Watermelon transgenic screening expression cassette and application thereof

Abstract

The invention discloses a watermelon transgenic screening expression cassette and application thereof, and belongs to the technical field of biology. According to the invention, an expression cassette of a TagRFP gene driven by a d35s promoter is constructed on a pCambia1300 vector, and a recombinant vector is obtained. A recombinant vector is introduced into watermelon cells by utilizing an agrobacterium-mediated genetic transformation technology, and a red fluorescent protein TagRFP gene is integrated into a watermelon genome, so that transgenic watermelons can be screened and identified by taking red fluorescence as an identifier in the whole growth period of watermelon genetic transformation. Compared with a traditional screening method, the screening method has the advantages that the screening process is greatly simplified, and the transgenic watermelon and the non-transgenic watermelon are clearly distinguished, so that the genetic transformation efficiency of the watermelon is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a transgenic screening expression cassette for watermelon and its application. Background Art

[0002] Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) is the fifth most consumed fresh fruit globally. Due to the wide diversity of its shape, size, flesh color, flavor, aroma, texture, and nutritional components, watermelon has become one of the model crops for fruit research. In the genetic research of watermelon, researchers have used the CRISPR / Cas9 system to precisely edit the genomic sequence and successfully obtained Clpds mutants, which has greatly promoted the research on watermelon gene function. However, compared with other species, the transgenic screening system in the genetic transformation process of watermelon is not yet mature enough, affecting the genetic transformation efficiency and being the main bottleneck restricting the progress of watermelon scientific research and molecular breeding. In the transgenic process, choosing a suitable screening marker is crucial for improving the genetic transformation efficiency. A suitable screening marker can significantly increase the success rate of genetic transformation, helping researchers more effectively introduce foreign genes into watermelon plants, thereby accelerating the breeding process of excellent varieties. Therefore, there is an urgent need to construct an efficient and reliable transgenic screening expression cassette to accelerate the genetic improvement process of watermelon and promote the continuous progress and development of molecular breeding technology. Summary of the Invention

[0003] The purpose of the present invention is to provide an efficient and reliable transgenic screening expression cassette for watermelon. The present invention has developed a watermelon genetic transformation screening vector using the red fluorescent protein TagRFP gene as a screening marker. Through the Agrobacterium-mediated genetic transformation method, the watermelon genetic transformation screening vector is introduced into watermelon, and the transgenic watermelon is screened through red fluorescence. This system is stable and efficient during the watermelon transformation process, can be used to screen and identify transgenic watermelon, greatly simplifies the entire screening process, and significantly improves the accuracy of identification.

[0004] In the first aspect, the present invention provides a transgenic screening expression cassette for watermelon, and the expression cassette contains a screening marker, and the screening marker is the red fluorescent protein TagRFP.

[0005] Furthermore, the transgenic screening expression cassette for watermelon of the present invention further contains a promoter and a terminator for initiating and terminating the red fluorescent protein TagRFP gene;

[0006] The promoter is the d35s promoter;

[0007] The terminator is the Nost terminator.

[0008] Furthermore, the nucleotide sequence of the transgenic screening expression cassette of the watermelon of the present invention is as shown in SEQ ID NO.1.

[0009] In a second aspect, the present invention provides a genetic transformation screening vector for watermelon, and the genetic transformation screening system for watermelon contains the transgenic screening expression cassette of watermelon.

[0010] Furthermore, the genetic transformation screening vector for watermelon of the present invention is obtained by ligating the transgenic screening expression cassette of watermelon to the plant expression vector pCambia1300.

[0011] In a third aspect, the present invention provides a method for cultivating transgenic watermelon. The genetic transformation screening vector for watermelon is used for Agrobacterium-mediated genetic transformation to obtain transgenic watermelon.

[0012] Furthermore, in the cultivation method of the present invention, the type of the Agrobacterium is EHA105.

[0013] Furthermore, in the cultivation method of the present invention, the material for genetic transformation is the wild-type material ZTC.

[0014] In a fourth aspect, the present invention relates to the application of the transgenic screening expression cassette of watermelon or the genetic transformation screening vector for watermelon in gene editing of watermelon.

[0015] In a fifth aspect, the present invention relates to the application of the transgenic screening expression cassette of watermelon or the genetic transformation screening vector for watermelon in the rapid screening of watermelon plants containing transgenic components and watermelon plants without transgenic components.

[0016] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0017] (1) The genetic transformation screening vector pd35s-TagRFP-Nost for watermelon of the present invention is not only applicable to specific foreign genes, but also can be used as a general tool to conveniently transfer various foreign genes into watermelon plants, broadening the application scope of genetic engineering technology in watermelon breeding.

[0018] (2) By integrating the TagRFP fluorescent protein gene, transgenic watermelon plants can emit red fluorescence throughout the tissue culture stage, enabling direct and rapid identification of transgenic plants and greatly improving the screening efficiency.

[0019] (3) When screening using tools such as fluorescence microscopes and portable fluorescence detection devices, no physical damage will be caused to transgenic watermelon plants, ensuring the non-destructiveness of the screening process and being beneficial for subsequent research and application.

[0020] (4) Researchers only need to simply combine the target foreign gene with the pd35s-TagRFP-Nost vector to form a new recombinant vector and introduce it into watermelon recipient cells, then they can achieve the cultivation of transgenic watermelons. This process is relatively simple and clear, reducing the operation difficulty of genetic engineering technology.

[0021] (5) The recombinant vector pd35s-TagRFP-Nost of the present invention provides a new technical means for watermelon breeding, which helps to accelerate the introduction and screening of genes with excellent traits and promote the improvement and innovation of watermelon varieties.

[0022] (6) Through an efficient screening method, it can be ensured that only the plants that have successfully transferred the target foreign gene are used for subsequent research and application, thereby improving the safety and reliability of transgenic crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the red fluorescence characteristics during the co-cultivation stage of watermelon cotyledons.

[0024] Figure 2 It is the red fluorescence characteristics during the recovery stage of watermelon positive shoots.

[0025] Figure 3 It is the red fluorescence characteristics during the rooting culture stage of watermelon. DETAILED DESCRIPTION OF THE INVENTION

[0026] Next, the technical solutions of the present invention will be described in conjunction with the embodiments. However, the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0027] Example 1

[0028] This example provides a method for constructing the recombinant vector pd35s-TagRFP-Nost.

[0029] In this example, primers were designed using Primer design and other tools (takarabio.com) to construct the vector.

[0030] In this example, based on the plant expression vector pCambia1300, the 35s promoter of cauliflower mosaic virus was used to initiate the red fluorescent tag gene TagRFP, and the recombinant vector pd35s-TagRFP-Nost was obtained. The 35s promoter was amplified with p35sF and p35sR, and the red fluorescent tag gene TagRFP-Nost element was amplified with RFP-F and RFP-R. The d35s and TagREP-Nost were ligated together by homologous recombination to obtain the d35s-TagRFP-Nost expression cassette sequence, as shown in SEQ ID NO.1. Specific primers p35sF, p35sR, RFP-F, and RFP-R were designed and synthesized, as shown in SEQ ID NOs. 2-5 respectively (Table 1).

[0031] Table 1 Specific primer sequences

[0032]

[0033] The target vector pCambia1300 was digested with the restriction endonuclease EcoRI (NEB) to linearize it, preparing for subsequent cloning steps. The d35s-TagRFP-Nost expression cassette sequence was amplified using the primers shown in SEQ ID NOs. 2-5. The amplified fragment and the linearized pCambia1300 vector after enzymatic digestion were both purified and recovered to ensure the accuracy of subsequent experiments. According to the instructions of the ClonExpress One Step Cloning Kit (C113-01, Nanjing Novoprotein Scientific Co., Ltd.), the purified fragment and the linearized vector were subjected to homologous recombination. After ligation, the obtained recombinant plasmid pd35s-TagRFP-Nost was transformed into Escherichia coli DH5α competent cells for subsequent amplification and screening. Finally, primers were designed to identify positive clones, and the primer sequences were as shown in SEQ ID NOs. 6-7. Through PCR amplification, clones containing the correct inserted fragment were screened. To further confirm the accuracy of the inserted sequence, the positive clones were subjected to sequencing analysis. The plasmid required for the experiment was obtained.

[0034] Example 2

[0035] This example provides the genetic transformation of watermelon.

[0036] The genetic transformation material used in this example is the wild-type material ZTC.

[0037] Take Escherichia coli DH5α carrying the positive plasmid and extract the positive plasmid. Introduce the positive plasmid into the competent cells of Agrobacterium tumefaciens EHA105 (purchased from Shanghai Weidi Biotechnology Co., Ltd.). Culture the successfully transformed EHA105 strain by shaking until its OD value reaches the range of 0.8 to 1.0, and then prepare it for the infection experiment.

[0038] Collect mature watermelon seeds, soak the seeds in water at 55°C. After the seed coats are softened, gently remove the seed coats to ensure the integrity of the embryos. Disinfect the dehulled seeds. In the laminar flow hood, soak the seeds in 75% alcohol solution for about 30 seconds, and then rinse the seeds with sterile water to remove the alcohol. Then soak the seeds in 3% sodium hypochlorite solution for about 10 minutes, and rinse the seeds with sterile water again to remove the residual disinfectant. Place the disinfected seeds on the medium (agar 3 g / L) for culture. The culture temperature is 28°C, and the culture time is 24 - 36 h. After the watermelon seeds germinate, evenly divide the two cotyledons into 8 pieces on the laminar flow hood as explants for Agrobacterium infection. Take 10 mL of MS liquid medium (M519, concentration 4.43 g / L), add 500 μL of Agrobacterium liquid as the infection solution, and use a 20 mL syringe to vacuum-infect the explants for 10 min. Then, air-dry the cotyledons and transfer them to the co-culture medium (M519 4.43 g / L, sucrose 30 g / L, 6-BA 1.5 g / L, agar 3 g / L). Transfer the medium to a dark environment and culture at a constant temperature of 28°C for 3 d. Then, transfer the swollen cotyledon pieces to the recovery medium (M519 4.43 g / L, sucrose 30 g / L, 6-BA 1.5 g / L, ticarcillin 200 mg / L, agar 3 g / L) for culture for 10 - 20 d, and use a portable fluorescent protein excitation light source (LUYOR-3415) to observe the fluorescent signal of the watermelon buds, and screen out the positive buds with red fluorescence. Transfer the screened positive bud seedlings to a new recovery medium. After they have the morphology of a complete plant, transfer them to the rooting medium (M519 4.43 g / L, sucrose 30 g / L, IAA 0.8 g / L, ticarcillin 200 mg / L, agar 3 g / L) until the positive buds grow and develop into a complete plant. After transplantation, transgenic watermelon plants are obtained.

[0039] As Figure 1 shown, after the watermelon cotyledons are infected and transferred to the co-culture medium, under the excitation light irradiation of 440 - 460 nm, obvious red fluorescent signals can be observed, indicating that Agrobacterium carrying the recombinant plasmid has successfully achieved genetic transformation of the explants. After the co-culture stage is over, the red fluorescent signals of the positive buds in the recovery stage are as Figure 2 shown, further confirming the success of genetic transformation. As Figure 3As shown, after the positive shoots were transferred to the rooting medium and developed into complete plants, the red fluorescence signal could still be observed, proving the stability and sustainability of the genetic transformation.

[0040] For different exogenous genes, the recombinant vector pd35s-TagRFP-Nost provided by the present invention is also applicable and can be introduced into watermelon plants with the help of genetic engineering technology. The TagRFP fluorescent protein gene contained in this recombinant vector, as a visual marking means, can be used to perform efficient screening by observing the red fluorescence released by the plant during the entire tissue culture stage of transgenic watermelon. Researchers only need to simply combine the target exogenous gene with the pd35s-TagRFP-Nost vector to form a new recombinant vector. Afterwards, this newly constructed recombinant vector is introduced into the receptor cells of the watermelon through Agrobacterium-mediated transformation or other efficient gene delivery methods, so that the transgenic watermelon plant obtained can show red fluorescence. Using tools such as fluorescence microscopes and portable fluorescence detection equipment, non-destructive and rapid screening and identification of transgenic watermelons can be easily achieved. Therefore, the recombinant vector pd35s-TagRFP-Nost introduced by the present invention has created an efficient and simple new way for the introduction and screening of other exogenous genes in watermelon.

[0041] The embodiments described above are part of the embodiments of the present invention, but not all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A watermelon transgenic screening expression cassette, characterized in that: The expression cassette comprises a screening marker, which is a red fluorescent protein TagRFP gene.

2. The watermelon transgenic screening expression cassette according to claim 1, wherein the expression cassette further comprises a promoter and a terminator for starting and stopping the red fluorescent protein TagRFP gene; The promoter is d35s promoter; The terminator is the Nost terminator.

3. The watermelon transgenic screening expression cassette according to any one of claims 1 to 2, characterized in that: The nucleotide sequence of the expression cassette is shown in SEQ ID NO.

1.

4. A watermelon genetic transformation screening vector, characterized in that: The invention comprises the watermelon transgenic screening expression cassette according to any one of claims 1 to 3.

5. The watermelon genetic transformation screening vector according to claim 4, characterized in that: The watermelon transgenic screening expression cassette according to any one of claims 1 to 3 is connected to the plant expression vector pCambia1300.

6. A method for cultivating transgenic watermelon, characterized in that: The watermelon genetic transformation screening vector described in claim 5 is genetically transformed by Agrobacterium-mediated genetic transformation to obtain transgenic watermelon.

7. The cultivation method according to claim 6, characterized in that: The type of Agrobacterium is EHA105.

8. The cultivation method according to claim 6, characterized in that: The genetically transformed material is the wild-type material ZTC.

9. Use of the watermelon transgenic screening expression cassette according to any one of claims 1 to 3 or the watermelon genetic transformation screening vector according to any one of claims 4 to 5 in watermelon gene editing.

10. Use of the watermelon transgenic screening expression cassette according to any one of claims 1 to 3 or the watermelon genetic transformation screening vector according to any one of claims 4 to 5 in the rapid screening of watermelon plants containing transgenic components and watermelon plants without transgenic components.

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