A highly efficient genetic transformation method for grape seeds that does not rely on tissue culture

Abstract

The invention discloses a highly efficient genetic transformation method for grape seeds that is not dependent on tissue culture, and relates to the fields of agricultural biotechnology and plant genetic engineering. The genetic transformation comprises the following steps: sterilizing grape seeds and then performing skin cutting, and then pre-culturing until the abdomen shell cracks; placing grape seeds with abdomen shell cracks in an impregnation solution for impregnation treatment and then planting them in a culture matrix to culture transgenic grape plants; the impregnation solution contains a recombinant engineering strain capable of expressing heterologous genes. The invention uses grape seeds as test materials, and successfully introduces exogenous genes into the genome of grapes through an Agrobacterium-mediated method, thereby achieving genetic transformation of grapes and obtaining transgenic grape plants. The transformation method of the invention effectively overcomes the defects of traditional genetic transformation technology, can achieve genetic transformation without relying on tissue culture technology, and has high transformation efficiency and convenient operation.

Description

Technical Field

[0001] The invention relates to the fields of agricultural biotechnology and plant genetic engineering, and in particular to a highly efficient genetic transformation method for grape seeds which is not dependent on tissue culture. Background Art

[0002] Grape (Vitis L.), belonging to the Vitaceae family, is one of the most widely planted economic fruit trees in the world. It is known for its unique taste and high nutritional value and is regarded as the first of the "four major fruits in the world". Grapes are not only nutritious and widely used, but also have bright colors, fragrant aromas, and delicious tastes. They are suitable for fresh consumption, winemaking, juice making, and dried fruit production. Among them, the 'Cabernet Sauvignon' variety is known for its easy cultivation, high juice yield, and excellent sugar-acid ratio. The wine brewed from it has a ruby ​​red color, a mellow taste, and a unique flavor. It is deeply favored by consumers and has extremely high economic value. However, the traditional hybrid breeding method has a long cycle and low efficiency, and it is difficult to meet the needs of rapidly breeding new varieties. In contrast, genetic engineering technology provides a more efficient way to breed new varieties. Although the research on grape transgenic technology has been relatively extensive, the research on grape seed transgenic under non-tissue culture (abbreviated as non-tissue culture) conditions is still blank.

[0003] In the Agrobacterium-mediated genetic transformation of grapes, commonly used receptor materials generally include leaves, petioles, anthers, filaments, stem tips, and stem segments. These materials come from different growth stages of grapes and have obvious temporal and spatial differences. In order to improve the transformation efficiency and create new varieties, it is crucial to select ideal transformation materials. Ideal materials should meet the following conditions: first, the treatment method is uniform and the repeatability is high; second, the degree of differentiation is low, the cell totipotency is strong, and there is the potential to develop into plants; third, it is easy to obtain, reducing the interference of material collection on the experiment; fourth, the quantity is sufficient, which is conducive to successful transformation. Grape seeds are excellent in quantity, uniformity, repeatability, easy to obtain, and cell totipotency, and are ideal genetic transformation materials. However, traditional grape Agrobacterium genetic transformation requires tissue culture, which has a long cycle, high contamination rate, and easy death of plants. Therefore, developing a non-tissue culture-dependent grape genetic transformation method is particularly critical for creating new varieties. Summary of the invention

[0004] The purpose of the present invention is to provide a grape seed efficient genetic transformation method that is not dependent on tissue culture to solve the problems existing in the above-mentioned prior art. The method effectively overcomes the defects of traditional genetic transformation technology, can achieve genetic transformation on the basis of not relying on tissue culture technology, and has high transformation efficiency and convenient operation.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for efficient genetic transformation of grape seeds that is independent of tissue culture, comprising the following steps:

[0007] The grape seeds are sterilized and then peeled, and then pre-cultured until the belly shell cracks;

[0008] placing grape seeds with cracked abdomen shells in an impregnation solution for impregnation treatment, and then planting them in a culture matrix to culture transgenic grape plants;

[0009] The infecting solution contains a recombinant engineered strain capable of expressing a heterologous gene.

[0010] Furthermore, the disinfection treatment includes alcohol disinfection and sodium hypochlorite solution disinfection.

[0011] Furthermore, the grape seeds are soaked in gibberellin before being sterilized.

[0012] Furthermore, the skin cutting process refers to scratching the back seed coat and beak of the grape seeds.

[0013] Furthermore, the dyeing solution also includes acetosyringone.

[0014] Furthermore, the concentration of acetosyringone is 100 mM.

[0015] Furthermore, the impregnation solution is obtained by adding acetosyringone to a bacterial solution containing the recombinant engineered strain and culturing in dark.

[0016] Furthermore, the dark culture time is 2 hours.

[0017] Furthermore, the OD of the bacterial solution 600 =0.6.

[0018] Furthermore, the vacuum impregnation treatment and the shaking infection are performed, the time for the vacuum impregnation treatment is 6 minutes, and the time for the shaking infection is 14 minutes.

[0019] The present invention discloses the following technical effects:

[0020] The present invention uses grape seeds as test materials and successfully introduces exogenous genes into the grape genome through an Agrobacterium-mediated method, thereby achieving genetic transformation of the grape and obtaining transgenic grape plants.

[0021] The genetic transformation method of the present invention is directly operated on grape seeds without tissue culture, and has the following advantages:

[0022] (1) Simple operation. It avoids a series of tedious processes required in the plant tissue culture process, such as medium preparation, explant disinfection, callus induction, subculture, co-cultivation, screening, regeneration bud induction, and rooting.

[0023] (2) Short growth cycle. It only takes 35-40 days to obtain transgenic grape seedlings, which is shorter than the traditional tissue culture method (85 days).

[0024] (3) High repeatability and universality. The material processing method is unified, simple, and easy to repeat.

[0025] (4) High economic benefits. The present invention adopts a method of directly using Agrobacterium liquid to infect grape seeds for genetic transformation, and uses a nutrient matrix to culture grape plants. Compared with the traditional plant tissue culture transformation process, this method significantly reduces the input of manpower and material resources, thereby greatly reducing the overall cost of genetic transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 The flowchart of seed transformation of the present invention is shown in the figure; wherein A is the activation of the engineering bacterial solution of the VvBBM gene carrier and the preparation of the infection solution; B is obtaining grape seeds, treating them in 2.5g / L GA3 and then cutting the skin; C is clamping the grape seeds that have been pre-cultured until the abdomen shell cracks into the infection solution; D is vacuuming the grape seeds and manually shaking them for infection; E is planting the grape seeds that have been dried of the bacterial solution into a high-temperature sterilized nutrient medium to cultivate them into complete plants;

[0028] Figure 2 The statistical results of seed germination of wild-type grape (UT) and transgenic grape (OE-VvBBM);

[0029] Figure 3 Plant height, root length, and total root number of UT and OE-VvBBM plants grown from seeds for 38 days;

[0030] Figure 4 The images are of UT and OE-VvBBM detected under a fluorescence confocal microscope;

[0031] Figure 5 PCR identification diagram of grape plants; among them, #1, #3, #5, #7, #12 and #13 showed positive bands; NE was the ddH2O negative control group; PO was the VvBBM plasmid positive control group;

[0032] Figure 6The expression level detection results of OE-VvBBM transgenic plants; a is the typical phenotype of overexpression plants; b is the relative expression level detection result of VvBBM;

[0033] Figure 7 Western blot (A) and PCR amplification (B) detection of VaMIEL1. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] The culture medium preparation method used in the following examples is as follows:

[0040] LB liquid medium: 10g / L sodium chloride + 10g / L peptone + 5g / L yeast extract.

[0041] LB solid medium: 10g / L sodium chloride + 10g / L peptone + 5g / L yeast extract + 15g / L agar.

[0042] The present invention has developed a highly efficient and rapid non-tissue culture-dependent Agrobacterium-mediated genetic transformation method for grape seeds. The transformation flow chart is shown in Figure 1 The method is described in detail as follows:

[0043] Example 1

[0044] 1. Construction of plant expression vector of VvBBM gene

[0045] Using the cDNA of somatic cells of 'Chardonnay' grapes as a template, primers 2300-BBM-Bam-F and 2300-BBM-Sal-R were used for PCR amplification to obtain a PCR product (SEQ ID NO.1). The vector pHZM03 was digested with SalI and SacI restriction endonucleases, respectively, and the reaction conditions were 30℃15min, 37℃30min, and 65℃20min. The purified and recovered PCR products and the digested products were subjected to homologous recombination reaction. The PCR reaction program was 55℃30min, 30℃20min. The recombinant solution was transformed into kanamycin (Kan)-resistant LB solid medium and inverted in a 37℃ constant temperature incubator for overnight culture. Pick a single colony, inoculate it into LB medium containing KAN, place it in a 37℃ constant temperature shaker, 180rpm, 16h, and extract the plasmid. Determine the concentration and purity. Perform enzyme digestion detection. Take 20μL of plasmid and send it to the company for sequencing.

[0046] SEQ ID NO.1:

[0047]

[0048] 2300-BBM-Bam-F:5'-ATGGCTTCCATGAACAACTGGT-3' (SEQ ID NO.2);

[0049] 2300-BBM-Sal-R: 5'-TGTATCATTCCATACTGTGAAGTGT-3' (SEQ ID NO. 3).

[0050] 2. Preparation of Agrobacterium

[0051] Select the correct recombinant plasmid and add it to the competent cell EHA105, mix well, and transfer it to the electroporation cup. Start the Eppendorf4308 electroporation instrument for electroporation transformation, and set the transformation parameters: 1.8KV, 5ms. Then, incubate in a shaker at 28℃, 180rpm for 2h, and apply Kan + Plate with Rif and culture in a 28°C incubator for 3 days. Pick the positive monoclonal colony and culture it in a 28°C shaker at 180 rpm for 48 hours.

[0052] Pipette 1mL of bacterial solution and add it to 50mL containing 50mg / L Kan + and 25 mg / L rifamycin (Rif) in LB (or YEP) liquid medium at 28°C with shaking at 180 rpm until OD 600 Reach 1.0; mix the bacterial solution with 30% glycerol in a 1:1 ratio and store in a -80℃ refrigerator for later use.

[0053] 3. Preparation of Receptor for Genetic Transformation of 'Cabernet Sauvignon' Grape Seeds

[0054] Healthy and plump 'Cabernet Sauvignon' seeds were selected, ultrasonically treated in a 2.5g / L gibberellin (GA3) solution for 2min, soaked at 55℃ for 13min, and then shaken in a shaker at 28℃ and 180rpm for 24h, then treated with 75% alcohol by volume for 30s, rinsed with sterile water 3 times, and then treated with sodium hypochlorite with an effective chlorine mass fraction of 1.5% for 15min, rinsed with sterile water 3 times, and in an ultra-clean workbench, the seed coat on the back of the seeds was cut with a knife, and the beak was scratched forcefully, and pre-cultured until the abdominal shell cracked.

[0055] 4. Genetic transformation of grape seeds

[0056] (1) Prepare the infection solution. Activate the Agrobacterium obtained in step 2, collect the cells, and resuspend them in sterile water to an OD of 600 =0.6, and add 100 mM acetosyringone at the same time, then place it in a shaker at 180 rpm and culture it in the dark for 2 hours to obtain the infection solution for use.

[0057] (2) Infection. In a laboratory with a sterile environment (i.e., not operated on a clean bench), Cabernet Sauvignon seeds that had been pre-cultured until there were obvious shell cracks on the abdomen were placed in the infection solution. After vacuuming for 6 minutes and shaking with the wrist for 14 minutes, the bacterial solution was dried with filter paper and then planted in a high-temperature sterilized nutrient matrix. The seeds were cultured at 25°C, 16 h light / 8 h dark for 25 days, and then fluorescence detection was performed. At the same time, seeds that were not infected with Agrobacterium were set as negative controls.

[0058] 5. Identification of transgenic grape plants

[0059] Under non-tissue culture conditions, by the 20th day, a total of 54 grape plants had germinated, with a germination rate of 75% ( Figure 2 ). On the 40th day, 14 plants with good growth and robustness were selected for identification of transgenic grape plants.

[0060] (1) The grape plants were observed using a fluorescence confocal microscope, and the positive seedlings were initially screened for those with fluorescent signal excitation. The wild-type grape (UT) and transgenic grape plants (OE-VvBBM) were detected under a fluorescence confocal microscope as shown in the figure. Figure 4 shown.

[0061] (2) Seedlings with fluorescent signals were selected for PCR detection. DNA from grape plant leaves was extracted using the DNAsecure Plant Kit (TIANGEN) as a template. Semi-BBM-F: 5'-GTAAGCAAGAGCAAGACCCTGATG-3' (SEQ ID NO. 4) and 2300-R: 5'-CTCGACCAGGATGGGCACC-3' (SEQ ID NO. 5) were used for PCR identification. Untransformed 'Cabernet Sauvignon' plants were used as controls. The results showed that all six grape plants had the target band, indicating that the VvBBM gene had been successfully inserted into the grape genome ( Figure 5 ).

[0062] (3) We selected 14 transformed grape plants for fluorescence quantitative PCR detection and found that the expression levels of VvBBM genes in transgenic grape plants were significantly higher than those in control plants ( Figure 6 ).

[0063] (4) By comparing wild-type grapes and transgenic grape plants, it was found that ( Figure 3 ), the growth vigor of transgenic grape plants was significantly higher than that of untransformed plants.

[0064] Based on the above results, it is preliminarily determined that under non-sterile conditions, the transformation rate can reach 42.85% using Cabernet Sauvignon seeds as the receptor material for transformation.

[0065] Example 2 Application of genetic transformation to other genes under non-tissue culture conditions

[0066] The target gene was successfully transferred into the 'Chardonnay' grape plant by using the same transformation method as in Example 1, using Agrobacterium EHA105 as the bioengineering strain and the plant expression vector pHZM03 carrying the transcription factor VaMIEL1 (SEQ ID NO.6) and the screening reporter gene GFP. On the 40th day after transformation, protein and genomic DNA were extracted from the grape plant, and tested by Western blotting and PCR amplification experiments, and specific protein bands and PCR bands were successfully detected ( Figure 7 ). Western blotting was performed again on the 60th day, and the same band was detected, thus confirming the long-term stable expression of the transferred gene in the grape plants.

[0067] SEQ ID NO.6:

[0068] .

[0069] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for efficient genetic transformation of grape seeds that does not rely on tissue culture, characterized in that: The following steps are involved: The grape seeds were ultrasonically treated in a 2.5 g / L gibberellin solution for 2 min, soaked at 55°C for 13 min, and then shaken at 28°C and 180 rpm for 24 h. They were then sterilized and skinned, and then pre-cultured until the abdominal shell cracked. placing grape seeds with cracked abdomen shells in an impregnation solution for impregnation treatment, and then planting them in a culture matrix to culture transgenic grape plants; The impregnation solution contains a recombinant engineered strain capable of expressing a heterologous gene; The skin cutting process refers to scratching the back seed coat and beak of the grape seeds; The impregnation treatment includes vacuum impregnation treatment and shaking impregnation treatment, the time of the vacuum impregnation treatment is 6 minutes, and the time of the shaking impregnation treatment is 14 minutes; The impregnation solution is obtained by adding acetosyringone to a bacterial solution containing the recombinant engineered strain and culturing in dark; The concentration of the acetosyringone is 100 mM; The OD of the bacterial solution 600 =0.6; The expression vector used by the recombinant engineering strain to express the heterologous gene is pHZM03.

2. The method for efficient genetic transformation of grape seeds independent of tissue culture according to claim 1, characterized in that: The disinfection treatment includes alcohol disinfection and sodium hypochlorite solution disinfection.

3. The method for efficient genetic transformation of grape seeds independent of tissue culture according to claim 1, characterized in that: The dark culture time is 2h.

Images