screening method" design, unexpected decontamination and high positive rate effects were achieved: Conventional transformation screening often uses direct application of co-cultures onto single-layer screening plates containing antibiotics, which easily leads to excessive proliferation of Agrobacterium and false positives in Alternaria ginseng transformation. This invention innovatively adopts a double-layer screening strategy of "after a thin layer of fungi (5-8 Alternaria ginseng colonies, with few hyphae and hyphal area accounting for 30%-60% of the total area of IM medium) and a few Agrobacterium (no more than 10), pouring in a layer of PDA solid medium containing hygromycin to cover it". This innovative design, which breaks away from conventional physical isolation and single-concentration screening, utilizes hygromycin to slowly diffuse from the upper PDA solid medium to the lower IM medium, creating a gradient distribution of antibiotic concentration from high to low between the two media. This unexpectedly and completely blocks the excessive proliferation of Agrobacterium. At the same time, the slow penetration of hygromycin provides sufficient recovery time for the transformed Alternaria ginseng, increasing the true positive rate of the screened clones to over 90% (compared to less than 10% in conventional methods), and greatly reducing false positive interference.
[0032] (2) The precisely defined parameter system overcomes the infection barrier: This invention does not use the conventional broad parameter range, but strictly locks in the Agrobacterium-induced OD. 600 The value is 0.5-0.6, and the concentration of the mycelial suspension containing spores is approximately 1×10⁻⁶. 5 spores / mL - 1×10 7The specific narrow range of spores / mL was used. This parameter combination, validated through extensive trial and error, achieves the optimal balance between Agrobacterium infection activity and fungal tolerance, even with the thick cell wall barrier of this specific fungus. This avoids fungal cell death due to over-infection or transformation failure due to insufficient infection. It provides a reliable technical means for transforming and validating *Alternaria ginseng* strains. Attached Figure Description
[0033] The above is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.
[0034] Figure 1 Screening chart of hygromycin B concentrations inhibiting the growth of Alternaria ginseng (0 / 25 / 50 / 100: PDA medium containing hygromycin B concentrations of 0 / 25 / 50 / 100 μg / mL; first row at plate formation; second row 5 days after plate formation).
[0035] Figure 2 The image shows the verification results of ApX-overexpressing Alternaria ginseng transformed with GFP / HygB amplification (M: Marker; 1-3: GFP colony PCR bands; 4-6: HygB colony PCR bands).
[0036] Figure 3 Fluorescence validation image of ApX-overexpressing Alternaria ginseng (Ap: wild-type Alternaria ginseng strain; ApX-GFP: pCAMBIA1300-35S-GFP-ApX-overexpressing Alternaria ginseng strain; TRANS: bright field; GFP: green fluorescence detection; MERGE: fusion).
[0037] Figure 4 The fluorescence verification image for Comparative Example 1 (Ap: wild-type Alternaria ginseng strain; ApX-GFP: pCAMBIA1300-35S-GFP-ApX overexpressing Alternaria ginseng strain; TRANS: bright field; GFP: green fluorescence detection; MERGE: fusion).
[0038] Figure 5 The fluorescence verification diagram for Comparative Example 2 (Ap: wild-type Alternaria ginseng strain; ApX-GFP: pCAMBIA1300-35S-GFP-ApX overexpressing Alternaria ginseng strain; TRANS: bright field; GFP: green fluorescence detection; MERGE: fusion). Detailed Implementation
[0039] Example 1
[0040] (1) Screening of hygromycin inhibitory concentrations in Alternaria ginseng
[0041] A 0.6 cm mycelial cake was formed on a PDA plate contaminated with *Alternaria ginseng* and then placed in PDA plates with hygromycin B concentrations of 0 μg / mL, 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. After incubation at 28℃, each group was divided into five replicates. Growth inhibition was observed after 5 days. Figure 1 The results showed that Alternaria ginseng did not grow in PDA plates containing hygromycin B at a concentration of 25 μg / mL. Therefore, this concentration (25 μg / mL) was selected as the screening concentration for overexpression transformation strains.
[0042] (2) Culture medium preparation method
[0043] ①MM liquid culture medium: K2HPO4 2.05 g / L, KH2PO4 1.45 g / L, NaCl 0.15 g / L, MgSO4·7H2O 0.50 g / L, CaCl2·2H2O 0.067 g / L, FeSO4·7H2O 0.0025 g / L, (NH4)2SO4 0.50 g / L, glucose 2.00 g / L.
[0044] ②IM liquid culture medium: K2HPO4 2.05 g / L, KH2PO4 1.45 g / L, NaCl 0.15 g / L, MgSO4·7H2O 0.50 g / L, CaCl2·2H2O 0.067 g / L, FeSO4·7H2O 0.0025 g / L, (NH4)2SO4 0.50 g / L, glucose 2.00 g / L, 0.5% (w / v) glycerol 5.00 mL, adjust pH to 5.3.
[0045] ③IM solid medium: Based on the above IM liquid medium components (K2HPO4 2.05 g / L, KH2PO4 1.45 g / L, NaCl 0.15 g / L, MgSO4·7H2O 0.50 g / L, CaCl2·2H2O 0.067 g / L, FeSO4·7H2O 0.0025 g / L, (NH4)2SO4 0.50 g / L, glucose 2.00 g / L, 0.5% (w / v) glycerol 5.00 mL), add 15.00 g / L agar and adjust the pH to 5.3.
[0046] (3) Preparation of Alternaria ginseng mycelial suspension
[0047] Add 10 mL of sterile, enzyme-free water to the surface of the PDA solid culture medium contaminated with Alternaria ginseng. Gently scrape the mycelium from the surface of the medium using a spreader (be careful not to scrape the medium itself) until the number of Alternaria ginseng spores in the mycelial suspension reaches approximately 1 × 10⁻⁶. 6 spores / mL.
[0048] (4) Activation, induction culture and co-culture transformation of Agrobacterium
[0049] Single colonies of the transformed pCAMBIA1300-35S-GFP-ApX Agrobacterium were picked and inoculated into 20 mL of the MM liquid medium prepared above (containing 20 μL of 50 mg / mL kanamycin), and incubated at 28 ℃ and 220 rpm / min for 48 h for activation.
[0050] After activation, the bacterial culture was mixed with IM induction liquid medium at a ratio of 1:9, with a total volume of 20 mL, and placed in a 100 mL centrifuge tube. 20 μL of 50 mg / mL kanamycin and 20 μL of 0.2 M acetylsalicylic acid were added to the system, and the mixture was induced in the dark at 28 °C to OD. 600 It reaches 0.5~0.6.
[0051] The induced Agrobacterium tumefaciens solution was mixed with an equal volume of the prepared Alternaria ginseng mycelial suspension, and then evenly spread on IM solid medium and incubated in the dark at 28 °C.
[0052] (5) Transformation screening and GFP validation
[0053] After co-culturing for 3 days, when a thin layer of *Alternaria ginseng* colonies and sporadic *Agrobacterium* colonies have grown on the culture medium (i.e., 5-8 *Alternaria ginseng* colonies are observed, with the mycelial area of the colonies accounting for 30%-60% of the total area of the IM medium, and the number of *Agrobacterium* plaques not exceeding 10. In this example, 6 *Alternaria ginseng* colonies were observed, with the mycelial area of the colonies accounting for approximately 50% of the total area of the IM medium, and the number of *Agrobacterium* plaques 5), a layer of PDA solid medium containing hygromycin B 25 μg / mL was poured onto the IM medium for double-layer screening.
[0054] Once Alternaria ginseng colonies have grown again on the culture medium, their hyphae are picked for verification.
[0055] PCR amplification was performed using the designed primers for verification.
[0056] GFP identification was performed using primers GFP-F (5'-CTTCAAAGCAAGTGGATTGATG-3', SEQ ID NO:1) and GFP-R (5'-CGTCGCCGTCCAGCTCGACCAG-3', SEQ ID NO:2). Hygromycin resistance identification was performed using primers Hyg BF (5'-CTGTCGAGAAGTTTCTGATCG-3', SEQ ID NO:3) and Hyg BR (5'-CTGATAGAGTTGGTCAAGACC-3', SEQ ID NO:4).
[0057] The results showed that the target band of pCAMBIA1300-35S-GFP-ApX overexpressing Alternaria ginseng was successfully obtained (see [link]). Figure 2 ).
[0058] Subsequently, further observation using a fluorescence microscope revealed obvious green fluorescence (GFP signal), confirming that pCAMBIA1300-35S-GFP-ApX had been successfully expressed in *Alternaria ginseng* (see [link to original text]). Figure 3 ).
[0059] It should be noted that the number of Alternaria ginseng spores in the mycelial suspension in the above experiment was approximately 1 × 10⁻⁶. 6 spores / mL, actually 1×10 5 spores / mL - 1×10 7 spores / m can achieve a similar effect.
[0060] Comparative Example 1
[0061] The only difference between this comparative example and Example 1 is the method of "step (5) conversion screening". The other steps and parameters are completely the same.
[0062] In this comparative example, after co-culture, instead of using a double-layer culture medium to cover the culture, the co-culture was washed with sterile water and then directly spread onto a single-layer solid PDA medium containing the same concentration (25 μg / mL) of hygromycin B for screening.
[0063] Experimental Results: After 5 days of culture, severe Agrobacterium overgrowth (appearing as a viscous bacterial thicket) was observed on the monolayer screening plate, and the growth of *Alternaria ginseng* was severely inhibited. Fifty suspected resistant fungal clones that barely grew were selected for PCR amplification verification (primers same as in Example 1), and the results showed no target gene band. Fluorescence microscopy also failed to detect GFP signal, confirming that the monolayer screening method cannot obtain effective transformed strains (see reference). Figure 4In contrast, Example 1 used a two-layer screening method, and of the 30 resistant clones selected, 28 were PCR positive, with a true positive rate as high as 93.3%, and the plates were clean and free of Agrobacterium contamination. Figure 3 ).
[0064] Comparative Example 2
[0065] The difference between this comparative example and Example 1 lies in the concentration of the ginseng Alternaria mycelial suspension and the induction conditions of Agrobacterium.
[0066] In this comparative example, the concentration of the prepared spore suspension was too high (approximately 1 × 10⁻⁶). 8 (spores / mL), and Agrobacterium was not strictly controlled in terms of induced OD value (directly used activated to OD). 600 Overnight cultures of >1.0 were co-cultured in equal volumes, and the remaining double-layer screening steps were the same as in Example 1.
[0067] Experimental results: After double-layer screening, almost no hygromycin-resistant *Alternaria ginseng* colonies grew on the plates. This is because when excessively high fungal spore concentrations are mixed with overly aged *Agrobacterium*, the *Agrobacterium* T-DNA transfer efficiency decreases significantly, and it fails to effectively penetrate the fungal barrier, leading to complete transformation failure. Figure 5 ).
[0068] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims and their equivalents, this invention also intends to include these modifications and variations. The above description is merely a preferred embodiment of the invention and is not intended to limit the invention in any way. Any simple modifications, equivalent changes, or alterations made by those skilled in the art using the disclosed technical content fall within the protection scope of this invention.
Claims
1. A method for Agrobacterium-mediated transformation of ginseng Alternaria gene, characterized in that, include: (1) Co-cultivation: The recombinant Agrobacterium containing the target gene, after activation and induction culture, was co-cultured with a suspension of Alternaria ginseng mycelium on IM medium. (2) Screening: After co-culturing, when 5-8 Alternaria ginseng colonies are observed, the mycelial area of the colonies accounts for 30%-60% of the total area of the IM medium, and the number of Agrobacterium plaques does not exceed 10, pour a layer of PDA solid medium containing screening antibiotics onto the IM medium for double-layer screening. (3) Verification: Once the ginseng Alternaria colonies have grown again, their hyphae will be selected for transformation verification.
2. The method for Agrobacterium-mediated transformation of ginseng Alternaria gene according to claim 1, characterized in that, In (1), the recombinant Agrobacterium containing the target gene after induction culture, OD 600 =0.5-0.6; the number of Alternaria ginseng spores in the mycelial suspension is 1×10⁻⁶. 5 spores / mL - 1×10 7 spores / mL.
3. The method for Agrobacterium-mediated transformation of ginseng Alternaria gene according to claim 2, characterized in that, In (1), the number of Alternaria ginseng spores in the mycelial suspension is 1 × 10⁻⁶. 6 spores / mL.
4. The method for Agrobacterium-mediated transformation of ginseng Alternaria gene according to claim 2, characterized in that, In step (1), the induction culture includes: mixing the activated recombinant Agrobacterium bacterial suspension with IM induction liquid medium at a ratio of 1:9 to make 20 mL, adding 20 μL of 50 mg / mL kanamycin and 20 μL of 0.2 M acetylsyl syringone, and inducing in the dark at 28 ℃ to OD. 600 =0.5-0.
6.
5. The method for Agrobacterium-mediated transformation of ginseng Alternaria gene according to claim 2, characterized in that, In (1), the preparation method of the Alternaria ginseng hyphae suspension is as follows: sterile, enzyme-free water is placed on a PDA solid culture medium covered with Alternaria ginseng, and the hyphae on the surface of the culture medium are scraped off using a spreader, so that the number of Alternaria ginseng spores in the hyphae suspension is 1×10⁻⁶. 6 spores / mL.
6. The method for Agrobacterium-mediated transformation of Alternaria ginseng gene according to claim 1, characterized in that, In (2), the screening antibiotic is hygromycin B, and the screening concentration is 25 μg / mL.
7. The method for Agrobacterium-mediated transformation of ginseng Alternaria gene according to claim 1, characterized in that, In (1), the target gene is labeled with GFP; In step (3), the transformation verification includes PCR primer amplification and fluorescence microscopy observation.
8. The method for Agrobacterium-mediated transformation of ginseng Alternaria gene according to claim 7, characterized in that, In (3), the PCR primers include: GFP forward primer 5'-CTTCAAAGCAAGTGGATTGATG-3'; GFP reverse primer 5'-CGTCGCCGTCCAGCTCGACCAG-3'; Hygromycin B forward primer 5'-CTGTCGAGAAGTTTCTGATCG-3'; Hygromycin B reverse primer 5'-CTGATAGAGTTGGTCAAGACC-3'.
9. The method for Agrobacterium-mediated transformation of ginseng Alternaria gene according to claim 1, characterized in that, The gene transformation method is a gene overexpression transformation method.
10. The method for Agrobacterium-mediated transformation of ginseng Alternaria gene according to claim 9, characterized in that, The recombinant Agrobacterium containing the target gene is EHA105 containing pCAMBIA1300-35S-GFP-ApX, where ApX is the target gene of Alternaria ginseng.