Method for introducing target DNA fragment into plant and application thereof

Abstract

The invention provides a method for introducing a target DNA fragment into a plant and application of the method, and the method comprises the following steps: carrying out electric fusion on a plant protoplast and an erecta minium protoplast containing the target DNA fragment in a nuclear genome, so as to realize the purpose of introducing the target DNA fragment into the plant. According to the invention, through early condition exploration, a cell fusion system of the phytoneum palmatum and the plant is established, and cell fusion and stable culture of subsequent fusion cultures can be well realized. According to the method, the target DNA fragment is transferred by fusion of the strignum leptosphaereum and the arabidopsis thaliana, and the method is expected to play a key role in application fields such as plant genome synthesis and plant customized transformation research, and has a relatively important application value.

Description

Technical Field

[0001] The invention belongs to the technical field of cell fusion, and specifically relates to a method for introducing a target DNA fragment into a plant and an application thereof. Background Art

[0002] Cell fusion technology is an emerging cell engineering technology developed in the 1960s. Cell fusion, also known as cell hybridization, protoplast fusion or somatic cell hybridization, refers to the process of asexually fusing different types of cells into mononuclear or multinuclear hybrid cells by artificial methods under in vitro conditions through cell mediation and culture. Cell fusion technology can be roughly divided into three categories: chemically induced fusion, biologically induced fusion and physically induced fusion.

[0003] Plant gene transformation technology refers to the method of introducing foreign genes into plant cells or tissues to obtain transgenic plants. There are two main types of existing plant foreign gene transformation technologies: the first type is the gene transfer method mediated by organisms, such as Agrobacterium-mediated method and plant virus-mediated method.

[0004] Agrobacterium-mediated transformation is a transformation method that uses Agrobacterium to transform recipient cells and insert the target gene into the recipient cell genome. This method is currently the most studied, most clearly defined, most widely used and mature plant transgenic technology. Currently, the commonly used method for plant transformation is Agrobacterium-mediated genetic transformation. Different cloning vectors can be used to transform fragments less than 100kb, but this method has disadvantages such as low genetic affinity, low transformation efficiency, low expression stability after transformation, and long transformation cycle. The plant virus-mediated method mainly inserts the exogenous target gene into a virus, uses the virus to transfect plants, and thus transfers the exogenous gene into plant cells.

[0005] The second category is the direct DNA introduction method. The direct DNA insertion method refers to a method that does not require the transformation of a plasmid vector to achieve the insertion of exogenous target genes, such as the gene gun method, the electric shock method, the polyethylene glycol method, etc.

[0006] Currently, the existing methods for transforming exogenous genes in plants have strict restrictions on the size of the transformed fragments. Therefore, providing a new method for introducing DNA fragments into plant cells has important application prospects. Summary of the invention

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for introducing a target DNA fragment into a plant and its application. The present invention conducts electric field-induced fusion of the protoplasts of Physcomitrella patens and plants, and utilizes the fusion of Physcomitrella patens and plants to achieve the transfer of DNA fragments, which is expected to play a key role in application fields such as plant genome synthesis and plant customized transformation research, and has relatively important significance.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for introducing a DNA fragment into a plant, the method comprising: electro-fusing a Physcomitrella patens protoplast containing the target DNA fragment in its nuclear genome with a protoplast of the plant to achieve introduction of the target DNA fragment into the plant.

[0010] In the present invention, the method for introducing a target DNA fragment into a plant can be used to introduce a target gene fragment into crops such as Arabidopsis, tomato or rice.

[0011] In the present invention, the target DNA fragment refers to a DNA sequence with important functional traits, which is first integrated into the nuclear genome of Physcomitrella patens and then fused with plant cells to achieve transfer to plants. The present invention achieves the fusion of Physcomitrella patens and Arabidopsis thaliana, providing a technical reference for the efficient transformation of plant cells with DNA fragments.

[0012] At present, most of the basic research or production input related to cell fusion is based on the same type of cells or cells of the same species, such as plant cells, animal cells or microbial cells. Although cell fusion is not bounded by species, the fusion between cells of different organisms is still difficult, including the selection of fusion materials, the setting of fusion conditions and the observation of fusion effects. The present invention has established a cell fusion system suitable for Physcomitrella patens and Arabidopsis thaliana through early condition exploration, which can better achieve stable cultivation of cell fusion and subsequent fusion cultures, and has relatively important significance.

[0013] Preferably, the method comprises the following steps:

[0014] Protoplasts of the Physcomitrella patens and the Arabidopsis thaliana are prepared respectively, the protoplasts are resuspended and washed with an electrofusion solution, and then the protoplasts are resuspended with a mannitol solution to obtain a mixed cell solution;

[0015] The mixed cell fluid is added to a fusion electrode for electrofusion, and the protoplasts after electrofusion are cultured and screened to obtain target fusion cells, thereby introducing the target DNA fragment into the plant.

[0016] Preferably, the electrofusion solution comprises, by concentration, 0.5-0.8 mol / L mannitol and 0.05-0.15 mmol / L CaCl2.

[0017] In the present invention, the concentration of mannitol in the electrofusion solution is 0.5-0.8 mol / L, for example, it can be 0.5 mol / L, 0.6 mol / L, 0.7 mol / L or 0.8 mol / L, etc.; the concentration of CaCl2 is 0.05-0.15 mmol / L, for example, it can be 0.05 mmol / L, 0.07 mmol / L, 0.09 mmol / L, 0.1 mmol / L, 0.13 mmol / L or 0.15 mmol / L, etc.

[0018] Preferably, the amount of protoplasts in the mixed cell fluid is (1.8-2)×10 5 / mL, for example, it can be 1.8×10 5 / mL, 1.9×10 5 / mL or 2×10 5 Pieces / mL, etc.

[0019] Preferably, the conditions for the electric fusion are: alternating current 30-40V (for example, 30V, 32V, 34V, 36V, 38V or 40V, etc.), direct current 500-600V (for example, 500V, 520V, 540V, 560V, 580V or 600V, etc.), AC action time 10-20s (for example, 10s, 12s, 14s, 16s, 18s or 20s, etc.), DC pulse time 15-25μs (for example, 15μs, 17μs, 19μs, 20μs, 21μs, 23μs or 25μs, etc.), the number of DC pulses is 2-3 (for example, 2 or 3), and the DC pulse interval is 0.3-0.7s (for example, 0.3s, 0.4s, 0.5s, 0.6s or 0.7s, etc.).

[0020] Preferably, the Physcomitrella patens protoplasts are prepared by a method comprising the following steps:

[0021] Disperse the Physcomitrella patens in the protonema state in a catalytic enzyme solution, place it in a dark room for static culture, and digest the cell wall; after the culture, filter and centrifuge, collect the precipitate, resuspend the protoplasts with a mannitol solution, filter again, and wash the protoplasts with a mannitol solution to obtain the Physcomitrella patens protoplasts.

[0022] Preferably, the mass concentration of the lysine solution is 1.5-2.5%, for example, it can be 1.5%, 1.8%, 2%, 2.2%, 2.4% or 2.5%, etc.

[0023] Preferably, the temperature of the static culture is 20-25°C, for example, 20°C, 22°C, 24°C or 25°C, and the time of the static culture is 1.5-2.5h, for example, 1.5h, 1.8h, 2h, 2.2h, 2.4h or 2.5h, etc.

[0024] Preferably, the filtration uses a 40-45 μm filter, for example, it can be 40 μm, 41 μm, 42 μm, 43 μm, 44 μm or 45 μm.

[0025] Preferably, the centrifugal force of the centrifugation is 150-180g, for example, 150g, 160g, 170g or 180g, etc., and the centrifugal time is 5-10min, for example, 5min, 6min, 7min, 8min, 9min or 10min, etc.

[0026] Preferably, the concentration of mannitol in the mannitol solution is 0.2-0.6 mol / L, for example, it can be 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L or 0.6 mol / L.

[0027] Preferably, the plant protoplasts are prepared by a method comprising the following steps:

[0028] The plant callus tissue is dispersed in T3 enzymatic hydrolysate, cultured in a dark room, and the cell wall is digested; after the culture, the solution is filtered and centrifuged, the precipitate is collected, the protoplasts are resuspended in W5 solution, filtered again, the protoplasts are washed in W5 solution, and finally stored in a mannitol solution.

[0029] In the present invention, the T3 enzymatic solution includes, by concentration, 0.8-1.2% Cellulase (for example, 0.8%, 0.9%, 1%, 1.1% or 1.2%, etc.), 0.1-0.3% Macerozyme (for example, 0.1%, 0.2% or 0.3%, etc.), 0.05-0.15% Pectinase (for example, 0.05%, 0.07%, 0.09%, 0.1%, 0.13% or 0.15%, etc.), 8-12mM CaCl2 (for example, 8mM, 9mM, 10mM, 11mM, 12mM, etc.), 18-22mM MES (for example, 18mM, 19mM, 20mM, 21mM, 22mM, etc.), 0.3-0.5M D-Mannitol (for example, it can be 0.3M, 0.4M or 0.5M, etc.), 18-22mM KCl (for example, it can be 18mM, 19mM, 20mM, 21mM or 22mM, etc.) and 0.05-0.15% BSA (for example, it can be 0.05%, 0.07%, 0.09%, 0.1%, 0.13% or 0.15%, etc.).

[0030] In the present invention, the W5 solution includes, by concentration, 150-160mM NaCl (for example, 150mM, 155mM or 160mM, etc.), 5-8mM KCl (for example, 5mM, 6mM, 7mM or 8mM, etc.), 120-130mM CaCl2 (for example, 120mM, 125mM or 130mM, etc.) and 1-3mM MES (for example, 1mM, 2mM or 3mM, etc.).

[0031] Preferably, the culture rotation speed is 70-80 rpm, for example, 70 rpm, 75 rpm or 80 rpm, etc., and the culture time is 1-2 h, for example, 1 h, 1.5 h or 2 h, etc.

[0032] Preferably, the filtration uses a 40-45 μm filter, for example, it can be 40 μm, 41 μm, 42 μm, 43 μm, 44 μm or 45 μm.

[0033] Preferably, the centrifugal force of the centrifugation is 80-120g, for example, 80g, 90g, 100g, 110g or 120g, etc., and the centrifugal time is 5-10min, for example, 5min, 6min, 7min, 8min, 9min or 10min, etc.

[0034] Preferably, the plant is selected from any one of Arabidopsis thaliana, tomato and rice.

[0035] Preferably, the culture screening is performed using a method comprising the following steps:

[0036] The protoplasts after electrofusion are placed in a dark room at 20-25°C (for example, 20°C, 22°C, 24°C or 25°C, etc.) and cultured for 20-24h (for example, 20h, 21h, 22h, 23h or 24h, etc.), and observed under the GFP fluorescence channel. Green fluorescent cells with chloroplasts are fused cells that have achieved fusion.

[0037] In a second aspect, the present invention provides application of the method for introducing a target DNA fragment into a plant as described in the first aspect in plant gene transformation.

[0038] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] (1) This invention has achieved a breakthrough in the fusion of Physcomitrella patens and Arabidopsis cells, laying a key technical foundation for the transfer of plant DNA fragments and the synthesis of plant artificial chromosomes. In the future, it is expected to be expanded to the development of exogenous DNA transfer technology for various plant cells.

[0041] (2) Although cell fusion is not bounded by species, the fusion between cells of different organisms is still difficult, including the selection of fusion materials, the setting of fusion conditions, and the observation of fusion effects. The present invention has established a relatively mature cell fusion system through the exploration of early experimental conditions. Using the chassis cells constructed by itself, the fusion effect of Physcomitrella patens and Arabidopsis cells can be better observed, and the stable culture of subsequent fusion cultures can be achieved, laying a foundation for further research on exogenous DNA transfer technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the counting result of Arabidopsis protoplasts after 2 hours of enzymatic hydrolysis in Example 1.

[0043] Figure 2 This is the counting result of the protoplasts of Physcomitrella patens after 2 hours of enzymatic hydrolysis in Example 1.

[0044] Figure 3 This is the protoplast after electric field induced fusion in Example 1.

[0045] Figure 4 This is the cell culture after electric field induced fusion in Example 1.

[0046] Figure 5 This is the protoplast after electric field induced fusion in Example 2.

[0047] Figure 6 This is the protoplast after electric field induced fusion in Example 3.

[0048] Figure 7 This is the Arabidopsis thaliana carrying the mCherry red fluorescent protein constructed in Example 4.

[0049] Figure 8 It is the Physcomitrella patens carrying the GFP green fluorescent protein in Example 4.

[0050] Fig. 9 This is the protoplast after electric field induced fusion in Example 4.

[0051] Fig.10 This is the cell culture after electric field induced fusion in Example 4.

[0052] Fig.11 This is the counting result of Arabidopsis protoplasts after 2 hours of enzymatic hydrolysis in Example 5.

[0053] Fig.12 This is the cell culture after polyethylene glycol PEG induced fusion in Comparative Example 1.

[0054] Fig.13 This is the protoplast after electric field induced fusion in Comparative Example 2.

[0055] Fig.14 This is the protoplast after electric field induced fusion in Comparative Example 3.

[0056] Fig.15 This is the protoplast after electric field induced fusion in Comparative Example 4. DETAILED DESCRIPTION

[0057] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0058] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0059] In the following Examples 1-3 and Example 5, the Arabidopsis callus cells used were all transformed with green fluorescent protein (GFP) in advance and successfully expressed the green fluorescent protein.

[0060] In Example 4, the Arabidopsis callus was transformed with mCherry red fluorescent protein, and the red fluorescent protein was successfully expressed; the protonema of Physcomitrella patens was transformed with GFP in the nuclear genome by polyethylene glycol (PEG)-mediated method, and was successfully expressed.

[0061] Example 1

[0062] Fusion of Physcomitrella patens protonema and Arabidopsis thaliana callus cells (electric field induced fusion method)

[0063] Add 5 mL of T3 enzymatic solution (Test 3: 1% Cellulase, 0.2% Macerozyme, 0.1% Pectinase, 10 mM CaCl2, 20 mM MES, 0.4 M D-Mannitol, 20 mM KCl, 0.1% BSA), carefully pick up 0.5g Arabidopsis callus (pS1300-VenusC-OST1-HPG) with sterilized tweezers, disperse it in a small round culture dish filled with T3 enzymatic solution, and place it in a 25℃ dark room shaker (shaker: Minquan, MQD-S3NR) at 75rpm for 2h; then place a 40μm filter on a new 50mL centrifuge tube, slowly add the digestion mixture (enzymatic solution: W5 solution volume ratio is 1:1), centrifuge at 100g (centrifuge: Xiangyi, H1850R) for 5min; slowly and carefully remove the supernatant, suspend the protoplasts with 2mL W5 solution (154mM NaCl, 5mM KCl, 125mM CaCl2, 2mM MES), and filter it again with a 40μm filter; add to 10mL W5 solution, centrifuge at 100g for 3 minutes, repeat washing twice; carefully remove the supernatant, add 1mL W5 solution to resuspend the protoplasts; take 20μL of the suspension and add it to the cell counter to count the protoplasts (microscope: OLYMPUS, DP74). The counting results of Arabidopsis protoplasts after 2h of enzymatic hydrolysis are shown in the figure. Figure 1 As shown in the figure, the experimental results show that after 2 hours of enzymatic hydrolysis of 0.5g Arabidopsis callus, protoplasts with good morphology and large number can be obtained. The number of protoplasts obtained is about 1.5×10 6 Pieces / mL.

[0064] Add 25 mL of 2% collapsing enzyme to a sterilized 50 mL centrifuge tube; take 4 dishes of Physcomitrella patens (wild-type protonema state, 2 g), disperse them and place them in a 25°C darkroom shaker for 2 h to digest the cell wall; place a 40 μm filter on a new 50 mL centrifuge tube, rinse the filter with 1 mL of 0.6 mol / L mannitol solution, slowly add the digestion mixture to the filter, and rinse the centrifuge tube with 0.6 mol / L mannitol solution to 40 mL; after filtration, centrifuge at 150 g for 10 min; slowly and carefully remove the supernatant with a pipette, do not move violently to cause the protoplasts to resuspend, and some supernatant can be left; suspend the protoplasts with 2 mL of 0.6 mol / L mannitol solution, and filter again with a 40 μm filter; add 0.6 mol / L mannitol solution to 40 mL, centrifuge at 150 g for 10 min, and repeat washing twice; carefully remove the supernatant, add 1 mL Resuspend the protoplasts in 0.6 mol / L mannitol solution; take 10 μL of the suspension and add it to the cell counter to count the protoplasts. The counting results of the protoplasts of Physcomitrella patens after 2 hours of enzymatic hydrolysis are as follows: Figure 2 As shown in the figure, the experimental results show that after 2 hours of enzymatic hydrolysis of 2g of Physcomitrella patens, protoplasts with good morphology and large number can be obtained. The number of protoplasts obtained is about 1.2×10 5 Pieces / mL.

[0065] Mix the two protoplasts in a ratio of 1:1 between Physcomitrella patens and Arabidopsis thaliana, centrifuge at 100 g for 10 min; discard the supernatant, gently resuspend with 1 mL of electrofusion solution (0.6 mol / L mannitol + 0.1 mmol / L CaCl2), centrifuge for 10 min, and repeat washing twice; add 1 mL of 0.6 mol / L mannitol solution to resuspend the cells; draw 20 μL of the mixed cell solution onto the fusion electrode (0.5 mm spacing) and perform electrofusion (AC 35 V, DC 550 V, AC action time 15 s, DC pulse time 20 μs, DC pulse number 3, DC pulse interval 0.5 s) (cell fusion instrument: NEPA GENE, ECFG21); draw the electrofused cell solution onto 3 mL of PIM (protoplast induction The operation was repeated in a 6-well cell culture dish containing liquid culture medium (PIM, protoplast induction medium) until 200 μL of cell solution was electrofused (note to set up a control) and cultured at 20°C in the dark.

[0066] The green fluorescence of Arabidopsis callus (no chloroplasts) and the chloroplasts of wild-type Physcomitrella patens (no fluorescence) were used to indicate the cell fusion between the two. After the electric field induced fusion, the cell state was normal, and green fluorescent cells with chloroplasts (fused cells) could be observed under the GFP fluorescence channel (microscope: OLYMPUS, SZX16), such as Figure 3 As shown, Figure 3 This is the protoplast after electric field induced fusion; after a period of culture, the cells cluster and proliferate normally, such as Figure 4 As shown, Figure 4 This is the cell culture after electric field-induced fusion.

[0067] Example 2

[0068] This embodiment provides a method for fusing Physcomitrella patens protoplasts and Arabidopsis thaliana callus cells, wherein the preparation methods of Physcomitrella patens protoplasts and Arabidopsis thaliana protoplasts refer to Example 1, and the steps and parameters of electric field induced fusion are as follows:

[0069] Mix the two protoplasts in a ratio of 1:1 between Physcomitrella patens and Arabidopsis thaliana, centrifuge at 100 g for 10 min; discard the supernatant, gently resuspend with 1 mL of electrofusion solution (0.5 mol / L mannitol + 0.15 mmol / L CaCl2), centrifuge for 10 min, and repeat washing twice; add 1 mL of 0.5 mol / L mannitol to resuspend the cells; draw 20 μL of the mixed cell solution onto the fusion electrode (0.5 mm spacing) for electrofusion (30 V AC, 500 V DC, 10 s AC action time, 15 μs DC pulse time, 3 DC pulses, and 0.3 s DC pulse interval) (cell fusion instrument: NEPA GENE, ECFG21); draw the electrofused cell solution into a 6-well cell culture dish containing 3 mL of PIM liquid culture medium, repeat the operation until 200 μL of cell solution is electrofused (note to set a control), and culture at 20°C in the dark.

[0070] The green fluorescence of Arabidopsis callus (no chloroplasts) and the chloroplasts of wild-type Physcomitrella patens (no fluorescence) were used to indicate the cell fusion between the two. After the electric field induced fusion, the cell state was normal, and green fluorescent cells with chloroplasts (fused cells) could be observed under the GFP fluorescence channel (microscope: OLYMPUS, SZX16), such as Figure 5 As shown, Figure 5 This is the protoplast after electric field induced fusion.

[0071] Example 3

[0072] This embodiment provides a method for fusing Physcomitrella patens protoplasts and Arabidopsis thaliana callus cells, wherein the preparation methods of Physcomitrella patens protoplasts and Arabidopsis thaliana protoplasts refer to Example 1, and the steps and parameters of electric field induced fusion are as follows:

[0073] Mix the two protoplasts in a ratio of 1:1 of Physcomitrella patens: Arabidopsis thaliana, centrifuge at 100g for 10min; discard the supernatant, gently resuspend with 1mL electrofusion solution (0.8mol / L mannitol + 0.05mmol / L CaCl2), centrifuge for 10min, and repeat washing twice; add 1mL 0.8mol / L mannitol to resuspend the cells; draw 20μL of mixed cell solution to the fusion electrode (0.5mm spacing) for electrofusion (AC 40V, DC 600V, AC action time 20s, DC pulse time 25μs, DC pulse number 3, DC pulse interval 0.7s) (cell fusion instrument: NEPA GENE, ECFG21); draw the electrofused cell solution into a 6-well cell culture dish containing 3mL PIM liquid culture medium, repeat the operation until 200μL cell solution is electrofused (note to set a control), and culture at 20℃ in the dark.

[0074] Fusion effect: The green fluorescence of Arabidopsis callus (no chloroplasts) and the chloroplasts of wild-type Physcomitrella patens (no fluorescence) were used to indicate the cell fusion between the two. After the electric field induced fusion, the cell state was normal, and green fluorescent cells with chloroplasts (fused cells) could be observed under the GFP fluorescence channel (microscope: OLYMPUS, SZX16). Figure 6 As shown, Figure 6 This is the protoplast after electric field induced fusion.

[0075] Example 4

[0076] This example provides a method for fusing Physcomitrella patens protonema and Arabidopsis thaliana callus cells, with reference to Example 1.

[0077] In this example, Arabidopsis thaliana carrying mCherry red fluorescent protein was constructed. Figure 7 ) and Physcomitrella patens carrying the green fluorescent protein GFP ( Figure 8 ) as the fusion parent. Under the fluorescence channel, the fused cells will be observed to have both red and green fluorescence.

[0078] After electric field-induced fusion, the cells are in normal state, and cells (fused cells) with fluorescence in both RFP and GFP fluorescence channels can be observed (microscope: OLYMPUS, SZX16). Fig. 9 As shown, Fig. 9The protoplasts were fused by electric field induction. After 30 days of culture, the cells clustered and proliferated normally, and cells with fluorescence under both RFP and GFP fluorescence channels could be observed. Fig.10 As shown, Fig.10 This is the cell culture after electric field-induced fusion.

[0079] The fusion cells observed so far can express both parental proteins simultaneously, indicating that the fusion is successful, and demonstrating that the target DNA fragment can be transferred through fusion.

[0080] Example 5

[0081] This embodiment provides a method for fusing protoplasts of Physcomitrella patens and Arabidopsis callus cells, wherein the steps for preparing protoplasts of Arabidopsis callus are as follows, and the remaining cell fusion steps refer to Example 1.

[0082] Add 5 mL of T3 enzymatic solution (Test 3: 1.2% Cellulase, 0.2% Macerozyme, 0.1% Pectinase, 10 mM CaCl2, 20 mM MES, 0.4 M D-Mannitol, 20 mM KCl, 0.1% BSA), carefully pick up 0.5g Arabidopsis callus (pS1300-VenusC-OST1-HPG) with sterilized tweezers, disperse it in a small round culture dish filled with T3 enzymatic solution, and place it in a 25℃ dark room shaker (shaker: Minquan, MQD-S3NR) at 75rpm for 2h; then place a 40μm filter on a new 50mL centrifuge tube, slowly add the digestion mixture (enzymatic solution: W5 solution volume ratio is 1:1), centrifuge at 100g (centrifuge: Xiangyi, H1850R) for 5min; slowly and carefully remove the supernatant, suspend the protoplasts with 2mL W5 solution (154mM NaCl, 5mM KCl, 125mM CaCl2, 2mM MES), and filter it again with a 40μm filter; add to 10mL W5 solution, centrifuge at 100g for 3 minutes, repeat washing twice; carefully remove the supernatant, add 1mL W5 solution to resuspend the protoplasts; take 20μL of the suspension and add it to the cell counter to count the protoplasts (microscope: OLYMPUS, DP74). The counting results of Arabidopsis protoplasts after 2h of enzymatic hydrolysis are shown in the figure. Fig.11 As shown in the figure, the experimental results show that after 2 hours of enzymatic hydrolysis of 0.5g Arabidopsis callus, protoplasts with good morphology and large number can be obtained. The number of protoplasts obtained is about 1.46×10 6 Pieces / mL.

[0083] Comparative Example 1

[0084] This comparative example provides a method for fusing Physcomitrella patens protoplasts and Arabidopsis callus cells. This comparative example adopts the polyethylene glycol PEG induced fusion method, wherein the preparation methods of Physcomitrella patens protoplasts and Arabidopsis protoplasts refer to Example 1. The specific steps are as follows:

[0085] The two protoplasts were mixed in a ratio of 1:1 between Physcomitrella patens and Arabidopsis thaliana, centrifuged at 100 g for 10 min, the supernatant was discarded, and the mixture was gently resuspended in 1 mL of W5. An equal volume of PEG-Ca was added. 2+ The solution was immediately gently inverted to mix, and allowed to stand at room temperature for 10 min; 1 mL of high pH-high Ca 2+ The solution was placed at room temperature for 5 min; centrifuged at 100 g for 5 min, and the supernatant was discarded; 1 mL of high pH-high Ca 2+ Solution, gently flick the centrifuge tube to resuspend, then centrifuge at 100g for 3 min, and discard the supernatant; add 1mL PIM liquid culture medium, gently flick the centrifuge tube to resuspend, then centrifuge at 100g for 2 min, and discard the supernatant; add 3mL PIM liquid culture medium to a 6-well cell culture dish to rinse, aspirate the PIM solution in the 6-well culture dish to resuspend the cells, and then transfer them to the corresponding culture dish and culture them at 20℃ in the dark.

[0086] Fusion effect: The green fluorescence of Arabidopsis callus (no chloroplasts) and the chloroplasts of wild-type Physcomitrella patens (no fluorescence) were used to indicate the cell fusion between the two. After PEG-induced fusion, the cell morphology was abnormal, and very few green fluorescent cells with chloroplasts (fused cells) were observed under the GFP fluorescence channel (microscope: OLYMPUS, SZX16). After a period of culture, the cells aggregated, but the number of dead cells was high. Fig.12 As shown, Fig.12 This is the cell culture after polyethylene glycol PEG-induced fusion.

[0087] By comparing Example 1 with Comparative Example 1, it can be seen that compared with electrofusion, polyethylene glycol fusion affects the cell state.

[0088] Comparative Example 2

[0089] This comparative example provides a method for fusing Physcomitrella patens protoplasts and Arabidopsis callus cells, wherein the preparation methods of Physcomitrella patens protoplasts and Arabidopsis protoplasts refer to Example 1, and the steps and parameters of electric field induced fusion are as follows:

[0090] Mix the two protoplasts in a ratio of 1:1 between Physcomitrella patens and Arabidopsis thaliana, centrifuge at 100 g for 10 min; discard the supernatant, gently resuspend with 1 mL of electrofusion solution (0.8 mol / L mannitol + 0.05 mmol / L CaCl2), centrifuge for 10 min, and repeat washing twice; add 1 mL of 0.8 mol / L mannitol to resuspend the cells; draw 20 μL of the mixed cell solution onto the fusion electrode (0.5 mm spacing) for electrofusion (AC 15 V, DC 300 V, AC action time 10 s, DC pulse time 15 μs, DC pulse number 1, DC pulse interval 0.5 s) (cell fusion instrument: NEPA GENE, ECFG21); draw the electrofused cell solution into a 6-well cell culture dish containing 3 mL of PIM liquid culture medium, repeat the operation until 200 μL of cell solution is electrofused (note to set a control), and culture at 20°C in the dark.

[0091] Fusion effect: The green fluorescence of Arabidopsis callus (no chloroplasts) and the chloroplasts of wild-type Physcomitrella patens (no fluorescence) were used to indicate the cell fusion between the two. After the electric field induction, the cells aggregated into clusters, but no green fluorescent cells with chloroplasts (fused cells) were observed under the GFP fluorescence channel (microscope: OLYMPUS, SZX16). Fig.13 As shown, Fig.13 This is the protoplast after electric field induced fusion.

[0092] Comparative Example 3

[0093] This comparative example provides a method for fusing Physcomitrella patens protonema and Arabidopsis thaliana callus cells. The difference between the method and comparative example 2 is that the parameters of the electric field induced fusion are as follows:

[0094] The alternating current was 50 V, the direct current was 650 V, the AC action time was 10 s, the DC pulse time was 15 μs, the number of DC pulses was 1, and the DC pulse interval was 0.5 s (cell fusion instrument: NEPA GENE, ECFG21).

[0095] After fusion, the cells aggregated into clusters, but no green fluorescent cells with chloroplasts (fused cells) were observed under the GFP fluorescence channel (microscope: OLYMPUS, SZX16). Fig.14 As shown, Fig.14 This is the protoplast after electric field induced fusion.

[0096] Comparative Example 4

[0097] This comparative example provides a method for fusing Physcomitrella patens protonema and Arabidopsis thaliana callus cells. The difference between the method and Example 1 is that the electrofusion solution is 1.5 mol / L mannitol + 0.3 mmol / L CaCl2).

[0098] After fusion, no green fluorescent cells with chloroplasts (fused cells) were observed under the GFP fluorescence channel (microscope: OLYMPUS, SZX16). Fig.15 As shown, Fig.15 It is the protoplast after electric field induced fusion. By comparing the results of Comparative Examples 2 and 3 with those of Implementation 1, it can be seen that the parameters of electric field induced fusion affect the final fusion effect. When the parameters of the alternating current in the conditions of electric fusion are outside the range of 30-40V and the parameters of the direct current are outside the range of 500-600V, the fusion effect is poor. The concentration range of the electric fusion solution in the present invention is optimized. It can be seen from the fusion results of Comparative Example 4 that when the concentration of mannitol is outside 0.5-0.8mol / L or the concentration of CaCl2 is outside 0.05-0.15mmol / L, the electric fusion effect is poor.

[0099] In summary, the present invention utilizes electric field induced fusion method to fuse Physcomitrella patens protoplasts hydrolyzed by collapsing enzyme solution and Arabidopsis callus protoplasts hydrolyzed by T3 solution, the operation process is simple and fast, fused cells can be observed through a microscope, subsequent cell regeneration culture can form cell clusters, and the cell state is normal. Compared with the polyethylene glycol (PEG) induced fusion method, the fusion efficiency between cells is improved. The Physcomitrella patens and Arabidopsis cell fusion system established by the present invention shows important application prospects in transferring stress resistance traits, carrying out crop improvement, realizing distant recombination and creating new species.

[0100] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for introducing a target DNA fragment into a plant, characterized in that: The method comprises: electrofusion of the Physcomitrella patens protoplasts containing the target DNA fragment in the cell nuclear genome and the protoplasts of the plant to achieve the introduction of the target DNA fragment into the plant.

2. The method according to claim 1, characterized in that: The method comprises the following steps: preparing protoplasts of the Physcomitrella patens and the plant respectively, resuspending and washing the protoplasts with an electrofusion solution, and then resuspending the protoplasts with a mannitol solution to obtain a mixed cell solution; The mixed cell fluid is added to a fusion electrode for electrofusion, and the protoplasts after electrofusion are cultured and screened to obtain target fusion cells, thereby introducing the target DNA fragment into the plant.

3. The method according to claim 2, characterized in that The electrofusion solution comprises, by concentration, 0.5-0.8 mol / L mannitol and 0.05-0.15 mmol / L CaCl2; Preferably, the amount of protoplasts in the mixed cell fluid is (1.8-2)×10 5 Pieces / mL.

4. The method according to any one of claims 1 to 3, characterized in that The conditions of the electrofusion are: alternating current 30-40V, direct current 500-600V, AC action time 10-20s, DC pulse time 15-25μs, number of DC pulses 2-3, and DC pulse interval 0.3-0.7s.

5. The method according to any one of claims 1 to 4, characterized in that The Physcomitrella patens protoplasts are prepared by a method comprising the following steps: Disperse the Physcomitrella patens in the protonema state in a catalytic enzyme solution, place it in a dark room for static culture, and digest the cell wall; after the culture, filter and centrifuge, collect the precipitate, resuspend the protoplasts with a mannitol solution, filter again, and wash the protoplasts with a mannitol solution to obtain the Physcomitrella patens protoplasts.

6. The method according to claim 5, characterized in that The mass concentration of the collapsed enzyme solution is 1.5-2.5%; Preferably, the temperature of the static culture is 20-25°C, and the time of the static culture is 1.5-2.5h; Preferably, the filtration uses a 40-45 μm filter; Preferably, the centrifugal force of the centrifugation is 150-180g, and the centrifugal time is 5-10min; Preferably, the concentration of mannitol in the mannitol solution is 0.2-0.6 mol / L.

7. The method according to any one of claims 1 to 6, characterized in that The plant protoplasts are prepared by a method comprising the following steps: The plant callus tissue is dispersed in T3 enzymatic hydrolysate, and cultured in a dark room to digest the cell wall; after the culture, the plant callus tissue is filtered and centrifuged to collect the precipitate, and the protoplasts are resuspended with W5 solution, filtered again, and the protoplasts are washed with W5 solution to obtain the plant protoplasts.

8. The method according to claim 7, characterized in that The culture speed is 70-80 rpm, and the culture time is 1-2 h; Preferably, the filtration uses a 40-45 μm filter; Preferably, the centrifugal force of the centrifugation is 80-120 g, and the centrifugal time is 5-10 min.

9. The method according to any one of claims 1 to 8, characterized in that The plant is selected from any one of Arabidopsis thaliana, tomato and rice.

10. Use of the method for introducing a target DNA fragment into a plant according to any one of claims 1 to 9 in plant gene transformation.

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