Agrobacterium tumefaciens-mediated banana leaf transformation method

Abstract

The invention discloses an agrobacterium tumefaciens-mediated banana leaf transformation method, and belongs to the technical field of plant molecular biology. The method comprises the following steps: selecting fresh banana leaves and disinfecting; transforming the plasmid with the target gene into an agrobacterium competent cell; selecting positive agrobacterium monoclone, transferring the positive agrobacterium monoclone into an antibiotic-containing LB liquid culture medium, and culturing; resuspending the thalli by using the infection suspension to obtain an infection solution; after hole pricking treatment is conducted on banana leaves, the infection liquid permeates into leaf tissues through vacuum infection; after infection, culturing for 3-4 days under a room-temperature dark condition; finally, the expression condition of the target gene is verified through RT-PCR. According to the method, distilled water is sprayed to the leaves after vacuum infection and vacuumizing, plant withering and death are avoided, transient expression of exogenous genes in the banana leaves is achieved, operation is easy and convenient, the period is short, conversion efficiency is high, a reliable technical platform is provided for banana functional gene research and disease-resistant mechanism analysis, and the method is suitable for large-scale popularization and application. The method has important scientific research and application values.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biotechnology, and more specifically, relates to an Agrobacterium-mediated transformation method for banana leaves. Background Technology

[0002] banana( Musa spp. ) is a member of the Musaceae family ( Musaceae Musa genus ( Musa L. Bananas are an important fruit and a major food crop, widely cultivated in tropical and subtropical regions, and have significant economic value globally. Rich in potassium, calcium, magnesium, vitamin C, and dietary fiber, bananas are highly effective in boosting immunity and regulating intestinal function. Therefore, bananas are in high demand worldwide and have become an indispensable part of the daily diet in many countries.

[0003] Currently, banana genetic transformation technologies mainly employ protoplast transformation, gene gun methods, and Agrobacterium-mediated transformation. Among these, Agrobacterium-mediated transformation is the most widely used plant genetic transformation method, but its transformation efficiency in bananas has remained low. As a monocotyledonous plant, bananas exhibit significant differences in cell wall structure and physiological characteristics compared to dicotyledonous plants, leading to low Agrobacterium infection efficiency. Furthermore, the long growth cycle, low reproduction coefficient, and complex genetic background of bananas have slowed progress in transgenic research.

[0004] Transient expression is a technique for obtaining short-term, high-level expression of a target gene. Compared to stable expression, transient expression requires less time, but it does not integrate the target gene into the host plant's gamete chromosome and cannot be stably inherited by the next generation. Due to its advantages such as simplicity, low cost, short cycle time, and good transformation efficiency, it has been widely used in biological research. Among these techniques, Agrobacterium-mediated transient expression is the most common.

[0005] Currently, research on transient expression systems in bananas is limited. Existing methods mostly employ gene gun methods or PEG-mediated protoplast transformation, which suffer from problems such as complex operation, low transformation efficiency, and long cycle times. Therefore, developing an efficient and simple Agrobacterium-mediated transient expression method suitable for bananas is of great significance for functional gene research and molecular biology studies in bananas. Summary of the Invention

[0006] The purpose of this invention is to provide an Agrobacterium-mediated transformation method for banana leaves that is simple to operate, highly efficient, and has a short cycle, enabling efficient and transient expression of exogenous genes in banana leaves, and providing a technical platform for banana functional gene research.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention provides an Agrobacterium-mediated transformation method for banana leaves, comprising the following steps: (1) Select green leaves from banana plants that are growing well and free from pests and diseases, disinfect the leaves with alcohol and place them in clean water for later use. (2) The plasmid carrying the exogenous target gene was transformed into Agrobacterium competent cells, cultured in a constant temperature incubator at 28℃, single clones were picked, and positive Agrobacterium carrying the target gene was obtained by PCR detection; (3) Select positive Agrobacterium single clones and transfer them into LB liquid medium containing the corresponding antibiotics. Incubate overnight in a shaker. Centrifuge the shaken bacterial solution at room temperature, discard the supernatant, collect the bacterial cells, resuspend them in the infection suspension to obtain the infection solution, and let it stand at 28°C for 3 hours. (4) Select the banana leaves from step (1), gently poke a hole at the leaf tip with the needle of a syringe, then put the leaves into the infiltration solution prepared in step (3), and then vacuum infiltrate the banana leaves so that the infiltration solution penetrates into the banana leaves to obtain infiltrated banana leaves. Then, culture the banana leaves in the dark at room temperature for 3 to 4 days. (5) Remove the banana leaves after in vitro culture in step (4), freeze them with liquid ammonia, extract RNA, reverse transcribe to obtain cDNA, and then perform RT-PCR detection to confirm the expression of the target gene.

[0008] Furthermore, in step (1), the size of the leaf is 5~20cm.

[0009] Further, in step (2), the competent Agrobacterium cells are Agrobacterium strain GV3101, and the plasmid is pTCK303-eGFP vector.

[0010] Furthermore, in step (2), the exogenous target gene is FoSSP1, a gene in fungi that encodes effector proteins.

[0011] Furthermore, in step (3), the LB liquid culture medium is an LB liquid culture medium with rifampicin and kanamycin resistance.

[0012] Furthermore, in step (3), the centrifugation conditions are 5000 rpm and 5 min.

[0013] Further, in step (3), the contamination suspension specifically includes 10 mM of 2-(N-morpholine)ethanesulfonic acid, 10 mM of magnesium chloride and 100 μM of acetylsuccinone.

[0014] Furthermore, in step (3), the optical density OD of the dyeing solution at a wavelength of 600 nm is 0.8.

[0015] Furthermore, in step (4), the vacuum impregnation pressure is 0.07 MPa and the time is 15 min.

[0016] Furthermore, in step (4), the banana leaves are treated with distilled water spray after vacuum impregnation.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) Based on the current situation that it is difficult to efficiently and quickly express genes transiently in bananas, this invention proposes a method for achieving transient overexpression of target genes on the outer edge of banana leaves by vacuum-mediated by Agrobacterium, which realizes efficient transient expression of target genes in banana leaves.

[0018] (2) This invention uses vacuum treatment on banana leaf tissue to make Agrobacterium tumefaciens liquid flow into leaf tissue by vacuum pressure, which reduces damage to the leaf and enables large-area infection of the whole leaf by Agrobacterium tumefaciens. It also enables transient expression of exogenous genes in banana leaves, laying the foundation for molecular biology research on banana functional genes.

[0019] (3) The present invention treats the plant with high humidity after vacuuming. Since vacuuming may damage the plant leaves and cause them to wilt, the present invention sprays the leaves with distilled water after vacuuming and sprays a large amount of distilled water on the protective cover and covers the plant overnight to maintain the plant's moisture and prevent the plant from wilting and dying, thus facilitating the infection of Agrobacterium. Attached Figure Description

[0020] Figure 1 The gene containing the fungal gene encoding an effector protein obtained in Example 1 of this invention. FoSSP1 The image shows the PCR verification results of positive Agrobacterium.

[0021] Figure 2 This is a schematic diagram of the banana leaf treatment and infection process in Embodiment 3 of the present invention.

[0022] Figure 3 Example 3 of the present invention shows the condition of banana leaves after infection (a) and the transient expression of the gene encoding the effector protein in the fungus. FoSSP1 Then, the results of gene expression verification by semi-quantitative RT-PCR are shown in Figure (b). Detailed Implementation

[0023] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0024] Example 1: Obtaining positive Agrobacterium tumefaciens Genes containing exogenous genes (genes encoding effector proteins in fungi) FoSSP1The plasmid pTCK303-eGFP (whose nucleotides are shown in SEQ ID NO.1) was transformed into Agrobacterium GV3101 using the freeze-thaw method. The transformation process was as follows: (1) Take 3 μl (about 100-200 ng) of plasmid and add it to 100 μl of competent cells, and incubate on ice for 5 min.

[0025] (2) Transfer to liquid nitrogen, freeze for 5 min, and then incubate in a water bath at 37°C for 5 min; (3) Add 700 μl of antibiotic-free LB liquid medium, place in a shaker at 28°C, and incubate at 180 rpm for 2 h; (4) Centrifuge at 4500 rpm for 5 min at room temperature, discard most of the supernatant, and resuspend the precipitate in 100 μl of culture medium; (5) Spread the resuspension onto LB solid plates (containing 25 μg / ml rifampicin and 50 μg / ml kanamycin) and incubate at 28°C for 48 h; (6) Select single clones and perform colony PCR and agarose gel electrophoresis for verification. The results are as follows: Figure 1 As shown, the amplification of the target band indicates that positive Agrobacterium was successfully obtained.

[0026] Example 2: Preparation of Infection Solution The single colonies of Agrobacterium, which were verified as positive in Example 1, were transferred to 5-10 mL of LB liquid medium containing rifampicin and kanamycin (10 g / L sodium chloride, 5 g / L yeast extract, 10 g / L tryptone) and cultured overnight in a shaker (28°C, 200 rpm / min). The bacterial culture was then aliquoted into sterile centrifuge tubes, centrifuged at 5000 rpm for 5 minutes, the supernatant was discarded, and the bacterial cells were collected. The bacterial cells were resuspended in an infection suspension (10 mM 2-(N-morpholine) ethanesulfonic acid, 10 mM magnesium chloride, 100 μM acetylsyl syringone) to achieve an optical density (OD) of 0.8 at 600 nm. The culture was then incubated at room temperature in the dark for 3 hours.

[0027] Example 3: Banana leaf infection Select healthy, disease-free green leaves from banana plants. Disinfect the leaves and place them in clean water for later use. The leaves should be 5-20cm in size. Figure 2 As shown in a.

[0028] Take banana leaves and gently puncture the epidermis at the leaf margin on the back of the banana leaf with a disposable syringe needle. Then, immerse the selected banana leaves below the surface of the infection solution prepared in Example 2. Place the beaker along with the banana leaves in a vacuum container and use a vacuum pump to evacuate the container to a vacuum osmotic pressure of 0.07 MPa. Vacuum permeation under vacuum for 15 minutes allows the Agrobacterium infection solution to completely penetrate the leaf mesophyll tissue. Figure 2f). Then remove the leaves, spray them with distilled water, and spray a large amount of distilled water on the protective cover. Cover the plant overnight to maintain its moisture and prevent it from wilting and dying, thus facilitating infection by Agrobacterium.

[0029] Example 4: RNA extraction and RT-PCR detection of gene expression After the banana leaves infected in Example 3 were cultured in the dark at room temperature for 3-4 days, 3 leaves were randomly selected.

[0030] For each leaf, the injected portion was cut off and flash-frozen in liquid nitrogen. The leaf was then ground into a powder using a mortar and pestle and placed into a 2 mL centrifuge tube. RNA was extracted using the Eastp® Super Total RNA Extraction Kit. The quality of RNA extraction was assessed using a micro spectrophotometer and agarose gel electrophoresis. The RNA was reverse transcribed into cDNA. The expression of the exogenous gene was then detected using the cDNA as a template.

[0031] The results are as follows Figure 3 As shown in (b), exogenous genes were detected in all three banana leaves. FoSSP1 To prove the target gene FoSSP1 It was successfully expressed in banana leaves.

[0032] In summary, this invention addresses the challenge of transient gene expression in bananas by employing a vacuum-induced perforation and infection process on banana leaves. This method enables Agrobacterium-mediated transformation of target genes, establishing a transient gene expression mechanism based on Agrobacterium vacuum-mediated transformation in banana leaves. This approach offers advantages such as simple operation, short transformation cycle, and high transformation efficiency. This invention achieves transient expression of target genes in living banana material using the Agrobacterium vacuum infection method, saving on transformation cycle and cost. It lays the foundation for functional gene and molecular biology research in bananas and possesses high practical and scientific application value.

Claims

1. An Agrobacterium-mediated transformation method for banana leaves, characterized in that, Includes the following steps: (1) Select green leaves from banana plants that are growing well and free from pests and diseases, disinfect the leaves with alcohol and place them in clean water for later use. (2) The plasmid carrying the exogenous target gene was transformed into Agrobacterium competent cells, cultured in a constant temperature incubator at 28℃, single clones were picked, and positive Agrobacterium carrying the target gene was obtained by PCR detection; (3) Select positive Agrobacterium single clones and transfer them into LB liquid medium containing rifampicin and kanamycin. Incubate overnight in a shaker. Centrifuge the shaken bacterial solution at room temperature, discard the supernatant, collect the bacterial cells, and resuspend them in an infection suspension containing 10 mM 2-(N-morpholine) ethanesulfonic acid, 10 mM magnesium chloride and 100 μM acetylsyl syringone to obtain the infection solution. Let it stand at 28°C for 3 h. (4) Select the banana leaves from step (1), gently poke a hole at the leaf tip with the needle of a syringe, then put the leaves into the infiltration solution prepared in step (3), and then vacuum infiltrate the banana leaves so that the infiltration solution penetrates into the banana leaves to obtain infiltrated banana leaves. Then, culture the banana leaves in the dark at room temperature for 3 to 4 days. (5) Remove the banana leaves after in vitro culture in step (4), freeze them with liquid ammonia, extract RNA, reverse transcribe to obtain cDNA, and then perform RT-PCR detection to confirm the expression of the target gene.

2. The Agrobacterium-mediated transformation method for banana leaves according to claim 1, characterized in that, In step (1), the size of the leaf is 5~20cm.

3. The Agrobacterium-mediated transformation method for banana leaves according to claim 1, characterized in that, In step (2), the competent Agrobacterium cells are Agrobacterium strain GV3101, and the plasmid is pTCK303-eGFP vector.

4. The Agrobacterium-mediated transformation method for banana leaves according to claim 1, characterized in that, In step (2), the exogenous target gene is FoSSP1, a gene in fungi that encodes effector proteins.

5. The Agrobacterium-mediated transformation method for banana leaves according to claim 1, characterized in that, In step (3), the centrifugation conditions are 5000 rpm and 5 min.

6. The Agrobacterium-mediated transformation method for banana leaves according to claim 1, characterized in that, In step (3), the optical density OD of the dyeing solution at a wavelength of 600 nm is 0.

8.

7. The Agrobacterium-mediated transformation method for banana leaves according to claim 1, characterized in that, In step (4), the vacuum impregnation pressure is 0.07 MPa and the time is 15 min.

8. The Agrobacterium-mediated transformation method for banana leaves according to claim 1, characterized in that, In step (4), the banana leaves are sprayed with distilled water after vacuum inoculation.

Images