Method for preparing thorn-free ilex cornuta leaf protoplast and establishing instantaneous conversion system

Abstract

The invention discloses a method for preparing a stingless Chinese holly leaf protoplast and establishing an instantaneous conversion system, which comprises the following steps: (1) preparing a material and pretreating the material: picking new tender stingless Chinese holly leaves as the material for preparing the protoplast, and pretreating the material by dark treatment; (2) protoplast preparation and separation: soaking the tender leaves subjected to dark treatment with ethanol, washing with water, cutting off leaf veins, cutting the leaf parts into shreds, adding enzymatic hydrolysate for enzymolysis, filtering, centrifuging, precipitating, washing and resuspending; (3) constructing a transient expression subcellular localization vector; and (4) instantaneous transformation of the thorn-free holly mesophyll cell protoplast. Technical support is provided for molecular breeding means of the ilex cornuta.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, and in particular to a method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system. Background Art

[0002] Thornless holly (Ilex cornuta var.'fortunei') is a variant of holly, and its ornamental value is highly favored in the application of garden plants compared with holly. From the perspective of its leaves, the leaves of thornless holly are leathery and shiny, with a dark green color, which can provide a lasting green background for the garden landscape, especially in autumn and winter, to preserve the green for the garden, adding color and layering to the garden landscape.

[0003] Thornless holly blooms in April and May. The flowers are yellow-green, small and dense, and grow in the leaf axils. Its fruit is a drupe-shaped ball, red when ripe. Thornless holly has a long fruit-bearing period and is highly ornamental. The tree has a beautiful crown and upright branches. After pruning, it can be made into bonsai, pile landscape and other shapes to meet the needs of different garden landscape applications. It can be planted alone as the main landscape of the garden to show its unique tree shape, or it can be grouped with other plants to create a rich and diverse landscape effect. In summary, thornless holly has a high ornamental artistic value in the field of gardening and horticulture and is widely used.

[0004] With the widespread commercial use of enzymes and the continuous development of contemporary biological technology, people have prepared protoplasts from various tissues of various plants (roots, stems, leaves, callus, etc.), and also regenerated complete plants. In addition, protoplasts have unique advantages in plant molecular breeding when used as experimental materials. Protoplasts are cells with cell walls removed. Without cell walls, exogenous genes can more easily approach and enter the cell membrane, which greatly improves the efficiency of gene introduction; protoplasts can maintain high activity and have the ability to regenerate cell walls and perform cell division. Even if they are stimulated to a certain extent during the introduction of exogenous genes, protoplasts can still recover and develop normally, thereby ensuring that the transformed cells can continue to grow and differentiate to form complete transgenic plants. Protoplasts are often used in protein subcellular localization analysis, single-cell sequencing, overcoming distant hybridization barriers, gene editing and other fields. With the rapid development of biological genetic transformation and genetic engineering related technologies, the advantages of protoplasts as experimental materials will be further magnified. How to obtain a large number of highly active plant protoplasts is the most basic condition for conducting genetic transformation related research. Therefore, it is of great significance to explore how to obtain efficient preparation and separation of protoplasts and transient expression systems. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system. The present invention uses an enzymatic hydrolysis method to construct a thornless holly leaf protoplast preparation, separation and purification system; and uses a PEG method to construct a protoplast instantaneous transformation system, so that relevant gene function research can be carried out through the thornless holly leaf protoplasts to provide technical support.

[0006] A method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system comprises the following steps:

[0007] (1) Preparation of materials and pretreatment of materials: Pick new tender leaves of thornless holly as materials for preparing protoplasts, and the pretreatment of materials is dark treatment;

[0008] (2) Protoplast preparation and separation: The dark-treated young leaves were soaked in ethanol, rinsed with water, the leaf veins were removed, the leaf parts were cut into thin filaments, enzymatic hydrolysis was added, filtered, centrifuged, precipitated, washed and resuspended;

[0009] (3) Construction of transient expression subcellular localization vector;

[0010] (4) Transient transformation of protoplasts from thornless holly mesophyll cells.

[0011] The method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system of the present invention comprises the following steps: placing the young thornless holly leaves in a dark environment at 25° C. for 12 hours.

[0012] The method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system of the present invention, wherein the protoplast preparation and separation specifically comprises the following steps:

[0013] Weigh 1 g of dark-treated thornless young leaves of holly, soak them in 75% ethanol for 5 min, and then rinse with ddH 2 O was rinsed repeatedly for 3 times to wash off the ethanol on the residual leaves. The sterilized material was placed in a sterile culture dish, and the leaf veins were removed with a blade on a clean bench. The remaining leaf parts were cut into 0.1-0.3 mm filaments and placed in a beaker containing 5 mL of enzymatic hydrolyzate. The filtrate was placed in a dark environment of a 25°C constant temperature shaker for enzymatic hydrolysis at 50 r / min. The filtrate was filtered with a filter device, and the filtrate was centrifuged at 100 r / min for 3 min. The precipitate was washed with 1-3 mL of W5 solution and resuspended, and repeated 2-3 times.

[0014] The method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system of the present invention comprises the following steps: the enzymatic solution is a CPW salt solution containing 2.5% cellulase, 1.0% macerate, 0.5% pectinase, 0.4% snailase and 0.3 mol / L mannitol, and the pH value is 5.7.

[0015] The method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system of the present invention comprises a filter device comprising a frame and a filter screen fixed thereon, wherein the filter screen comprises two layers, the upper layer being a 70 μm nylon filter screen and the lower layer being a 40 μm nylon filter screen.

[0016] The method for preparing thornless holly leaf protoplasts and establishing a transient transformation system of the present invention, wherein the construction of the transient expression subcellular localization vector specifically comprises the following steps:

[0017] (A) The amplification primers pORE_R4-F and pORE_R4-R were designed based on the coding sequence of FPS2 of Ilex oleraceus for connecting to the subcellular localization vector pORE_R4;

[0018] (B) Using cDNA from thornless holly leaves as template and IcFPS2-pORE_R4-F / R as primers, the FPS2 gene fragment was amplified by PCR. The amplified fragment was purified and reconstructed into the pORE_R4 vector linearized with Nhel and Xhol.

[0019] (C) The recombinant pORE_R4 carrying the FPS2 gene fragment was heat-shock transformed into DH5α colon competent cells, and the correct recombinant clone was selected after double digestion with Nhel and Xhol and sequenced and verified, named pORE_R4-FPS2, containing 1170 base pairs;

[0020] (D) Sufficient amount of pORE_R4-FPS2 plasmid was extracted using the enhanced endotoxin-free plasmid extraction kit, with a mass concentration of >5 μg / μL, for protoplast transformation of thornless holly leaf cells.

[0021] The method for preparing thornless holly leaf protoplasts and establishing a transient transformation system of the present invention, wherein the transient transformation of the thornless holly leaf protoplasts specifically comprises the following steps:

[0022] The PEG-mediated method was used to transiently transform the protoplasts of thornless holly mesophyll cells with the constructed plasmid containing the target gene and GFP signal. 200 μL of thornless holly leaf protoplast suspension was first added into a 5 mL centrifuge tube, with a density of ≈2x10 6 g -1 , activity>80%, centrifuge and discard the supernatant, add 200μL of MMG solution and mix gently, then add 10μL of plasmid (concentration 0.5μg / mL) and 40% concentration PEG-CaCl with the same volume as the protoplasts. 2 Solution, a certain concentration of PEG, 0.2M mannitol, 100mM CaCl 2, gently blow and mix; then place it at room temperature, incubate in the dark for 10-15 minutes, and then add 2 volumes of W5 solution to terminate the reaction; centrifuge the transformed thornless holly leaf protoplasts at 100g for 2-3 minutes, discard the supernatant and resuspend it with 1mL W5 solution, repeat 3-4 times, wrap it with tin foil and place it in a 25℃ incubator in the dark for 24-36h; finally, add 5-10μL of fluorescent dye DAPI working solution, 0.1μg / mL, to the suspension of thornless holly leaf protoplasts after dark culture, and gently pipette and mix it, observe it under a laser confocal microscope, and observe the GFP fluorescence signal and DAPI fluorescence signal of the thornless holly leaf protoplasts.

[0023] The method for preparing thornless holly leaf protoplasts and establishing a transient transformation system of the present invention comprises the following steps: the MS medium is improved during the bud induction and rooting stages, and the formula of the improved MS medium is modified as follows based on the original MS medium: ammonium sulfate is used to replace ammonium nitrate, and 300 mg / L asparagine is added;

[0024] The newly grown tender leaves of the thornless holly are derived from sterile seedlings obtained by tissue culture, wherein the culture medium formula for inducing bud differentiation is: improved MS culture medium + 1.5 mg / L 6-BA + 0.25 mg / L NAA + 0.4 mg / L ascorbic acid + 0.1 g / L activated carbon; the rooting culture medium formula is: improved MS culture medium + 0.1 mg / L NAA + 0.1 mg / L IBA.

[0025] The method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system of the present invention, wherein the tissue culture also includes the collection and disinfection of explants: selecting the top of a thornless holly branch or a lateral bud as an explant, which is required to be healthy and free of diseases and insect pests, and using 75% alcohol and 0.5% sodium hypochlorite solution to disinfect the surface of the explant to avoid contamination.

[0026] The method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system of the present invention comprises the following culture conditions for tissue culture: culture temperature: 23-25°C, light: 12-16 hours / day, light intensity: 1000-2000 lux, and humidity: 60-70%.

[0027] The method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system of the present invention is different from the prior art in that:

[0028] The method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system uses young leaves grown from thornless holly (Ilexcornuta var.'fortunei') as materials, adopts a 4-factor 3-level orthogonal test L 9(3 4 ) The mass concentrations of related enzymes were analyzed: cellulase (1.5%, 2.0%, 2.5%), macerate (1.0%, 1.5%, 2.0%) and osmotic pressure regulator mannitol (0.20, 0.25, 0.30, 0.35, 0.40 mol / L) on the yield and activity of isolated thornless holly leaf protoplasts; in addition, a single variable was set to conduct experiments (enzymatic hydrolysis time 2.0, 2.5, 3.0, 3.5, 4.0 h) to screen the best enzymatic hydrolysis time for preparing thornless holly leaf protoplasts, so as to determine the optimal conditions for separating thornless holly leaf protoplasts; the thornless holly leaf protoplasts prepared and separated under the optimal conditions were used as materials, and the plasmid was transformed by polyethylene glycol (PEG4000)-mediated method to establish an instantaneous transformation system for obtaining thornless holly leaf protoplasts. L 9 (3 4 ) The results of orthogonal test showed that the optimal enzymatic hydrolysis conditions were: the leaves of thornless holly were hydrolyzed in an enzyme solution containing 2.50% cellulase, 1.00% maize enzyme, 0.50% pectinase, 0.40% snail enzyme, and 0.30 mol / L mannitol under dark conditions for 3.5 h (50 rpm, 25 ° C), and the yield could reach 11.75×10 6 g -1 , the activity was 68.30%. After the thornless holly leaf protoplasts were transformed with pORE_R4-FPS2 plasmid by PEG-mediated method, the green fluorescence signal and DAPI blue fluorescence signal in the cell nucleus could be observed under laser confocal microscope.

[0029] The invention determines the optimal enzymatic hydrolysis concentration and the transient transformation method of the thornless holly leaf protoplasts obtained by the enzymatic hydrolysis method, and establishes a protoplast transient transformation system for gene function analysis of the thornless holly.

[0030] The invention also establishes a rapid propagation system of thornless holly. Tissue culture is an important method for rapid propagation of thornless holly, and a large number of plants with consistent genetic traits can be obtained in a short time. Comparative experiments show that the best formula for inducing bud differentiation is: improved MS medium + 1.5 mg / L 6-BA + 0.25 mg / L NAA + 0.4 mg / L ascorbic acid + 0.1 g / L activated carbon, and the budding rate can reach 88.6%. The best rooting medium is: improved MS medium + NAA (naphthaleneacetic acid, 0.1 mg / L) + IBA (indolebutyric acid, 0.1 mg / L), and the rooting rate can reach 90.2%. The obtained sterile seedlings can be used for subsequent protoplast extraction.

[0031] The method for preparing thornless holly leaf protoplasts and establishing the transient transformation system of the present invention will be further described below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The present invention is a flow chart of protoplast preparation in the method of the present invention;

[0033] Figure 2 It is a schematic diagram of the structure of the filtering device in the method of the present invention;

[0034] Figure 3 is the plasmid map of pORE_R4-FPS2 in the method of the present invention;

[0035] Figure 4 The result diagram of different dark treatment combinations in the pretreatment process of the method of the present invention;

[0036] Figure 5 The protoplasts are prepared under the most suitable conditions in the method of the present invention;

[0037] Figure 6 This is a graph showing the results of a mannitol concentration gradient experiment in the method of the present invention;

[0038] Figure 7 Figure 2 is a protoplast diagram at different mannitol concentrations in the method of the present invention;

[0039] Figure 8 This is a diagram showing the results of an enzymatic hydrolysis time gradient experiment in the method of the present invention;

[0040] Fig. 9 Figure 2 is a protoplast diagram at different enzymatic hydrolysis times in the method of the present invention;

[0041] Fig.10 This is a diagram of the filtration results of a single-layer nylon mesh used for comparison in the method of the present invention;

[0042] Fig.11 This is a diagram of the filtering results of the self-made double-layer filtering device in the method of the present invention;

[0043] Fig.12 This is a diagram of the transient transformation of thornless holly mesophyll protoplasts with the R4-FPS2 vector mediated by PEG in the method of the present invention;

[0044] The Chinese counterparts of the English in the drawings of the present invention are as follows:

[0045] Yield: output;

[0046] Vitality: Vitality;

[0047] Protoplast yield: protoplast yield;

[0048] Protoplast viability: Protoplast viability;

[0049] D-Mannitol concentration: D-mannitol concentration;

[0050] Enzymolysis duration: Enzymolysis time. DETAILED DESCRIPTION

[0051] 1 Materials and methods

[0052] 1.1 Materials and reagents

[0053] The experimental materials were from the thornless holly (Ilex cornuta var.'fortunei') planted in Zhejiang Agriculture and Forestry University, and protoplasts were prepared by picking its new tender leaves. Cellulase (Cellulase R-10) and macrozyme (Macerozyme R-10), snail enzyme, pectinase, morpholineethanesulfonic acid (MES), mannitol and polyethylene glycol 4000 (PEG4000) were purchased from Yuanye Biological Company; RNA extraction kit, agarose gel DNA recovery kit, endotoxin-free plasmid extraction kit (enhanced) and rapid reverse transcription kit were purchased from Tiangen Biological Company; recombinant cloning kit and high-fidelity enzyme were purchased from Novozyme, and the rest of the reagents were conventional reagents in China.

[0054] Preparation of protoplast hydrolysate: weigh the enzymes (cellulase, macerator, snail enzyme and pectinase) of corresponding mass concentration and dissolve them in CPW salt solution (CaCl 2 1110mg / L, KCl 745mg / L, 2-morpholineethanesulfonic acid (MES) 3900mg / L, PvP8700mg / L, bovine serum albumin (BSA) 1000mg / L), then add corresponding concentration of mannitol, and adjust the pH value of the enzymatic hydrolyzate to 5.7.

[0055] 1.2 Preprocessing

[0056] Dark treatment: Place the young leaves of thornless holly in a dark environment at 25℃ for 12 hours and then perform enzymatic hydrolysis on them.

[0057] 1.3 Methods for protoplast preparation and separation

[0058] In this experiment, an orthogonal experimental design (L 9 (3 4 )), all other experiments controlled a single variable for design experiments.

[0059] Weigh 1g of young thornless holly leaves, soak them in 75% ethanol for 5 minutes, rinse them repeatedly with dd water for 3 times to clean the ethanol remaining on the leaves. Place the sterilized material in a sterile culture dish, use a blade to cut off the leaf veins on a clean bench, cut the remaining leaf parts into 0.1-0.3mm filaments, put them in a beaker containing 5mL of enzyme solution, and place them in a dark environment with a constant temperature (25℃) shaker (50r / min) for enzymatic hydrolysis. Filter with a filter device, centrifuge the filtrate at 100r / min for 3 minutes, wash the precipitate with 1-3mL W5 solution and resuspend it, and repeat 2-3 times. Figure 1 shown.

[0060] 1.4 Fabrication of protoplast filtration device

[0061] The filtering device comprises a frame and a filter screen fixed thereon, wherein the filter screen comprises two layers, the upper layer is a 70 μm nylon filter screen, and the lower layer is a 40 μm nylon filter screen.

[0062] Production method: Cut two layers of 70μm and 40μm nylon mesh into 3×3cm square pieces respectively, embed them into the filter plastic mold frame in turn, and cut off the excess parts, such as Figure 2 shown.

[0063] 1.5 Protoplast counting and vitality detection

[0064] The protoplast yield was counted by using a blood cell counting chamber. Each sample was statistically repeated three times, and the average value was taken to finally calculate the protoplast yield of thornless holly leaves.

[0065] Protoplast activity assay was performed by using fluorescein diacetate (FDA) at a concentration of 0.01% and counted.

[0066] 1.6 Construction of transient expression subcellular localization vector

[0067] The specific steps for constructing the positioning vector are:

[0068] (A) Based on the coding sequence of FPS2 of Ilex oleraceus, amplification primers for connecting to the subcellular localization vector pORE_R4 were designed: pORE_R4-F (5'-ATGCTGGACTATAATGTACCTGGAGGG-3')

[0069] pORE_R4-R(5'-CTTCTGCCTCTTGTATATCTTACCCAAAAAGGA-3').

[0070] (B) Using cDNA from thornless holly leaves as template and IcFPS2-pORE_R4-F / R as primers, the FPS2 gene fragment was amplified by PCR. The amplified fragment was purified and reconstructed into the pORE_R4 vector linearized with Nhel and Xhol.

[0071] (C) The recombinant pORE_R4 carrying the FPS2 gene fragment was heat-shock transformed into DH5α colon competent cells, and the correct recombinant clone was selected after double digestion with Nhel and Xhol and sequencing, and named pORE_R4-FPS2 ( Figure 3 , containing 1170 base pairs);

[0072] (D) A sufficient amount (mass concentration > 5 μg / μL) of pORE_R4-FPS2 plasmid was extracted using the Endotoxin-Free Plasmid Extraction Kit (Enhanced) for protoplast transformation of thornless holly leaf cells.

[0073] 1.7 Transient transformation of protoplasts from thornless holly mesophyll cells

[0074] The PEG-mediated method was used to transiently transform the protoplasts of thornless holly mesophyll cells with the constructed plasmid containing the target gene and GFP signal. First, 200 μL of thornless holly leaf protoplast suspension (density ≈ 2x10 6 g -1 , activity>80%), centrifuge and discard the supernatant, add 200 μL of MMG solution and mix gently, then add 10 μL of plasmid (0.5 μg / mL) and 40% concentration of PEG-CaCl in the same volume as the protoplasts. 2 Solution (a certain concentration of PEG, 0.2M mannitol, 100mM CaCl 2 ), gently blow and mix, then place it at room temperature, incubate in the dark for 10-15min, add 2 times the volume of W5 solution to terminate the reaction; centrifuge the transformed thornless holly leaf protoplasts at 100g for 2-3min, discard the supernatant and resuspend it with 1mL W5 solution (repeat 3-4 times), wrap it with tin foil and place it in a 25°C incubator in the dark for 24-36h. Finally, add 5-10μL of fluorescent dye DAPI working solution (0.1μg / mL) to the suspension of thornless holly leaf protoplasts after dark culture and gently pipette and mix, observe under a laser confocal microscope, and observe the GFP fluorescence signal and DAPI fluorescence signal of the thornless holly leaf protoplasts.

[0075] 2 Results and analysis

[0076] 2.1 Establishment of the rapid propagation system and sterile seedling culture of thornless holly

[0077] Tissue culture is an important method for rapid propagation of thornless holly, which can obtain a large number of plants with consistent genetic traits in a short time. First, select the top of the tender branch or lateral bud of thornless holly as the explant, which must be healthy and free of pests and diseases. Use 75% alcohol and 0.5% sodium hypochlorite solution to disinfect the surface of the explant to avoid contamination. Through 6 groups of comparative experiments, it was found that the best formula for inducing bud differentiation is: modified MS medium + 1.5mg / L 6-BA + 0.25mg / L NAA + 0.4mg / L ascorbic acid + 0.1g / L activated carbon, and the budding rate can reach 88.6%. The best rooting medium is: modified MS medium + NAA (naphthaleneacetic acid, 0.1mg / L) + IBA (indolebutyric acid, 0.1mg / L), and the rooting rate can reach 90.2%. Culture temperature: 23-25℃, light: 12-16 hours / day, light intensity 1000-2000lux, humidity: 60-70%. The sterile seedlings obtained above can be used for subsequent protoplast extraction.

[0078] Table 1 Effect of different hormone concentration ratios on the induction of thornless holly buds

[0079]

[0080]

[0081] Table 2 Induction of thornless holly buds by ascorbic acid

[0082]

[0083] Table 3 Effects of different hormone concentration ratios on the induction of thornless holly roots

[0084]

[0085] 2.2 Effect of pretreatment on protoplast isolation

[0086] Dark treatment comparison experiment: Place the young leaves of thornless holly in a dark environment at 25℃ for a certain period of time, and then perform enzymatic hydrolysis. Set up three treatments, 12h, 18h, and 24h, respectively, and use the untreated leaves as the control.

[0087] The results are as follows Figure 4 As shown in the figure, the protoplast yield under dark treatment was significantly higher than that of the control group. As the dark treatment time increased, its yield showed an upward trend. Dark treatment increased the yield of thornless holly leaf protoplasts because dark treatment caused the plant cell wall to become relatively loose, making it easier to be decomposed by enzymes such as cellulase and pectinase. The protoplast activity reached its maximum at 12 hours of treatment, and then decreased with the extension of time. This is because the material was kept away from light for a long time, causing damage to the plant cells.

[0088] 2.3 Effects of different enzyme concentration combinations on protoplast separation

[0089] The orthogonal experimental design (L 9 (3 4 )) were screened for different concentrations of enzyme solutions (cellulase, maceration enzyme, pectinase and snailase) for preparing and separating thornless holly mesophyll protoplasts. Newly grown tender leaves of thornless holly were selected from the campus of Zhejiang Agriculture and Forestry University. The concentration of mannitol as an osmotic pressure regulator in the enzyme solution was controlled at 0.30 mol / L, and the enzymolysis time was set to 3.5 h. The orthogonal experimental results showed (Table 4, Figure 5 ) It can be seen that among the 9 combined enzyme treatments, the combination with the lowest protoplast yield and the lowest protoplast activity was treatment 3, with an average yield of only 1.5×10 6 / g, and the average activity is only 56.70%.

[0090] Taking into account the yield and activity of thornless holly leaf protoplasts from different groups, combination treatment 7 was determined to be the optimal enzymatic solution for separating thornless holly leaf protoplasts: an enzymatic solution combination of 2.5% cellulase + 1.0% malonase + 0.5% pectinase + 0.4% snailase + 0.3 mol / L mannitol. The optimal enzyme solution combination 7 was used to prepare the separated thornless holly leaf protoplasts. Figure 5 shown.

[0091] Table 3 Different enzyme treatment combinations

[0092]

[0093] 2.4 Effect of different mannitol concentrations on protoplast preparation and separation

[0094] After determining the different enzyme concentrations of enzyme solution combination 7, based on this, 0.20-0.40 mol / L mannitol was added in sequence to enzymolyze the tender leaves of thornless holly. The enzymolysis time was controlled to 3.5 h. The statistical results of protoplast yield and activity are shown in Figure 6 , Figure 7 As shown in the figure, when the mannitol concentration is 0.20 mol / l, the yield of separated and purified protoplasts is the highest, but some of the thornless holly leaf protoplasts are swollen; when the mannitol concentration is 0.35 mol / l, the obtained protoplasts have the highest activity, but their state is not round and shrinks; in general, the mannitol concentration of 0.30 mol / L is the optimal concentration for the separation and purification system of thornless holly leaf cell protoplasts.

[0095] 2.5 Effect of different enzymatic hydrolysis times on protoplast separation

[0096] Through orthogonal gradient experiments, the best enzymatic hydrolysis solution combination has been selected, and on this basis, the enzymatic hydrolysis time required for separating the protoplasts of the thornless holly mesophyll was further explored. Figure 8 and 9 It can be seen that when the enzymatic hydrolysis time is before 3.5 h, the protoplast yield and activity of thornless holly leaf will gradually increase with the increase of enzymatic hydrolysis time, and the protoplast yield increases from 3.25×10 6 g -1 Increased to 14.50×10 7 g -1 , the activity increased from 61.36% to 81.32%. Among them, the protoplast yield of 3.5h of enzymatic hydrolysis was significantly higher than that of 2.5 and 3.0h. However, when the enzymatic hydrolysis time exceeded 3.5h, the protoplast activity and yield continued to decrease over time. When the enzymatic hydrolysis time was 2.0h, only individual protoplasts could be observed, so the protoplast yield and activity when the enzymatic hydrolysis time was 2.0h were not statistically analyzed. Based on the above results, we believe that the optimal time for preparing thornless holly leaf protoplasts using enzymatic hydrolysis is 3.5h.

[0097] 2.6 Effect of filtration device on protoplast separation

[0098] The study found that the thornless holly leaf protoplast suspension obtained by filtering the enzymatic hydrolysate containing thornless holly leaves with a traditional single-layer nylon mesh contained a large amount of leaf fragments and impurities after separation and purification. The number of protoplast suspension fragments obtained by separation and purification with the self-designed filtration device was greatly reduced, such as Fig.10 and 11 shown.

[0099] 2.7 Transient transformation of thornless hollyhock protoplasts using PEG

[0100] To further verify the transient transformation system of thornless holly leaf protoplasts established in the present invention, we used the transient transformation system of thornless holly leaf protoplasts to transiently transform the pORE_R4-FPS2 plasmid encoding the FPS2-GFP fusion protein into the protoplasts isolated and purified from thornless holly leaves. Fig.12 ) showed that in the thornless holly leaf protoplasts successfully transformed with the pORE_R4-FPS2 plasmid, GFP fluorescence signals could be detected and overlapped with the blue fluorescence of the fluorescent dye DAPI. The above results indicate that the FPS2-GFP fusion protein containing the target gene is expressed in the cell nucleus, which is consistent with the results of the previous bioinformatics prediction analysis. Based on this, it is proved that the transient transformation system of thornless holly leaf protoplasts established in this study is reliable.

[0101] 3 Discussion and Conclusion

[0102] The yield and activity of protoplasts are mainly affected by a variety of factors, including plant materials, enzymatic hydrolysis time, different enzyme component combinations and their contents.

[0103] First of all, the plant material used to prepare protoplasts is the most basic factor that determines the activity and yield of protoplasts. Different tissues of plant materials, such as leaves, roots, stems, callus, and flowers, can be used to prepare and isolate protoplasts, but different tissue materials have certain limitations in preparation. For example, the protoplasts isolated and prepared from callus are colorless and transparent, and there will be some difficulties in observation and subsequent transformation experiments; the protoplasts prepared from the petals of plants are often brightly colored and easy to observe under a microscope, but the extraction of protoplasts from petals is often limited by factors such as short flowering period. On the whole, plant leaves as preparation materials are not only easy to obtain, but also cause little damage to the plant itself during the sampling process. They are excellent materials for isolating and obtaining plant protoplasts, but in the process of shredding the leaves, cutting too fine will result in more leaf fragments after enzymatic hydrolysis; cutting too coarse will result in too low enzymatic hydrolysis efficiency and failure to obtain sufficient protoplasts. By conducting gradient experiments on factors such as enzyme solution concentration, dark pretreatment, osmotic pressure regulator concentration, and enzymolysis time, the present invention successfully separated and purified thornless holly leaf protoplasts with a rounded shape and good condition using thornless holly leaves as materials.

[0104] Secondly, in order to obtain plant protoplasts with high yield and activity, enzymatic hydrolysis time is also a very important influencing factor. First, in different plants, the time required for protoplast separation using enzymatic hydrolysis is significantly different. For example, it takes 2 hours to separate protoplasts from grape leaves, 4 hours to separate protoplasts from pineapple leaf bases, and 7 hours to separate protoplasts from kiwifruit, while the enzymatic hydrolysis time required to separate protoplasts from Malus octagonalis leaves is 10 hours; second, for different tissues of the same plant, the required enzymatic hydrolysis time is also quite different. For example, it takes about 5 hours to prepare protoplasts using tung flower bud cells, while it takes up to 10 hours to separate protoplasts from tung cotyledons. Therefore, how to determine the enzymatic hydrolysis time required for separating protoplasts from specific plants and their tissues is the key to ensuring the acquisition of protoplasts with high yield and good condition. At the same time, placing the enzymatic hydrolyzate containing plant tissues on a shaker for a period of time after the enzymatic hydrolysis is completed can further promote the release of protoplasts from it. If the enzymatic hydrolysis time is too short, the protoplasts will not be completely released, resulting in a decrease in the yield of the protoplasts finally obtained; if the enzymatic hydrolysis time is too long, the protoplast plasma membrane will be damaged, resulting in a decrease in the activity of the protoplasts and damage to the cell membrane. The present invention conducts a gradient experiment on the enzymatic hydrolysis time required by the thornless holly leaf tissue, and determines that the most suitable enzymatic hydrolysis time for preparing and separating the thornless holly leaf protoplasts is 3.5h.

[0105] Finally, in the process of enzymatic hydrolysis of plant protoplasts, the combination of enzymes of different types and concentrations in the enzymatic hydrolysis solution is also an important factor affecting the yield and activity of protoplasts. Because the cell wall components of each plant and its different tissues are quite different, the combination of enzyme solutions required for enzymatic hydrolysis of protoplasts is also different. For example, 1.5% cellulase and 0.5% pectinase are used to separate rose leaf protoplasts, and 2.0% cellulase R-10 and 1.0% analyte are used to separate rose leaf protoplasts. During the enzymatic hydrolysis process, if a lower amount of enzyme is used, the enzymatic hydrolysis time of the separated protoplasts will be significantly increased. When the enzymatic hydrolysis time is too long, the enzymatic hydrolysis solution will have a toxic effect on the protoplasts, resulting in a decrease in the yield and activity of the protoplasts; and using too high an enzyme concentration will affect the osmotic pressure balance of the protoplasts, further damaging the protoplast plasma membrane and reducing the activity of the protoplasts. In this experiment, the optimal conditions for separating protoplasts from thornless holly leaves were determined by orthogonal experiments with four factors and three levels on enzyme types and concentrations. Under these conditions, the yield of separated protoplasts was 11.75×10 6 g -1 .

[0106] By PEG method, foreign gene is introduced into the protoplast of plant, and it is an ideal transformation system to study plant genetic engineering by protoplast transient transformation. There are many factors affecting protoplast transient transformation, such as PEG solution concentration, protoplast incubation practice, protoplast transformation time, plasmid size and concentration, protoplast quantity and so on, which will affect the transient transformation efficiency. The present invention will contain the pORE_R4 plasmid of target gene and will be transformed into thornless holly leaf protoplasts transiently.

[0107] The present invention successfully constructed a protoplast separation and purification system and an instantaneous transformation system using thornless holly leaf protoplasts as materials. The enzyme solution of 2.5% cellulase + 1.0% malonase + 0.5% pectinase + 0.4% snail enzyme + 0.3 mol / L mannitol was used for enzymatic hydrolysis in the dark for 3.5 hours, and finally a yield of 11.75×10 6 g -1 , protoplast activity> 68.30% of thornless holly leaf protoplasts. At the same time, the pORE_R4 plasmid containing the target gene was transiently transformed into thornless holly leaf protoplasts using the PEG-mediated method, and GFP fluorescence in the cell nucleus could be observed under a laser confocal microscope. This also proves that thornless holly leaf protoplasts can be used to carry out subcellular localization and other related research in the future, providing technical support for thornless holly molecular breeding methods.

[0108] The embodiments described above are merely descriptions of preferred implementation 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 preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system, characterized in that: The steps include: (1) Preparation of materials and pretreatment of materials: Pick new tender leaves of thornless holly as materials for preparing protoplasts, and the pretreatment of materials is dark treatment; (2) Protoplast preparation and separation: The dark-treated young leaves were soaked in ethanol, rinsed with water, the leaf veins were removed, the leaf parts were cut into thin filaments, enzymatic hydrolysis was added, filtered, centrifuged, precipitated, washed and resuspended; (3) Construction of transient expression subcellular localization vector; (4) Transient transformation of protoplasts from thornless holly mesophyll cells.

2. The method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system according to claim 1, characterized in that: The dark treatment condition is: placing the young leaves of thornless holly in a dark environment at 25° C. for 12 hours.

3. The method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system according to claim 2, characterized in that: The protoplast preparation and separation specifically comprises the following steps: Weigh 1 g of dark-treated thornless holly leaves, soak them in 75% ethanol for 5 min, rinse them repeatedly with ddH2O for 3 times, wash off the ethanol remaining on the leaves, place the sterilized material in a sterile culture dish, cut the leaf veins with a blade on a clean bench, cut the remaining leaf parts into 0.1-0.3 mm filaments, put them in a beaker containing 5 mL of enzymatic hydrolyzate, place them in a dark environment of a 25°C constant temperature shaker for enzymatic hydrolysis, 50 r / min, filter them with a filter device, centrifuge the filtrate at 100 r / min for 3 min, wash the precipitate with 1-3 mL of W5 solution and resuspend it, and repeat 2-3 times.

4. The method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system according to claim 3, characterized in that: The enzymatic hydrolysis solution is a CPW salt solution, which contains 2.5% cellulase, 1.0% macerate, 0.5% pectinase, 0.4% snail enzyme and 0.3 mol / L mannitol, and the pH value is 5.

7.

5. The method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system according to claim 3, characterized in that: The filtering device comprises a frame and a filter screen fixed thereon, wherein the filter screen comprises two layers, the upper layer is a 70 μm nylon filter screen, and the lower layer is a 40 μm nylon filter screen.

6. The method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system according to claim 1, characterized in that: The construction of the transient expression subcellular localization vector specifically includes the following steps: (A) According to the coding sequence of FPS2 of Ilex oleraceus, amplification primers pORE_R4-F and pORE_R4-R were designed to connect to the subcellular localization vector pORE_R4; (B) Using cDNA from thornless holly leaves as template and IcFPS2-pORE_R4-F / R as primers, the FPS2 gene fragment was amplified by PCR. The amplified fragment was purified and reconstructed into the pORE_R4 vector linearized with Nhel and Xhol. (C) The recombinant pORE_R4 carrying the FPS2 gene fragment was heat-shock transformed into DH5α colon competent cells, and the correct recombinant clone was selected after double digestion with Nhel and Xhol and sequenced and verified, named pORE_R4-FPS2, containing 1170 base pairs; (D) Sufficient amount of pORE_R4-FPS2 plasmid was extracted using the enhanced endotoxin-free plasmid extraction kit, with a mass concentration of >5 μg / μL, for protoplast transformation of thornless holly leaf cells.

7. The method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system according to claim 6, characterized in that: The instantaneous transformation of the thornless holly mesophyll cell protoplasts specifically comprises the following steps: The PEG-mediated method was used to transiently transform the protoplasts of thornless holly mesophyll cells with the constructed plasmid containing the target gene and GFP signal. 200 μL of thornless holly leaf protoplast suspension was first added into a 5 mL centrifuge tube, with a density of ≈2x10 6 g -1 , activity>80%, centrifuge and discard the supernatant, add 200μL of MMG solution and mix gently, then add 10μL 0.5 μg / mL of plasmid, and 40% concentration of PEG-CaCl2 solution of equal volume with protoplasts, a certain concentration of PEG, 0.2Mmannitol, 100mMCaCl2, gently blow and mix; then place it at room temperature, incubate in the dark for 10-15min, add 2 times volume of W5 solution to terminate the reaction; 100g of the transformed thornless holly leaf protoplasts is centrifuged for 2-3min, the supernatant is discarded and then resuspended with 1mLW5 solution, repeated 3-4 times, wrapped with tin foil and placed in a 25°C incubator in dark for 24-36h; finally, 5-10 μL of fluorescent dye DAPI working solution, 0.1 μg / mL, is added to the suspension of the thornless holly leaf protoplasts after dark culture, and gently sucked and mixed with a pipette, and examined under a laser confocal microscope to observe the GFP fluorescent signal and DAPI fluorescent signal of the thornless holly leaf protoplasts.

8. The method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system according to claim 1, characterized in that: The MS medium was modified during the shoot induction and rooting stages. The formula of the modified MS medium was based on the original MS medium with the following changes: ammonium sulfate was used instead of ammonium nitrate, and 300 mg / L asparagine was added; The newly grown tender leaves of the thornless holly are derived from sterile seedlings obtained by tissue culture, wherein the culture medium formula for inducing bud differentiation is: improved MS culture medium + 1.5 mg / L 6-BA + 0.25 mg / L NAA + 0.4 mg / L ascorbic acid + 0.1 g / L activated carbon; the rooting culture medium formula is: improved MS culture medium + 0.1 mg / L NAA + 0.1 mg / L IBA.

9. The method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system according to claim 8, characterized in that: The tissue culture also includes the collection and disinfection of explants: selecting the top of the tender branch or the side bud of thornless holly as the explant, which is required to be healthy and free of diseases and insect pests, and using 75% alcohol and 0.5% sodium hypochlorite solution to disinfect the surface of the explant to avoid contamination.

10. The method for preparing thornless holly leaf protoplasts and establishing an instantaneous transformation system according to claim 9, characterized in that: The culture conditions of the tissue culture are: culture temperature: 23-25° C., light intensity: 12-16 hours / day, light intensity 1000-2000 lux, and humidity: 60-70%.

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