Plant living body nucleic acid efficient delivery method based on nanoparticles and application

Abstract

The invention discloses a nanoparticle-based plant living body nucleic acid efficient delivery method and application, and belongs to the technical field of biology and the field of agriculture. The functional nucleic acid and a salt compound required by plant growth are self-assembled to form carrier-free nano-particles, then the carrier-free nano-particles and a plant living body are co-incubated and cultured, and plant living body cells actively take in the carrier-free nano-particles, so that the functional nucleic acid is efficiently delivered into the plant living body. The carrier-free nanoparticles prepared by the invention contain functional nucleic acid and salt compounds, and have the advantages of simple preparation process, low cost, good biological safety and the like. In addition, delivery steps are simple and convenient, the method is suitable for various plant species and plant cells in different development stages, and non-destructive efficient gene delivery can be achieved. The technology not only effectively improves the efficiency and convenience of plant gene delivery, but also provides reliable support for application of plant genetic improvement tools, precision agriculture and the like, and has remarkable application prospects and market potential.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and agriculture, and specifically relates to a nanoparticle-based efficient plant nucleic acid delivery method and application. Background Art

[0002] Plant gene delivery technology is a key technology in the field of modern biotechnology. By accurately introducing genes or editing tools into plant cells, it has demonstrated its far-reaching application potential in many key areas such as crop genetic improvement, plant pathogen defense, nutrient and pesticide delivery, and plant health monitoring. In the field of biopharmaceuticals, this technology has revolutionized the transformation of plants into efficient biofactories for the production of therapeutic proteins and vaccines, opening up new avenues for low-cost, large-scale production. At the same time, it also gives us the ability to monitor the response of plants to environmental changes in real time, providing a solid scientific foundation for plant health and soil management. In addition, by precisely editing plant genetic information, plant traits can be purposefully improved, which promotes the sustainable development of agriculture, improves the nutritional value of crops, and has a positive and far-reaching impact on global food security and biodiversity conservation.

[0003] At present, the research and application of plant gene delivery technology has made significant progress and has become an important part of modern agricultural biotechnology. Traditional gene delivery methods, such as Agrobacterium-mediated transformation, are one of the most commonly used gene delivery methods. Foreign genes are introduced into plant cells through Agrobacterium. However, this method has obvious host specificity and is only applicable to certain dicotyledons. The scope of application is relatively limited. For many important crops, especially some monocotyledons, it is difficult to achieve efficient transformation through this method. In addition, Agrobacterium transformation usually requires a complex tissue culture process, which increases the time and cost of the experiment. Gene gun method is a physical gene delivery technology that can quickly introduce foreign genes into plant cells. Although this method can cover a wide range of crop species, its transformation efficiency is low, and it is easy to cause physical damage to plant cells during operation, affecting the growth and development of plants. In addition, this method usually requires high-cost equipment, which is not conducive to large-scale application.

[0004] In order to overcome the limitations of existing methods, the development of new and efficient plant gene delivery technologies has become a hot topic in current plant biotechnology research and a key issue that needs to be solved urgently. With the development of nanotechnology, delivery systems based on nanomaterials are expected to become effective carriers for plant gene delivery. For example, nanotechnologies such as carbon nanotubes and graphene nanocarriers can effectively deliver functional genes or small interfering RNA (siRNA) into plant cells to achieve genetically engineered plants without transgenic insertion. Although such methods have performed well in improving gene delivery efficiency and protecting nucleic acids from degradation, their preparation process usually involves complex chemical synthesis, which is costly and usually requires other auxiliary measures (such as ultrasound) to be introduced into plant cells, making it difficult to achieve large-scale agricultural applications.

[0005] The present invention proposes a method and application of efficient delivery of plant nucleic acid based on nanoparticles, which relates to the fields of biotechnology and agriculture. This method uses functional nucleic acids and salt compounds necessary for plant growth as raw materials, self-assembles to form carrier-free nanoparticles through the freeze concentration effect induced by low temperature, and co-incubates with intact plant cells, and actively absorbs plant living bodies to achieve efficient delivery of plant genes. This process does not require complex experimental operation parts and long-term plant tissue cultivation processes, and is suitable for plant tissues at each growth stage. The nanoparticles prepared by the present invention contain a variety of functional nucleic acids and salt compounds, and the preparation process is simple and cost-effective, while ensuring biosafety, and achieving efficient gene delivery to intact plant cells of various plant species. Therefore, the present invention provides a new plant nucleic acid delivery technology, which provides a more efficient, safe and simple solution for applications such as plant gene editing, the development of transgenic crops, and precision agriculture by overcoming the limitations of traditional methods, showing broad application prospects and market potential. Summary of the invention

[0006] The purpose of the present invention is to provide a method and application of efficient delivery of plant living nucleic acids based on nanoparticles, in which functional nucleic acids and essential salt compounds for plant growth are self-assembled into carrier-free nanoparticles, and active plant receptors are actively absorbed by nanoparticles through co-incubation of nanomaterials and plant receptors, thereby achieving efficient gene delivery in intact plant cells of various plant species.

[0007] To achieve the above object, the present invention adopts the following technical solution: A nanoparticle-based method for efficiently delivering nucleic acids into living plants, wherein functional nucleic acids are self-assembled with salt compounds required for plant growth to form carrier-free nanoparticles, which are then co-incubated and cultured with living plants. The living plant cells actively absorb the carrier-free nanoparticles, thereby achieving efficient delivery of functional nucleic acids into living plants.

[0008] The above-mentioned nanoparticle-based method for efficient delivery of plant living nucleic acid specifically comprises the following steps: (1) functional nucleic acids and salt compounds necessary for plant growth are mixed in an aqueous phase, and self-assembled into carrier-free nanoparticles through freeze concentration effect below freezing point; (2) uniformly mixing the carrier-free nanoparticles and the plant culture medium at a volume ratio of 1:1 to 1:100 to obtain a plant culture medium containing the carrier-free nanoparticles; (3) The living plant is placed in a plant culture medium containing the carrier-free nanoparticles and co-incubated for 1 to 72 hours, so that the carrier-free nanoparticles are fully in contact with the living plant and the carrier-free nanoparticles are actively taken up by the living plant cells, thereby achieving efficient delivery of the functional nucleic acid into the living plant.

[0009] Furthermore, the functional nucleic acid is any one or more combinations of mRNA, siRNA, shRNA, pDNA, T-DNA, cDNA, and mini-chromosome.

[0010] Furthermore, the plant is any one of Arabidopsis, rice, wheat, corn, tomato, soybean, cucumber, sugarcane, ginseng, and honeysuckle.

[0011] Furthermore, the above-mentioned salt compound is any one or more combinations of potassium nitrate, calcium nitrate, ammonium persulfate, ammonium chloride, superphosphate, calcium monophosphate, potassium sulfate, potassium chloride, calcium nitrate, calcium chloride, magnesium sulfate, magnesium chloride, calcium chloride, cobalt chloride, zinc sulfate, copper sulfate, and manganese sulfate.

[0012] Furthermore, the aqueous phase is a mixture of glycerol and water.

[0013] Furthermore, the plant culture medium is any one of sterile water, seedling medium, callus induction medium, and callus subculture medium.

[0014] Furthermore, the developmental stage of the living plant is any one of a seed germination stage, a sprout stage, a vegetative growth stage, a reproductive growth stage, a flowering stage, a fruiting stage, a mature stage, and a senescent stage.

[0015] Furthermore, the roots, stems, leaves, flowers, fruits, seeds, buds, callus tissues, tubers, root tubers or protoplasts of the living plants are placed in a plant culture medium containing carrier-free nanoparticles for co-incubation and culture.

[0016] The application of the above-mentioned nanoparticle-based method for efficient delivery of plant living nucleic acids includes applications in the fields of plant gene silencing, plant gene editing, crop genetic improvement, nutrition and pesticide delivery, biosensors, biopharmaceuticals, biological breeding, and plant gene therapy.

[0017] The significant advantages of the present invention are: (1) Simplified nanomaterial preparation process The present invention uses functional nucleic acid, salt compounds necessary for plant growth and glycerol as raw materials, and promotes their self-assembly to form carrier-free nanoparticles by standing at freezing temperature. This process is not only simple to operate, but also has high cost-effectiveness and biosafety.

[0018] (2) Efficient, safe and simple gene delivery method The carrier-free nanoparticles of the present invention effectively protect nucleic acids from degradation, thereby improving the efficiency of gene delivery. In addition, the co-incubation culture method is used to deliver functional nucleic acids to intact tissue cells of plants, which overcomes the obvious damage and operational complexity caused to plants by traditional plant gene delivery, which ensures the biosafety of gene delivery and improves the stability and reliability of the gene delivery process. At the same time, the salt compounds required for plant growth are used as raw materials, and the characteristics of plant growth actively absorbing nutrients are used to promote the active uptake of nanoparticles, thereby simplifying the operation process and achieving efficient delivery of plant genes.

[0019] (3) Wide applicability The carrier-free nanoparticle gene delivery system of the present invention is applicable to a variety of plant species, including dicotyledonous and monocotyledonous plants, significantly expanding the application scope of gene delivery, having a wide adaptability, and meeting the needs of genetic improvement of a variety of plants.

[0020] (4) Broad application prospects The gene delivery system of the present invention provides a theoretical basis and technical support for plant nucleic acid delivery and real-time plant monitoring, has important application potential and market prospects, and is expected to promote the development and application of plant genetic improvement tools and biosensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The present invention is a flow chart of a method for efficiently delivering nucleic acids in living plants based on nanoparticles.

[0022] Figure 2 This is Example 1, a nanoparticle-based efficient plant gene delivery system for Arabidopsis roots and target protein GFP fluorescence tracing.

[0023] Figure 3 This is the performance test of the nanoparticle-based efficient gene delivery system for Arabidopsis thaliana apical plants in Example 2.

[0024] Figure 4 This is a performance test of the nanoparticle-based Nipponbare rice callus plant gene efficient delivery system of Example 3.

[0025] Figure 5 This is Example 4, a nanoparticle-based efficient plant gene delivery system for Arabidopsis roots and a biosensor performance test.

[0026] Figure 6 This is a performance test of the nanoparticle-based Nipponbare rice callus plant gene efficient delivery system of Example 5.

[0027] Figure 7 This is a performance test of the nanoparticle-based efficient plant gene delivery system for Nipponbare rice callus and Minghui 86 rice callus according to Example 6.

[0028] Figure 8 This is the performance test of the nanoparticle-based efficient plant gene delivery system to wheat seeds in Example 7.

[0029] Fig. 9 This is the performance test of the nanoparticle-based efficient plant gene delivery system for cucumber seeds in Example 8. DETAILED DESCRIPTION

[0030] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0031] The ARF5-GFP vector in the present invention is obtained by connecting the ARF5 cDNA sequence shown in SEQ ID NO.1 to the pAN580 vector, and the Pt-GFP vector in the present invention is obtained by connecting the Ptilosarcus cDNA sequence shown in SEQ ID NO.2 to the pAN580 vector; the pAN580 vector in the present invention has been disclosed in [He Han, Yang Xipeng, Zhao Wanxin, et al. Establishment of pineapple leaf base protoplast isolation and transient transformation system [J]. Journal of Northwest A&F University (Natural Science Edition), 2024, 52(7): 81-88, 98.] and other documents, and it carries an EGFP tag (EGFP is a variant of GFP, and observing GFP fluorescence in the embodiments of the present invention refers to observing EGFP fluorescence). The pYLCRISPR / Cas9Pubi-H vector in the present invention has been disclosed at https: / / www.addgene.org / (Plasmid: #66187), and it carries a Pubi promoter and a Cas9 tag. The pCambia1391Z vector in the present invention has been disclosed in documents such as [Xiong Feng. Cloning and expression analysis of PhRAX2 and PhRAX3 genes in petunia [D]. Anhui Agricultural University.] and has a GUS tag.

[0032] The method for obtaining 3-day-old Arabidopsis thaliana seedlings with complete roots in the present invention is as follows: after sterilizing Arabidopsis thaliana seeds, vernalizing at 4°C for 3 days, inoculating into sterile water, and culturing at 20°C for 3 days until roots and germination occur, thereby obtaining 3-day-old Arabidopsis thaliana seedlings with complete roots.

[0033] The method for obtaining rice callus in the present invention comprises the following steps: removing the seed coat of rice seeds, sterilizing the seeds, inoculating the seeds into a callus induction medium, and culturing the seeds at 28° C. in the dark for 21 days until callus grows out, thereby obtaining rice callus.

[0034] The formula of the callus induction medium of the present invention is: 4.43 g / L MS medium base salt, 4 mg / L 2,4-D, 2.8 g / L proline, 0.3 g / L acid hydrolyzed casein, 30 g / L sucrose, and 3.2 g / L plant gel; the pH value is 5.8.

[0035] The formula of the callus subculture medium in the present invention is: 4.43 g / L MS medium base salt, 4 mg / L 2,4-D, 2.8 g / L proline, 0.3 g / L acid hydrolyzed casein, and 30 g / L sucrose; the pH value is 5.8.

[0036] Embodiment 1: The functional nucleic acid delivery process of this embodiment is as follows Figure 1 As shown, the functional nucleic acid used is ARF5-GFP vector, the applied plant species is Arabidopsis thaliana, and the applied plant organ is root tip. The specific steps are as follows: S1: Dissolve the ARF5-GFP vector in sterile water to prepare an ARF5-GFP aqueous solution with a concentration of 100 ng / μl; S2: KNO 3 Dissolve in sterile water to prepare a 300 mM KNO solution. 3 Aqueous solution; S3: 3 μl of 100 ng / μl ARF5-GFP aqueous solution, 54 μl of 300 mM KNO 3 The aqueous solution was mixed evenly with 3 μl of 10 vol% sterile glycerol aqueous solution, and allowed to stand in liquid nitrogen for 2 min to obtain an aqueous solution of carrier-free nanoparticles 1; S4: mixing the aqueous solution of the carrier-free nanoparticles 1 and sterile water at a volume ratio of 1:100 to obtain a culture medium containing the carrier-free nanoparticles 1; S5: The root tips of 3-day-old intact rooted Arabidopsis seedlings were immersed in a culture medium containing the carrier-free nanoparticles 1, and co-incubated and cultured at 20° C. to obtain treated intact rooted Arabidopsis seedlings; S6: The treated Arabidopsis thaliana seedlings with roots were placed on a glass slide, and GFP fluorescence was observed using a confocal microscope.

[0037] Embodiment 2: The functional nucleic acid used in this example is the ARF5-GFP vector, the plant species used is Arabidopsis thaliana, and the plant organ used is the stem apex growth point. The specific steps are as follows: S1: Dissolve the ARF5-GFP vector in sterile water to prepare an ARF5-GFP aqueous solution with a concentration of 100 ng / μl; S2: KNO 3 Dissolve in sterile water to prepare a 300 mM KNO solution. 3 Aqueous solution; S3: 3 μl of 100 ng / μl ARF5-GFP aqueous solution, 54 μl of 300 mM KNO 3 The aqueous solution was mixed evenly with 3 μl of 10 vol% commercially available sterile glycerol aqueous solution, and allowed to stand in liquid nitrogen for 2 min to obtain an aqueous solution of carrier-free nanoparticles 1; S4: mixing the aqueous solution of the carrier-free nanoparticles 1 and sterile water at a volume ratio of 1:100 to obtain a culture medium containing the carrier-free nanoparticles 1; S5: The shoot tip growth point of a 3-day-old intact rooted Arabidopsis seedling is immersed in a culture medium containing the carrier-free nanoparticle 1, and the culture is co-incubated at 20° C. to obtain a treated intact rooted Arabidopsis seedling; S6: The treated Arabidopsis thaliana seedlings with roots were placed on a glass slide, and GFP fluorescence was observed using a confocal microscope.

[0038] Embodiment 3: The functional nucleic acid used in this example is the pAN580 vector, the plant species used is rice, and the plant organ used is callus tissue. The specific steps are as follows: S1: Dissolve the pAN580 vector in sterile water to prepare a pAN580 aqueous solution with a concentration of 100 ng / μl; S2: KNO 3 Dissolve in sterile water to prepare a 300 mM KNO solution. 3 Aqueous solution; S3: 3 μl of 100 ng / μl pAN580 aqueous solution, 54 μl of 300 mM KNO 3 The aqueous solution was mixed evenly with 3 μl of 10 vol% commercial sterile glycerol aqueous solution, and allowed to stand in liquid nitrogen for 2 min to obtain an aqueous solution of carrier-free nanoparticles 2; S4: mixing the aqueous solution of the carrier-free nanoparticles 2 and the callus subculture medium at a volume ratio of 1:50 to obtain a culture medium containing the carrier-free nanoparticles 2; S5: immersing the rice callus in a culture medium containing the carrier-free nanoparticles 2, and co-incubating and culturing at 28° C. to obtain treated rice callus; S6: Extract RNA from the treated rice callus tissue and amplify the GFP fragment using PCR technology to detect the functional nucleic acid delivery.

[0039] Embodiment 4: The functional nucleic acid used in this example is the Pt-GFP vector, the plant species used is Arabidopsis thaliana, and the plant organ used is the root tip. The specific steps are as follows: S1: Dissolve the Pt-GFP vector in sterile water to prepare a Pt-GFP aqueous solution with a concentration of 100 ng / μl; S2: 3 μl of 100 ng / μl Pt-GFP aqueous solution, 55.5 μl of 0.9 wt% sterile physiological saline and 1.5 μl of 10 vol% commercially available sterile glycerol aqueous solution were mixed evenly, and allowed to stand in liquid nitrogen for 2 min to obtain a carrier-free nanoparticle 3 aqueous solution; S4: uniformly mixing the aqueous solution of the carrier-free nanoparticles 3 and sterile water at a volume ratio of 1:50 to obtain a culture medium containing the carrier-free nanoparticles 3; S5: immersing the root tips of 3-day-old intact rooted Arabidopsis seedlings into a culture medium containing carrier-free nanoparticles 3, and incubating and culturing at 20° C. to obtain treated intact rooted Arabidopsis seedlings; S6: The treated Arabidopsis thaliana seedlings with roots were placed on a glass slide, and GFP fluorescence was observed using a confocal microscope.

[0040] Embodiment 5: The functional nucleic acid used in this example is the pYLCRISPR / Cas9Pubi-H vector, the plant species used is rice, and the plant organ used is callus tissue. The specific steps are as follows: S1: Dissolve the pYLCRISPR / Cas9Pubi-H vector in sterile water to prepare a pYLCRISPR / Cas9Pubi-H aqueous solution with a concentration of 100 ng / μl; S2: KNO 3 Dissolve in sterile water to prepare a 300 mM KNO solution. 3 Aqueous solution; S3: 5 μl of 100 ng / μl pYLCRISPR / Cas9Pubi-H aqueous solution, 52 μl of 300 mM KNO 3 The aqueous solution was mixed evenly with 3 μl of 10 vol% commercially available sterile glycerol aqueous solution, and allowed to stand in liquid nitrogen for 2 min to obtain a carrier-free nanoparticle 4 aqueous solution; S4: mixing the aqueous solution of the carrier-free nanoparticles 4 and the callus subculture medium at a volume ratio of 1:50 to obtain a culture medium containing the carrier-free nanoparticles 4; S5: immersing the rice callus in a culture medium containing the carrier-free nanoparticles 4, and co-incubating and culturing at 28° C. to obtain treated rice callus; S6: Extract RNA from the treated rice callus tissue and amplify the partial fragment of Cas9 by PCR technology to detect the functional nucleic acid delivery.

[0041] Embodiment 6: The functional nucleic acid used in this example is the pCAMBIA1391Z vector, the plant species used is rice, and the plant organs used are seeds. The specific steps are as follows: S1: Dissolve the pCAMBIA1391Z vector in sterile water to prepare a pCAMBIA1391Z aqueous solution with a concentration of 100 ng / μl; S2: KNO 3 Dissolve in sterile water to prepare a 300 mM KNO solution. 3 Aqueous solution; S3: 3 μl of 100 ng / μl pCAMBIA1391Z aqueous solution, 45 μl of 300 mM KNO 3 The aqueous solution was mixed evenly with 12 μl of 10 vol% commercially available sterile glycerol aqueous solution, and allowed to stand in liquid nitrogen for 2 min to obtain an aqueous solution of carrier-free nanoparticles 5; S4: uniformly mixing the aqueous solution of the carrier-free nanoparticles 5 and the callus induction medium at a volume ratio of 1:50 to obtain a medium containing the carrier-free nanoparticles 5; S5: After sterilizing the rice seeds, immersing them in a culture medium containing the carrier-free nanoparticles 5, and co-incubating and culturing them at 28° C. to obtain treated rice seeds; S6: Use Gus staining solution to observe the delivery of Gus gene in treated rice seeds.

[0042] Embodiment 7: The functional nucleic acid used in this example is the pCAMBIA1391Z vector, the plant species used is wheat, and the plant organ used is seeds. The specific steps are as follows: S1: Dissolve the pCAMBIA1391Z vector in sterile water to prepare a pCAMBIA1391Z aqueous solution with a concentration of 100 ng / μl; S2: KNO 3 Dissolve in sterile water to prepare a 300 mM KNO solution. 3 Aqueous solution; S3: 3 μl of 100 ng / μl pCAMBIA1391Z aqueous solution, 45 μl of 300 mM KNO 3 The aqueous solution was mixed evenly with 12 μl of 10 vol% commercially available sterile glycerol aqueous solution, and allowed to stand in liquid nitrogen for 2 min to obtain an aqueous solution of carrier-free nanoparticles 5; S4: uniformly mixing the aqueous solution of the carrier-free nanoparticles 5 and the callus induction medium at a volume ratio of 1:50 to obtain a medium containing the carrier-free nanoparticles 5; S5: After disinfecting the wheat seeds, immerse them in a culture medium containing the carrier-free nanoparticles 5, and incubate them at 28° C. for 72 hours to obtain treated wheat seeds; S6: Use Gus staining solution to observe the delivery of Gus gene in treated wheat seeds.

[0043] Embodiment 8: The functional nucleic acid used in this example is the pCAMBIA1391Z vector, the plant species used is cucumber, and the plant organ used is seeds. The specific steps are as follows: S1: Dissolve the pCAMBIA1391Z vector in sterile water to prepare a pCAMBIA1391Z aqueous solution with a concentration of 100 ng / μl; S2: KNO 3 Dissolve in sterile water to prepare a 300 mM KNO solution. 3 Aqueous solution; S3: 3 μl of 100 ng / μl pCAMBIA1391Z aqueous solution, 45 μl of 300 mM KNO 3 The aqueous solution was mixed evenly with 12 μl of 10 vol% commercially available sterile glycerol aqueous solution, and allowed to stand in liquid nitrogen for 2 min to obtain an aqueous solution of carrier-free nanoparticles 5; S4: uniformly mixing the aqueous solution of the carrier-free nanoparticles 5 and the callus induction medium at a volume ratio of 1:50 to obtain a medium containing the carrier-free nanoparticles 5; S5: After disinfecting the cucumber seeds, immerse them in a culture medium containing the carrier-free nanoparticles 5, and incubate them at 28° C. for 72 hours to obtain treated cucumber seeds; S6: Use Gus staining solution to observe the delivery of Gus gene in treated cucumber seeds.

[0044] Figure 2 This is Example 1, a nanoparticle-based efficient plant gene delivery system for Arabidopsis roots and target protein GFP fluorescence tracing. Figure 2 a is a map of the ARF5-GFP vector. Figure 2b is a TEM image of the carrier-free nanoparticle 1. It can be seen from the figure that due to the freezing crystallization extrusion concentration, the ARF5-GFP vector and KNO 3 Nanoparticles are formed with a size between 20 and 30 nm. Figure 2 c is the 24h stability TEM image of the aqueous solution of the carrier-free nanoparticles 1. It can be seen from the figure that the carrier-free nanoparticles 1 can be stably stored in the aqueous solution, and the size is about 50nm after storage for 24h. Figure 2 d is a confocal image of the delivery efficiency of carrier-free nanoparticles 1 to the root tips of Arabidopsis seedlings at different co-incubation culture times. It can be seen from the figure that when the culture medium containing carrier-free nanoparticles 1 is co-incubated with the root tips of Arabidopsis seedlings for 12 hours, obvious GFP fluorescence can be observed in the root tips of Arabidopsis seedlings, indicating that the carrier-free nanoparticles prepared by the present invention can efficiently deliver target proteins in the roots of Arabidopsis seedlings. According to the GFP fluorescence, the expression of the target protein fused with GFP in the roots of Arabidopsis seedlings can be tracked in real time, and the position, distribution and dynamic changes of the target protein in the cells can be accurately grasped. Figure 2 e is a diagram showing the effect of carrier-free nanoparticles 1 delivering the target gene from the root tip of Arabidopsis seedlings to the shoot apex growth point. As can be seen from the figure, the culture medium containing carrier-free nanoparticles 1 was co-incubated with the root tip of Arabidopsis seedlings for 24 hours (the Arabidopsis shoot apex growth point was not in contact with the carrier-free nanoparticles), and obvious GFP fluorescence appeared in the Arabidopsis shoot apex growth point, indicating that the carrier-free nanoparticles prepared by the present invention can track the localization and behavior of the target protein ARF5 in Arabidopsis plants in real time through long-distance transport, which is beneficial to revealing the function and mechanism of action of the target protein ARF5.

[0045] Figure 3 This is a performance test of the nanoparticle-based efficient plant gene delivery system for Arabidopsis thaliana shoot apex growth point in Example 2. Figure 3 a is a confocal image of the delivery efficiency of carrier-free nanoparticles 1 to the Arabidopsis shoot apex growth point at different co-incubation culture times. It can be clearly seen from the figure that when the culture medium containing carrier-free nanoparticles 1 is co-incubated with the Arabidopsis shoot apex growth point for 3 hours, the Arabidopsis shoot apex growth point has obvious GFP fluorescence, indicating that the carrier-free nanoparticles prepared by the present invention can efficiently deliver the target protein ARF5 expressed in fusion with GFP at the Arabidopsis shoot apex growth point. Figure 3b is a PCR amplification diagram of the gene delivery efficiency of the carrier-free nanoparticles 1 to the whole Arabidopsis seedlings at different co-incubation culture times. RNA was extracted from the whole Arabidopsis seedlings after co-incubation culture, and the GFP fragment was successfully amplified by PCR amplification (lane 1: co-incubated with the culture medium containing the carrier-free nanoparticles 1 for 24 hours, lane 2: co-incubated with the culture medium containing the carrier-free nanoparticles 1 for 12 hours, lane 3: co-incubated with the culture medium containing the carrier-free nanoparticles 1 for 6 hours, lane 4: co-incubated with the culture medium containing the carrier-free nanoparticles 1 for 3 hours, lane 4: co-incubated with the ARF5-GFP aqueous solution for 24 hours, lane 5: co-incubated with sterile water for 24 hours), indicating that the carrier-free nanoparticles prepared by the present invention can achieve efficient delivery of target genes in Arabidopsis plants.

[0046] Figure 4 This is a performance test of the nanoparticle-based Nipponbare rice callus plant gene efficient delivery system of Example 3. Figure 4 a is the map of pAN580 vector. Figure 4 b is the detection of the gene delivery efficiency amplification of the carrier-free nanoparticles 2 to the rice callus at different co-incubation time by PCR amplification. RNA was extracted from the whole rice callus after co-incubation, and the GFP fragment was successfully amplified by PCR amplification (lane 1: co-incubated with the culture medium containing the carrier-free nanoparticles 2 for 48 h, lane 2: co-incubated with the culture medium containing the carrier-free nanoparticles 2 for 24 h, lane 3: co-incubated with the culture medium containing the carrier-free nanoparticles 2 for 12 h, lane 4: co-incubated with the culture medium containing the carrier-free nanoparticles 2 for 6 h, lane 5: co-incubated with the pAN580 aqueous solution for 48 h, lane 6: co-incubated with the callus subculture culture medium for 48 h), indicating that the carrier-free nanoparticles prepared by the present invention can achieve efficient delivery of target genes in rice callus.

[0047] Figure 5 This is Example 4, a nanoparticle-based efficient plant gene delivery system for Arabidopsis roots and a biosensor performance test. Figure 5 a is a map of the Pt-GFP vector. Figure 5 b is a TEM image of the carrier-free nanoparticle 3. It can be seen from the figure that due to the freezing crystallization extrusion concentration, the Pt-GFP carrier and the NaCl in the physiological saline form nanoparticles with a size between 50 and 70 nm. Figure 5 c is the 24h stability TEM image of the aqueous solution of the carrier-free nanoparticles 3. It can be seen from the figure that the carrier-free nanoparticles 3 can be stably stored in the aqueous solution, and the size is between 15 and 30 nm after storage for 24 hours. Figure 5d is a confocal image of the delivery efficiency of carrier-free nanoparticles 3 to the root tips of Arabidopsis at different co-incubation culture times. When the culture medium containing carrier-free nanoparticles 3 was co-cultured with the root tips of Arabidopsis for 3 hours, obvious GFP fluorescence could be observed in the roots of Arabidopsis, indicating that the carrier-free nanoparticles prepared by the present invention can achieve conditional responsive expression in the roots of Arabidopsis in as short as 3 hours, suggesting that the carrier-free nanoparticles prepared by the present invention can achieve real-time monitoring of plant roots under specific conditions, and can be used as a plant sensor to detect various intracellular conditions, such as ion concentration and pH value.

[0048] Figure 6 This is a performance test of the nanoparticle-based Nipponbare rice callus plant gene efficient delivery system of Example 5. Figure 6 a is a map of the pYLCRISPR / Cas9Pubi-H vector and a flow chart of the preparation and application of carrier-free nanoparticles 4. Figure 6 b is a TEM image of the carrier-free nanoparticle 4. It can be seen from the figure that due to the freezing crystallization extrusion concentration, the pYLCRISPR / Cas9Pubi-H vector and KNO 3 Nanoparticles are formed with a size between 30 and 40 nm. Figure 6 c is a TEM image of the stability of the culture medium containing carrier-free nanoparticles 4. It can be seen from the figure that the carrier-free nanoparticles 4 can be stably preserved in the callus subculture culture medium, and the size is 40-70 nm after preservation for 24 hours. Figure 6 d is a PCR amplification diagram of the gene delivery efficiency of carrier-free nanoparticles 4 to rice callus at different co-incubation culture times. RNA was extracted from the whole rice callus after co-incubation and culture, and the Cas9 fragment could be successfully amplified by PCR amplification (lane 1: co-incubated with the culture medium containing carrier-free nanoparticles 4 for 48 hours, lane 2 is co-incubated with the culture medium containing carrier-free nanoparticles 4 for 24 hours, lane 3 is co-incubated with the culture medium containing carrier-free nanoparticles 4 for 12 hours, lane 4 is co-incubated with the culture medium containing carrier-free nanoparticles 4 for 6 hours, lane 5 is co-incubated with the pYLCRISPR / Cas9Pubi-H aqueous solution for 48 hours, and lane 6 is co-incubated with the secondary callus culture medium for 48 hours), indicating that the carrier-free nanoparticles prepared by the present invention can achieve efficient gene delivery in Nipponbare rice callus.

[0049] Figure 7 This is the performance test of the efficient gene delivery system for rice Nipponbare seeds and rice Minghui 86 seeds based on nanoparticles in Example 6. Figure 7 a is the map of pCAMBIA1391Z vector; Figure 7 b is the TEM image of the carrier-free nanoparticle 5. It can be clearly seen from the figure that due to the freezing crystallization extrusion concentration, the pCAMBIA1391Z vector and KNO3 Form nanoparticles with a size between 20 and 30 nm; Figure 7 c is a TEM image of the stability of carrier-free nanoparticles 5 in callus induction medium for 24 h. It can be seen from the figure that carrier-free nanoparticles 5 can also be stably preserved in callus induction medium, with a size between 20-40 nm; Figure 7 d is a Gus staining image of gene delivery of carrier-free nanoparticles 5 to Nipponbare rice seeds. During the co-incubation culture, the culture medium containing carrier-free nanoparticles 5 was replaced with fresh medium every 24 hours, and the delivery was continued for 10 days. On the eleventh day, the delivery of the Gus gene was observed with Gus staining solution. It can be observed that the embryo of the Nipponbare rice seeds co-incubated with carrier-free nanoparticles 5 has obvious Gus blue phenomenon, indicating that the plant gene delivery nanoparticles prepared by the present invention can achieve efficient gene delivery in Nipponbare rice callus tissue; Figure 7 e is the Gus staining image of gene delivery of carrier-free nanoparticles 5 to Minghui 86 rice seeds. During the co-incubation period, the culture medium containing fresh carrier-free nanoparticles 5 was replaced every 24 hours, and the delivery was continued for 10 days. On the eleventh day, the delivery of the Gus gene was observed with Gus staining solution. It can be observed that the embryo of Minghui 86 rice seeds co-incubated with carrier-free nanoparticles 5 had obvious Gus blue phenomenon, indicating that the carrier-free nanoparticles we prepared can achieve efficient gene delivery in Minghui 86 rice callus tissue.

[0050] Figure 8 This is a performance test of the plant gene efficient delivery system based on carrier-free nanoparticles 5 in wheat seeds in Example 7 (plasmid: indicates co-incubation and culture with pCAMBIA1391Z aqueous solution, NPs: indicates co-incubation and culture with a culture medium containing carrier-free nanoparticles 5). The delivery of the Gus gene was observed using a Gus staining solution, and it can be observed that the embryo of the wheat seeds co-incubated with the culture medium containing carrier-free nanoparticles 5 has an obvious Gus blue phenomenon, indicating that the carrier-free nanoparticles prepared by us can achieve efficient gene delivery in wheat seeds.

[0051] Fig. 9 This is a performance test of the plant gene efficient delivery system based on the carrier-free nanoparticles 5 for cucumber seeds in Example 8 (plasmid: indicates co-incubation and culture with the pCAMBIA1391Z aqueous solution, NPs: indicates co-incubation and culture with the culture medium containing the carrier-free nanoparticles 5). The delivery of the Gus gene was observed with a Gus staining solution, and it can be observed that the embryo of the cucumber seeds co-incubated with the culture medium containing the carrier-free nanoparticles 5 has an obvious Gus blue phenomenon, indicating that the carrier-free nanoparticles prepared by us can achieve efficient gene delivery in cucumber seeds.

[0052] In summary, the present invention provides a method and application of efficient delivery of plant nucleic acid based on nanoparticles, which first self-assembles functional nucleic acid and salt compounds necessary for plant growth to form nanoparticles. The prepared nanoparticles can be stably preserved in a plant culture medium, and can be co-incubated with the living plant only. Through active absorption by the living plant, efficient gene delivery to multiple parts of the living plant at different growth stages can be achieved without other auxiliary operations, and efficient gene delivery can also be achieved in multiple varieties of living plants. The method has broad application value in plant gene editing, gene delivery, plant sensors, and plant gene biofunctional research.

Claims

1. A method for efficiently delivering plant living nucleic acid based on nanoparticles, characterized in that: Functional nucleic acids and salt compounds required for plant growth are self-assembled to form carrier-free nanoparticles, which are then co-incubated and cultured with living plants. The living plant cells actively absorb the carrier-free nanoparticles, thereby achieving efficient delivery of functional nucleic acids into living plants.

2. The method for efficient delivery of plant living nucleic acid based on nanoparticles according to claim 1, characterized in that: The specific steps include: (1) Mixing functional nucleic acids and salt compounds necessary for plant growth in an aqueous phase, and self-assembling to form carrier-free nanoparticles through freeze concentration effect below freezing point; (2) uniformly mixing the carrier-free nanoparticles and the plant culture medium at a volume ratio of 1:1 to 1:100 to obtain a plant culture medium containing the carrier-free nanoparticles; (3) The living plant is placed in a plant culture medium containing carrier-free nanoparticles and co-incubated for 1 to 72 hours to allow the carrier-free nanoparticles to fully contact the living plant and be actively taken up by the living plant cells, thereby achieving efficient delivery of the functional nucleic acid into the living plant.

3. The method for efficient delivery of plant living nucleic acid based on nanoparticles according to claim 2, characterized in that: The functional nucleic acid is any one or more combinations of mRNA, siRNA, shRNA, pDNA, T-DNA, cDNA, and mini-chromosome.

4. The method for efficient delivery of plant living nucleic acid based on nanoparticles according to claim 2, characterized in that: The plant is any one of Arabidopsis, rice, wheat, corn, tomato, soybean, cucumber, sugarcane, ginseng and honeysuckle.

5. The method for efficient delivery of plant living nucleic acid based on nanoparticles according to claim 2, characterized in that: The salt compound is any one or more combinations of potassium nitrate, calcium nitrate, ammonium persulfate, ammonium chloride, superphosphate, calcium monophosphate, potassium sulfate, potassium chloride, calcium nitrate, calcium chloride, magnesium sulfate, magnesium chloride, calcium chloride, cobalt chloride, zinc sulfate, copper sulfate, and manganese sulfate.

6. The method for efficient delivery of plant living nucleic acid based on nanoparticles according to claim 2, characterized in that: The aqueous phase is a mixture of glycerol and water.

7. The method for efficient delivery of plant living nucleic acid based on nanoparticles according to claim 2, characterized in that: The plant culture medium is any one of sterile water, seedling medium, callus induction medium and callus subculture medium.

8. The method for efficient delivery of plant living nucleic acid based on nanoparticles according to claim 2, characterized in that: The development stage of the living plant is any one of a seed germination stage, a budding stage, a vegetative growth stage, a reproductive growth stage, a flowering stage, a fruiting stage, a mature stage, and a senescent stage.

9. The method for efficient delivery of plant living nucleic acid based on nanoparticles according to claim 2, characterized in that: The roots, stems, leaves, flowers, fruits, seeds, buds, callus tissues, tubers, root tubers or protoplasts of living plants are placed in a plant culture medium containing carrier-free nanoparticles for co-incubation and culture.

10. The use of the nanoparticle-based efficient plant nucleic acid delivery method according to claim 1, characterized in that: It includes applications in the fields of plant gene silencing, plant gene editing, crop genetic improvement, nutrient and pesticide delivery, biosensors, biopharmaceuticals, biological breeding, and plant gene therapy.

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