Establishment method of gene gun mediated vaccinium plant efficient transgenic system

Abstract

The invention provides a method for establishing a gene gun mediated efficient transgenic system of vaccinium plants. The method comprises the following steps: by taking tender leaves of a sterile vaccinium plant as explants, optimizing a treatment method of a hypertonic culture medium on the explants; the plasmid vector for transgenosis comprises a first expression vector containing transposase and a second expression vector for target gene integration; after integrating the target gene into the second expression plasmid, mixing the target gene with the first expression plasmid to prepare a DNA wrapped micro-elastic body; bombarding the explant by using the DNA coated micro-bombardment under a sterile condition through a gene gun; and putting the bombarded explant on a regeneration culture medium to induce germination, and carrying out pressure screening to obtain a positive transgenic plant. The method can effectively improve the transgenosis efficiency of the vaccinium plant, is not limited by species, can realize efficient genetic transformation of various wild blueberries, greatly shortens the obtaining period of the transgenic plant, and fills the key technical blank in the field.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, in particular to a method for establishing a high-efficiency transgenic system of Vaccinium plant mediated by a gene gun. Background Art

[0002] blueberry( Vaccinium spp.) is a member of the Ericaceae family ( Ericaceae ) Vaccinium ( Vaccinium ) is an important economic crop. As blueberries expand their market influence, plants of the same genus are also gaining increasing attention. Wild blueberries, such as the Vaccinium vitis-idaea of ​​Northeast my country, the European bilberry (Vaccinium truncatum) endemic to Europe, and the evergreen bilberry of the southern United States, are rich in active ingredients such as anthocyanins, polyphenols, and vitamins. They possess significant antioxidant, anti-inflammatory, and neuroprotective properties, and, like blueberries, hold great development potential.

[0003] Both blueberries and wild blueberries require genetic engineering for functional gene research and gene-editing breeding. However, traditional Agrobacterium-mediated transgenic techniques are species-specific, with significant variations in procedures and formulations between species. Traditional blueberry transgenic techniques are complex, with lengthy transformation cycles and difficult to master, resulting in low transformation efficiency. There is an urgent need for a simple, universal method.

[0004] Gene gun bombardment, a well-established physical transformation method, uses high-speed microprojectiles to deliver exogenous DNA directly into plant cells. It boasts significant advantages, including a wide host range, ease of use, and the ability to simultaneously transform multiple genes. Currently, this technology has achieved remarkable success in important crops such as rice, wheat, and cotton, resulting in the successful development of numerous transgenic varieties with improved traits. Compared to Agrobacterium-mediated transformation, gene gun technology overcomes host compatibility limitations, offers a simpler workflow, significantly shortens the transformation cycle, and the transformed tissue generally maintains good regeneration capacity. However, gene guns suffer from low transformation efficiency. This is because gene gun-mediated transformation lacks the protein factors of Agrobacterium that promote gene integration, resulting in extremely low transformation efficiency. The gene gun-mediated transgenic efficiency is approximately 0.04%, far below the experimental requirement of >3%. Therefore, increasing gene integration efficiency is key to the development of gene gun technology.

[0005] Transposases, typically encoded by transposons, recognize specific sequences at either end of the transposon, detaching it from adjacent sequences and reinserting it into a new DNA target site without requiring homology. Therefore, transposases can be used to facilitate DNA integration into plant genomes. The only known successful transposase for plant transcription is the ping-pong transposase in rice; reports on transposases in other plants are limited.

[0006] Blueberries not only have a unique flavor but are also rich in health-promoting ingredients like anthocyanins, offering promising prospects for development in health foods and pharmaceutical raw materials. While traditional hybridization has yielded numerous superior varieties, the limited gene pool makes it difficult for breakthrough varieties to emerge.

[0007] Therefore, it is necessary to develop and utilize the abundant wild blueberry resources and carry out gene-editing breeding to accelerate the commercialization of wild blueberries. Existing technologies have difficulty achieving efficient genetic transformation of blueberries and wild blueberries, severely restricting the development and utilization of this rare resource and the improvement of its varieties. The development of an efficient gene gun transformation system applicable to a variety of blueberries not only has important theoretical innovation value but also provides key technical support for improving the quality, efficiency, and sustainable development of the blueberry industry. Summary of the Invention

[0008] The present invention provides a gene gun-mediated method for establishing a high-efficiency transgenic system for Vaccinium plants. The method can effectively improve the efficiency of establishing the transgenic system for Vaccinium plants, achieve efficient genetic transformation of various wild blueberries, break through species restrictions, greatly shorten the period for obtaining transgenic plants, and address key technical gaps in this field.

[0009] The specific technical solutions are as follows: The present invention provides a method for establishing a high-efficiency transgenic system of Vaccinium plants mediated by a gene gun, comprising: (1) Using sterile young leaves of Vaccinium plant as explants, they were placed in hypertonic medium for pretreatment; (2) using gene gun technology to prepare DNA-encapsulated microbullets from the mixed plasmids, and bombarding the pretreated explants with the DNA-encapsulated microbullets under sterile conditions; The mixed plasmid is obtained by mixing a first expression vector containing a transposase and a second expression vector containing a target gene; (3) After the bombardment is completed, the bombarded explants are induced to sprout and transgenic positive plants are obtained.

[0010] In the present invention, the efficiency of establishing a transgenic system for Vaccinium plants is effectively improved through "hypertonic culture pretreatment + gene gun bombardment + subsequent tissue culture conditions"; the transformation rate is increased from the original 5‰ to 8.9% and 11.58%, the transformation cycle of Vaccinium dulcis is shortened from 8 months to 3-4 months, and the transformation cycle of Vaccinium waxleaf is shortened from 2-3 months to 1-2 months.

[0011] Furthermore, in step (1), the Vaccinium plant is Vaccinium vitis-idaea or Vaccinium waxleaf.

[0012] Furthermore, in step (1), the formula of the hypertonic culture medium is: WPM + 2%~4% sucrose + 7 g / L~9 g / L agar + 0.05 mg / L~1.0 mg / L CPPU + 0.05 mg / L~0.2 mg / L IBA + 0.2 mol / L~0.8 mol / L mannitol, pH =5.20±0.02.

[0013] Furthermore, in step (1), the pre-processing method is: Take some leaves of the explants and spread them flatly in hypertonic medium with the abaxial side facing upwards; and / or, the area of ​​the partial leaf is > (0.4 cm × 0.4 cm); And / or, the pretreatment time is 4 to 24 h; more preferably, the pretreatment time is 4 to 8 h or 6 to 8 h; more preferably, the pretreatment time is 8 h.

[0014] Furthermore, in step (2), the first expression vector comprises a first original expression vector and a transposase encoding gene inserted into the first original expression vector; The first original expression vector is selected from the plant overexpression vector: pCAMBIA1300. Other overexpression vectors may also be used, for example, including but not limited to pGreen, pAK320NEO and other series.

[0015] Furthermore, the first original expression vector is selected from pCAMBIA1300, pGreen or pAK320NEO; further preferably, the first original expression vector is selected from pCAMBIA1300.

[0016] And / or, the nucleotide sequence of the transposase encoding gene is shown as SEQ ID NO.1.

[0017] SEQ ID NO.1:

[0018] Furthermore, in step (2), the second expression vector comprises a second original expression vector and a target gene inserted into the second original expression vector; further preferably, the first original expression vector is selected from pCAMBIA1300.

[0019] The second original expression vector is selected from the plant overexpression vector: pCAMBIA1300. Other overexpression vectors may also be used, for example, including but not limited to pGreen, pAK320NEO and other series.

[0020] Furthermore, the second original expression vector is selected from pCAMBIA1300, pGreen or pAK320NEO; And / or, the molar ratio of the first expression vector to the second expression vector is 1:1-100; more preferably, the molar ratio of the first expression vector to the second expression vector is 1:1.

[0021] Furthermore, in step (2), the DNA-encapsulated microbullet comprises: gold powder, spermidine, calcium chloride and mixed plasmid; And / or, the molar ratio of the gold powder, spermidine, and calcium chloride is 1:3600-4200:8000-12000; and / or, the gas pressure of the gene gun bombardment is 1100-1200 psi; and / or, the vacuum degree of the gene gun bombardment is 23-25 ​​inches Hg; and / or, the bombardment distance of the gene gun bombardment is 3-9 cm; And / or, the number of bombardments of the gene gun bombardment is 1 to 2 times.

[0022] The core purpose of spermidine and calcium chloride is to promote the binding of DNA to gold powder. Calcium chloride provides calcium ions (Ca²⁺) to neutralize the negative charge of DNA molecules, reducing the repulsive force between DNA and negatively charged microcarriers, creating conditions for the two to bind. Spermidine, as a polyamine substance, further enhances the adsorption of DNA and microcarriers, helps DNA firmly attach to the surface of microcarriers, ensures that exogenous DNA can effectively enter the target cells along with the microcarriers during bombardment, and improves transformation efficiency.

[0023] Furthermore, in step (3), the bud induction comprises: subjecting the bombarded explant to dark culture, photoperiod induction culture, regeneration culture, screening culture, germination culture and rooting culture in sequence, to finally obtain transgenic buds; The dark culture comprises: first culturing the stably transformed cells in a hypertonic culture medium, and then transferring the cells to a regeneration culture medium without mannitol for further culturing; And / or, in dark culture, the formula of the hypertonic medium is: WPM + 2%-4% sucrose + 7 g / L-9 g / L agar + 0.05 mg / L-1.0 mg / L CPPU + 0.05 mg / L-0.2 mg / L IBA + 0.2 mol / L-0.8 mol / L mannitol, pH = 5.20±0.02; and / or, in dark culture, the culture time in the hypertonic medium is 16 to 18 h; And / or, in dark culture, the formula of the regeneration medium is: WPM + 3% sucrose + 8.5 g / L agar + 0.05 mg / L~0.8 mg / L CPPU + 0.05 mg / L~0.2 mg / L IBA, pH = 5.20±0.02; And / or, in dark culture, the culture time in the regeneration medium is 32 to 56 hours.

[0024] Furthermore, in step (3), the photoperiodic induction culture conditions are light culture at 24°C for 16 h and dark culture at 24°C for 8 h; And / or, the medium for photoperiod-induced culture is WPM + 3% sucrose + 8.5 g / L agar + 0.05 mg / L to 0.8 mg / L CPPU + 0.05 mg / L to 0.2 mg / L IBA + 10 mg / L Hyg, pH = 5.20 ± 0.02; And / or, the regeneration culture conditions are: light culture at 23-25°C for 14-18 hours; And / or, the culture medium formula for the regeneration culture is: WPM + 3% sucrose + 8.5 g / L agar + 0.05 mg / L~0.8 mg / L CPPU + 0.05 mg / L~0.2 mg / L IBA, pH=5.20±0.02.

[0025] Furthermore, in step (3), And / or, the screening culture conditions are: culturing under light at 23-25°C for 14-18 hours; And / or, the medium formula for the screening culture is: WPM + 3% sucrose + 8.5 g / L agar + 0.8 mg / L CPPU + 0.05 mg / L IBA + 15 mg / L Hyg, pH = 5.20±0.02; And / or, the germination culture conditions are: culturing under light at 23-25° C. for 14-18 h; And / or, the culture medium formula for the germination culture is: WPM + 3% sucrose + 8.5 g / L agar + 0.05 mg / L to 0.8 mg / L CPPU + 0.05 mg / L to 0.2 mg / L IBA + 15 mg / L Hyg, pH = 5.20±0.02; And / or, the rooting culture conditions are: culturing under light at 23-25° C. for 14-18 h; And / or, the culture medium formula for the rooting culture is: WPM + 3% sucrose + 8.5 g / L agar + 0.3 mg / L IAA + 1.0 mg / L KT + 15 mg / L Hyg, pH = 5.20±0.02.

[0026] Compared with the prior art, the present invention has the following beneficial effects: (1) The method of the present invention successfully explored the gene gun bombardment technology as a universal genetic transformation method for blueberry plants, providing a reliable experimental basis for the future use of gene gun-mediated transformation of blueberries with target genes, filling the key technical gap, and not only creating a new technology for functional gene transformation of blueberries, but also providing technical support for studying the role of various functional genes in blueberries.

[0027] (2) By using the transposase gene as an auxiliary plasmid, the efficiency of integrating the target gene into the genome is greatly improved, which fundamentally breaks through the limitation of low integration rate of the gene gun-mediated transgenic method.

[0028] (3) The experimental process was precisely optimized, from the time of hypertonic treatment of explants, the parameters of gene gun bombardment to regeneration screening, to achieve efficient transformation of Vaccinium explants. Transgenic adventitious buds can be obtained in 15-20 days, and transgenic buds can be obtained through subsequent regeneration and screening culture, greatly shortening the breeding cycle.

[0029] (4) In the method of the present invention, fresh leaves of Vaccinium plants are used as explants. These leaves are easy to obtain and simple to operate, which simplifies the transformation process.

[0030] (5) This method can greatly reduce the operational threshold and time cost of existing transgenic technology, save manpower and material resources for transgenic companies, reduce the overall cost by 1 / 3-1 / 2, and has great commercial promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The images of the buds of Vaccinium vitis-idaeae under the bright and dark fields of a Zeiss macro-variable magnification stereo fluorescence microscope in Example 1 are as follows; Among them, a is a picture of positive buds that have been successfully genetically modified after gene gun bombardment using the method described in this patent under a macro-variable magnification stereo fluorescence microscope in the bright field; b is a picture of positive buds that have been successfully genetically modified after gene gun bombardment using the method described in this patent under a macro-variable magnification stereo fluorescence microscope GFP dark field; c is a picture of positive buds that have been successfully genetically modified using other methods not described in this patent under the bright field; d is a picture of positive buds that have been successfully genetically modified using other methods not described in this patent under the GFP dark field; e is a picture of positive buds that have been successfully genetically modified after gene gun bombardment using the method of this embodiment; f is a picture of positive buds that have been successfully genetically modified after gene gun bombardment using the method of this embodiment under the dark field.

[0032] Figure 2 This is a picture of the bud of waxberry under the bright and dark fields of a Zeiss macro-variable magnification stereo fluorescence microscope in Example 5; Among them, a is a picture of a positive bud with successful transgenic under bright field; b is a picture of a positive bud with GFP under dark field; c is a picture of another positive bud that was successfully genetically modified under bright field; d is a picture of another positive bud that was successfully genetically modified under dark field.

[0033] Figure 3 The results show the efficiency of gene gun-mediated transgenic transformation of Vaccinium plants before and after plasmid transformation in Example 5, Treatment II of Example 4, and Example 6. DETAILED DESCRIPTION

[0034] In order to make those skilled in the art better understand the present invention, the technical solution of the present invention is clearly and completely described below in conjunction with specific embodiments. It should be noted that the following detailed description is exemplary and is only a part of the embodiments of the present invention, rather than all embodiments.

[0035] Based on the embodiments of the present invention, it should be pointed out that for those skilled in the art, several variations and improvements can be made without departing from the concept of the present invention, and these should also be regarded as the scope of protection of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

[0036] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this application belongs. The experimental materials used in the examples of the present invention are all conventional experimental materials in the art and can be purchased through commercial channels. Experimental methods without detailed conditions were performed according to conventional experimental methods or according to the operating instructions recommended by the supplier.

[0037] In the following examples of this application, "WPM" refers to a medium for plant tissue culture (full name: Lloyd & McCown Woody Plant Basal Medium with Vitamins (Phyto Tech, L449). "Sucrose" refers to sucrose; "Agar" refers to agar; "IBA" refers to indolebutyric acid; "CPPU" refers to chlorpyrifos; "Hyg" refers to hygromycin; "IAA" refers to indoleacetic acid; "KT" refers to kinetin; "GA" refers to gibberellin; "Trytone" refers to tryptone; and "Yeast Extract" refers to yeast extract.

[0038] The formula of hypertonic medium is: WPM + 3% Sucrose + 8.5 g / L Agar + 0.8 mg / L CPPU +0.05 mg / L IBA + 0.4 M mannitol, pH =5.20±0.02; The formula of regeneration medium is: WPM + 3% Sucrose + 8.5 g / L Agar + 0.8 mg / L CPPU +0.05 mg / L IBA, pH = 5.20 ± 0.02; The formula of photoperiod induction medium is: WPM + 3% Sucrose + 8.5 g / L Agar + 0.8 mg / LCPPU + 0.05 mg / L IBA + 10 mg / L Hyg, pH = 5.20 ± 0.02; The formulation of the screening medium was: WPM + 3% Sucrose + 8.5 g / L Agar + 0.8 mg / L CPPU + 0.05 mg / L IBA + 10 mg / L Hyg, pH = 5.20 ± 0.02; The formula of germination medium was: WPM + 3% Sucrose + 8.5 g / L Agar + 0.8 mg / L CPPU +0.05 mg / L IBA + 10 mg / L Hyg, pH = 5.20 ± 0.02; The formula of rooting medium is: WPM + 3% Sucrose + 8.5 g / L Agar + 0.3 mg / L IAA +1.0 mg / L KT + 15 mg / L Hyg, pH =5.20±0.02.

[0039] The formula of LB liquid medium is: NaCl 10 g / L + Trytone 10 g / L + Yeast Extract 5 g / L.

[0040] The gene gun used in the present invention is a product of the American Bio-RAD company, which refers to a high-speed helium gene gun PDS-1000 / He (Bio-Rad).

[0041] Buffer P1-P4 used to extract plasmids in the present invention is a product of Easy-Stay Biotechnology Co., Ltd. The original expression vector used in the present invention was purchased from Addgene and designed independently based on it.

[0042] Method for determining transformation efficiency: observe using a Zeiss macro-variable magnification stereo fluorescence microscope, and the blueberry regenerated shoots that show green fluorescence (GFP) under laser irradiation are transgenic shoots.

[0043] Transformation efficiency was calculated as: number of transgenic shoots / total number of explants. If more than one shoot from a single explant was transgenic, they were counted as one.

[0044] Example 1 This embodiment provides a method for establishing a high-efficiency transgenic system for Vaccinium plants mediated by a gene gun. The specific steps are as follows: (1) Using sterile young leaves of Vaccinium plant as explants, they were placed in hypertonic medium for pretreatment; The specific steps are as follows: (1-1) Explant Acquisition and Pre-culture. Three-week-old fresh leaves of Vaccinium vitis-idaea (the wild blueberry with the highest market share in my country, primarily grown in Northeast my country) were collected as explants under a sterile environment. The leaves should be 0.5 cm to 0.8 cm long and 0.4 cm to 0.6 cm wide and in good condition. The leaves were spread flat on regeneration medium plates for bud induction and pre-cultured for 2 days.

[0045] (1-2) Hypertonic pretreatment: Leaves pre-cultured for 2 days were transferred to a plate containing 0.4 M mannitol in a hypertonic medium and subjected to hypertonic pretreatment for 8 hours.

[0046] (2) Using gene gun technology, the mixed plasmid is made into DNA-coated microbullets, and the pretreated explants are bombarded with the DNA-coated microbullets under sterile conditions; the mixed plasmid is obtained by mixing a first expression vector containing a transposase and a second expression vector containing a target gene.

[0047] The specific steps are as follows: (2-1) Extracting plasmids. The first expression vector consists of the first original expression vector pCAMBIA1300 and a transposase encoding gene (nucleotide sequence shown in SEQ ID NO. 1) inserted into the first original expression vector. The transposase encoding gene is inserted into the multiple cloning site (MCS) between the CaMV 35S promoter and the NOS terminator of the first original expression vector pCAMBIA1300 and digested with SalI and HindIII. The second expression vector consists of the second original expression vector pCAMBIA1300 and a target gene (i.e., the commonly used green fluorescent protein gene GFP) inserted into the second original expression vector. The green fluorescent protein gene GFP is inserted into the multiple cloning site (MCS) between the CaMV 35S promoter and the NOS terminator of the second original expression vector and digested with SalI and BmHI. All of the above expression vectors were amplified from Escherichia coli.

[0048] The specific method is as follows: pick a single colony from the E. coli culture plate and inoculate it into 4 mL of LB liquid medium containing kanamycin, and culture it at 37°C and 200 rpm for 14 h; when the OD600 of the bacterial solution is in the range of 0.6-0.8, transfer the bacterial solution to a 1.5 mL or 2.0 mL centrifuge tube, centrifuge it at 12000 rpm (≥13000 g) at room temperature for 1 min, and discard the supernatant; add 200 μL of Buffer P1 to the centrifuge tube to fully resuspend the bacterial solution; add 200 μL of Buffer P2 to the centrifuge tube for lysis, gently invert 6-8 times, and the lysis time should not exceed 5 min. The lysed solution becomes clear and viscous; then add 200 μL of Buffer P3 to the centrifuge tube, gently invert 6-8 times, and after thorough mixing, place the centrifuge tube in a high-speed centrifuge and centrifuge it at 12000 rpm (≥13000 g) at room temperature for 10 min; aspirate the supernatant after centrifugation for 2 Place the Hipure DNA adsorption column in a 1.5 mL collection tube and centrifuge at 12,000 rpm (≥13,000 g) at room temperature for 30 seconds. Remove the column and discard the waste solution. Return the column to the collection tube, add 500 μL of Buffer P4 (containing anhydrous ethanol), and centrifuge at 12,000 rpm (≥13,000 g) at room temperature for 30 seconds. Remove the column and discard the waste solution. Repeat the wash cycle once. Return the column to the collection tube and centrifuge at 12,000 rpm (≥13,000 g) at room temperature for 1 minute. Place the column in a clean 1.5 mL centrifuge tube and let it sit for 1-2 minutes. Then, add 40 μL of elution buffer to the center of the column membrane. Let it sit at room temperature for 1-2 minutes, then centrifuge at 12,000 rpm (≥13,000 g) at room temperature for 1 minute. Take 2 μL of the extracted plasmid for concentration measurement. Store the remaining plasmid at -20°C.

[0049] (2-2) Prepare the gold powder required for gene gun bombardment. The gold powder used in this example has a diameter of 0.8-1.5 μm. The amount of gold powder used for gene gun bombardment is 1.5 mg per time. 30 mg of gold powder is sufficient for approximately 20 bombardments. Place the gold powder in a 1.5 mL sterilized EP tube. Add 1000 μL of 70% analytical grade ethanol to the tube and vortex vigorously for 3-5 minutes to evenly clean the gold powder surface with the ethanol. Let it stand at room temperature for 15 minutes. Centrifuge at 12,000 rpm for 5 seconds and discard the ethanol. After the alcohol wash, add 1000 μL of sterile deionized water, let it stand for 1 minute, and then centrifuge to remove the supernatant. Wash two to three times. Add 500 μL of 50% glycerol to obtain the treated gold powder.

[0050] (2-3) Prepare the microprojectiles required for gene gun bombardment. First, the auxiliary plasmid DNA (i.e., the first expression vector containing the transposase) and the target vector DNA (i.e., the second expression vector containing the target gene) were mixed in a 1:1 ratio, and then 25 μL of gold powder that had been treated in advance was taken, and 10 μL of 0.10 M spermidine, 20 μL of plasmid DNA, and 25 μL of 2.5 M calcium chloride were added to the gold powder suspension in sequence; each time a reagent was added, it was thoroughly mixed and oscillated for 2 minutes, and immediately placed on ice for 1 minute after oscillation; centrifuged at 12,000 rpm in a high-speed centrifuge for 10 seconds and the supernatant was discarded; 140 μL of 70% ethanol was added, and the mixture was mixed by pipetting with a pipette tip and oscillated for 1 minute; ultrasonicated for 5 seconds, centrifuged at 12,000 rpm for 10 seconds, and the supernatant was discarded; 140 μL of anhydrous ethanol was added, and the mixture was mixed by pipetting with a pipette tip and oscillated for 1 minute, centrifuged at 12,000 rpm for 10 seconds and the supernatant was discarded; finally, 5 μL of 100% Ethanol was added to ensure that the gold powder precipitate was completely resuspended to obtain DNA-coated microbullets with a plasmid concentration of 3000 ng / gun, and finally placed on ice for use.

[0051] (2-4) Gene gun bombardment. In a clean bench, the surface of the gene gun and the vacuum chamber were wiped clean with 75% ethanol and sterilized with UV light for 30 minutes. Gene gun bombardment was performed under strict aseptic conditions within the clean bench. Prior to bombardment, the barrier net and tray were sterilized in an autoclave, and the cleavable membrane and carrier membrane were disinfected by soaking in anhydrous ethanol for 15 minutes. In this example, a PDS-1000 / He gene gun manufactured by Bio-Rad was used to perform gene gun-mediated genetic transformation on Vaccinium vitis-idaea leaves after hypertonic treatment.

[0052] The specific bombardment process is as follows: after opening the clean bench, place the barrier net, fixed ring, cleavable membrane and carrier membrane on sterilized filter paper and blow dry in the clean bench; open the main valve of the helium cylinder, adjust the pressure of the helium cylinder to 1100psi higher than the rupture pressure of the cleavable membrane, and then turn on the transformer switch and the gene gun vacuum pump switch; install the cleavable membrane on the bracket, and then screw the bracket clockwise onto the gas accelerator and tighten it with a wrench; install the carrier membrane on the fixed ring, use the gun tip to fully resuspend the gold powder microbombs coated with DNA, and absorb the microbombs prepared in advance. Spread the bullet evenly in the center of the crackable membrane, and then place it on the clean bench to blow dry; after drying, place the blocking net on the blocking net holder, and place the fixed ring assembled with the carrier membrane on the top of the blocking net, so that the side with the gold powder particles faces the blocking net, and then tighten the lid and insert it into the first layer of the bombardment chamber; place the culture medium plate containing the explant material on the tray. At this time, the explant material is concentrated and spread flat in the center of the culture medium plate, keep the bombardment distance at 3 cm, and close the door; first press the "VAC" button on the gene gun, and when the vacuum degree reaches 25 inches Hg, quickly press the "HOLD" button, then press and hold the "FIRE" button and keep it still. When you hear a "pop" sound, quickly release the button, the bombardment is completed, press the "VENT" button after completion, and after the vacuum meter returns to zero, take out the bombarded sample; the bombardment number is 1 time.

[0053] (3) After the bombardment is completed, the bombarded explants are induced to sprout and transgenic plants are obtained.

[0054] The specific steps are as follows: (3-1) After the bombardment is completed, the bombarded explant material is quickly covered and placed on a hypertonic medium for 16 hours of dark culture to stabilize the transformed cells. The explant material is then transferred to a regeneration medium without mannitol and cultured in the dark for another 48 hours. Regeneration is then induced under light conditions by photoperiods, culturing at 24°C for 16 hours in the light and 24°C for 8 hours in the dark. After culturing under light for another 5-7 days, the explant material is transferred to a regeneration medium containing hygromycin for regeneration culture at 24°C for 16 hours in the light. (3-2) During the regeneration culture of explant leaves to induce shoot growth, transgenic buds will gradually appear after 15 to 20 days. At this point, the explant leaves with transgenic buds should be transferred to a new regeneration medium plate containing hygromycin to allow the buds to continue growing and developing into transgenic buds. Once a large number of adventitious buds have grown and expanded, the explant material should be transferred to a rooting medium containing hygromycin and cultured at 24°C with 16 hours of light. Meanwhile, the culture medium plate should be changed every 15 to 20 days. Through this series of operations, positive plants will eventually be obtained.

[0055] The specific conversion rate of the method of this embodiment was determined, and the results are shown in Example 2.

[0056] Example 2 The difference between Example 2 and Example 1 is that the hypertonic treatment time of the Vaccinium myrtillus leaves (i.e., the explant material to be bombarded) (i.e., the treatment time of 8 hours in step (1-2) of Example 1) is designed in a gradient manner, and the remaining steps are exactly the same as in Example 1.

[0057] The specific contents are as follows: The hypertonic treatment time was set to seven gradients: 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h. The air pressure was 1100 psi, the vacuum was maintained at 25 inches Hg, the plasmid dosage was 3000 ng / gun, the bombardment distance was maintained at 3 cm, the bombardment number was 1, and the Vaccinium vitis-idaea leaf tissue explants after hypertonic treatment were bombarded with a gene gun.

[0058] The results are shown in Table 1.

[0059] Table 1 Effect of hypertonic treatment time on GFP gene expression

[0060] The expression of green fluorescent protein was detected by Zeiss macro-zoom stereo fluorescence microscope, and it was found that there was a direct correlation between the hypertonic treatment time and the expression of green fluorescent protein.

[0061] Table 1 shows that the expression efficiency in Vaccinium vulgaris leaf tissue fluctuated with increasing hypertonic treatment time. Transformation efficiency peaked at 9.10%-10.73% within the 6-8 hour range, significantly higher than other treatment times. For treatment times less than 6 hours, transformation efficiency gradually increased with increasing treatment time, indicating that a certain duration of hypertonic treatment has a positive impact on cellular status. However, when hypertonic treatment time exceeded 8 hours, reaching 10 hours, GFP expression dropped sharply to 2.44%. By 12 hours, GFP expression was absent compared to other treatments of the same duration, suggesting that prolonged hypertonic treatment may cause cell damage or metabolic inhibition. By setting seven gradients of hypertonic treatment time and combining the final GFP expression in leaf tissue, we determined that 8 hours is the optimal hypertonic treatment time for gene gun-mediated genetic transformation of Vaccinium vulgaris leaf tissue.

[0062] Example 3 The difference between Example 3 and Example 1 is that the amount of plasmid in the microbullets used for gene gun bombardment (i.e., the amount of plasmid used in steps (2-3) of Example 1 is 3000 ng / gun) is designed in a gradient manner. The remaining steps are exactly the same as in Example 1.

[0063] The specific contents are as follows: under the premise of hypertonic treatment time of 8 h, air pressure of 1100 psi, vacuum degree of 25 inchesHg, bombardment distance of 3 cm, and bombardment number of times; the plasmid dosage was set to three gradients of 1500 ng / gun, 3000 ng / gun, and 4500 ng / gun, and the leaf tissue explants of Vaccinium vitis-idaea treated with hypertonicity were bombarded with gene gun.

[0064] Table 2 Effect of plasmid dosage on GFP gene expression

[0065] The expression of green fluorescent protein was detected by Zeiss macro-zoom stereo fluorescence microscope, and it was found that there was a direct correlation between the amount of plasmid used and the expression of green fluorescent protein.

[0066] It can be seen from Table 2 that the transformation efficiency reached the highest (10.73%) when the plasmid dosage was 3000 ng / gun, which was significantly higher than 1500 ng / gun (6.67%) and 4500 ng / gun (5.91%).

[0067] Therefore, 3000 ng / gun is the optimal parameter, and 1500-3000 ng / gun is recommended as the effective range. Using a plasmid dosage of 3000 ng / gun, combined with 6-8 h of hypertonic treatment, can synergistically improve gene gun transformation efficiency.

[0068] Example 4 The difference between Example 4 and Example 1 is that the bombardment distance is designed in a gradient, and the remaining steps are exactly the same as Example 1.

[0069] The specific contents are as follows: Under the premise of plasmid dosage of 3000 ng / gun, hypertonic treatment time of 8 h, air pressure of 1100 psi, vacuum degree maintained at 25 inches Hg, and bombardment number of times, the bombardment distance was set to three gradients: 3 cm (treatment I), 6 cm (treatment II), and 9 cm (treatment III), with 3 cm being the closest distance that the equipment can be set to; the leaf tissue explants of Vaccinium vitis-idaea treated with hypertonicity were bombarded by gene gun.

[0070] Table 3 Effect of bombardment distance on GFP gene expression

[0071] The expression of green fluorescent protein was detected by Zeiss macro-zoom stereo fluorescence microscope, and it was found that there were obvious differences in the expression of green fluorescent protein after bombardment of blueberry leaf tissue at three bombardment distances.

[0072] Table 3 shows that a bombardment distance of 6 cm achieved the highest transformation efficiency (11.58%), surpassing both 3 cm (10.73%) and 9 cm (4.82%). The transformation efficiency at 3 cm (10.73%) remained high, but decreased slightly compared to 6 cm. The transformation efficiency at 9 cm (4.82%) dropped sharply, reaching only 37.36% of that at 6 cm, indicating that long-range bombardment significantly impacted gene delivery efficiency. The optimal bombardment distance for gene gun-mediated genetic transformation of Vaccinium vitis-idaea leaf tissue is 6 cm. Based on these results, it is recommended that gene gun bombardment be conducted under the synergistic conditions of 6-8 h of hyperosmotic treatment, a plasmid dosage of 3000 ng / gun, and a bombardment distance of 6 cm to maximize transformation efficiency.

[0073] Example 5 The difference between Example 5 and Example 1 is that: other blueberries of the genus Vaccinium such as Vaccinium vitis-idaea are genetically transformed using a gene gun, and the operation steps are the same as those in Example 1.

[0074] By the above method, positive transgenic shoots of wax-leaved blueberry were Figure 2 As shown in Figure 2, the transformation rate of positive transgenic shoots of wax-leaved blueberry is as follows: Figure 3 As shown, the specific conversion rate is 8.9%.

[0075] Example 6 In Example 6, we used a recombinant plant transgenic plasmid from before transformation to genetically transform Vaccinium vitis-idaea by gene gun-mediated transformation. The only difference between this procedure and Example 1 is that the first expression vector consists solely of the original first expression vector, pCAMBIA1300. All other steps remain the same as in Example 1.

[0076] Experimental results show that the conversion efficiency of this method is about 5‰.

[0077] This data is in stark contrast to the conversion rates achieved by the modified vector-mediated transgenic method in Vaccinium myrtillus and Vaccinium truncatum: the conversion rate of the modified vector in Vaccinium myrtillus (i.e., obtained in Example 5) reached 8.9%, and the conversion cycle was shortened from the previous 2-3 months to 1-2 months; in Vaccinium myrtillus (i.e., obtained in Treatment II of Example 4), it was as high as 11.58%, and the conversion cycle was shortened from the previous 8 months to 3-4 months.

[0078] This shows that the transformation efficiency mediated by the recombinant plasmid before the transformation is much lower than that of the vector after the transformation. Figure 3 shown.

Claims

1. A method for establishing a high-efficiency transgenic system for Vaccinium plants mediated by a gene gun, characterized in that: include: (1) Using sterile young leaves of Vaccinium plant as explants, they were placed in hypertonic medium for pretreatment; (2) using gene gun technology to prepare DNA-encapsulated microbullets from the mixed plasmids, and bombarding the pretreated explants with the DNA-encapsulated microbullets under sterile conditions; The mixed plasmid is obtained by mixing a first expression vector containing a transposase and a second expression vector containing a target gene; (3) After the bombardment is completed, the bombarded explants are induced to sprout and transgenic positive plants are obtained.

2. The establishment method according to claim 1, characterized in that In step (1), the formula of the hypertonic culture medium is: WPM + 2%~4% sucrose + 7 g / L~9 g / L agar + 0.05 mg / L~1.0 mg / L CPPU + 0.05 mg / L~0.2mg / L IBA + 0.2 mol / L~0.8 mol / L mannitol, pH = 5.20±0.

02.

3. The establishment method according to claim 1, wherein: In step (1), the pretreatment method is: Take some leaves of the explants and spread them flatly in hypertonic medium with the abaxial side facing upwards; and / or, the area of ​​the partial leaf is > (0.4 cm × 0.4 cm); And / or, the pretreatment time is 4 to 24 hours.

4. The establishment method according to claim 1, wherein: In step (2), the first expression vector comprises a first original expression vector and a transposase encoding gene inserted into the first original expression vector; and / or, the first original expression vector is selected from pCAMBIA1300, pGreen or pAK320NEO; And / or, the nucleotide sequence of the transposase encoding gene is shown as SEQ ID NO.

1.

5. The establishment method according to claim 1, wherein: In step (2), the second expression vector comprises a second original expression vector and a target gene inserted into the second original expression vector; The second original expression vector is selected from pCAMBIA1300, pGreen or pAK320NEO; And / or, the molar ratio of the first expression vector to the second expression vector is 1:1-100.

6. The establishment method according to claim 1, wherein: In step (2), the DNA-encapsulated microbullet comprises: gold powder, spermidine, calcium chloride and mixed plasmids; And / or, the molar ratio of the gold powder, spermidine, and calcium chloride is 1:3600-4200:8000-12000; and / or, the gas pressure of the gene gun bombardment is 1100-1200 psi; and / or, the vacuum degree of the gene gun bombardment is 23-25 ​​inches Hg; and / or, the bombardment distance of the gene gun bombardment is 3-9 cm; And / or, the number of bombardments of the gene gun bombardment is 1 to 2 times.

7. The establishment method according to claim 1, wherein: In step (3), the bud induction comprises: subjecting the bombarded explant to dark culture, photoperiod induction culture, regeneration culture, screening culture, germination culture and rooting culture in sequence, to finally obtain transgenic buds; The dark culture comprises: first culturing the stably transformed cells in a hypertonic culture medium, and then transferring the cells to a regeneration culture medium without mannitol for further culturing; And / or, in dark culture, the formula of the hypertonic medium is: WPM + 2%-4% sucrose + 7.5 g / L-9.0 g / L agar + 0.05 mg / L-1.0 mg / L CPPU + 0.05 mg / L-0.2 mg / L IBA + 0.2 mol / L-0.8 mol / L mannitol, pH = 5.20±0.02; and / or, in dark culture, the culture time in the hypertonic medium is 16 to 18 h; And / or, in dark culture, the formula of the regeneration medium without mannitol is: WPM + 2%-4% sucrose + 7.5g / L-9.0g / L agar + 0.05mg / L-0.8mg / L CPPU + 0.05mg / L-0.2mg / L IBA, pH=5.20±0.02; And / or, in dark culture, the culture time in the regeneration medium is 32 to 56 hours.

8. The establishment method according to claim 7, characterized in that: In step (3), the photoperiod induction culture conditions are light culture at 24°C to 25°C for 16 h to 18 h, and dark culture at 24°C to 25°C for 8 h to 10 h; and / or, the medium for photoperiod-induced culture is WPM + 2%-4% sucrose + 7.5 g / L-9.0 g / L agar + 0.05 mg / L-0.8 mg / L CPPU + 0.05 mg / L-0.2 mg / L IBA + 8 mg / L-12 mg / L Hyg, pH = 5.20 ± 0.02; And / or, the regeneration culture conditions are: light culture at 23-25°C for 14-18 hours; And / or, the culture medium formula for the regeneration culture is: WPM + 2%~4% sucrose + 7.5 g / L~9.0 g / L agar + 0.05 mg / L~0.8 mg / L CPPU + 0.05 mg / L~0.2 mg / L IBA, pH=5.20±0.

02.

9. The establishment method according to claim 7, wherein: In step (3), And / or, the screening culture conditions are: culturing under light at 23-25°C for 14-18 hours; And / or, the culture medium formula for the screening culture is: WPM + 2%-4% sucrose + 7.5 g / L-9.0 g / L agar + 0.05 mg / L- 0.8 mg / L CPPU + 0.05 mg / L-0.2 mg / L IBA + 12 mg / L-18 mg / L Hyg, pH = 5.20±0.02; And / or, the germination culture conditions are: culturing under light at 23-25° C. for 14-18 h; And / or, the culture medium formula for the germination culture is: WPM + 2%-4% sucrose + 7.5 g / L-9.0 g / L agar + 0.05 mg / L- 0.8 mg / L CPPU + 0.05 mg / L-0.2 mg / L IBA + 12 mg / L-18 mg / L Hyg, pH = 5.20±0.02; And / or, the rooting culture conditions are: culturing under light at 23-25° C. for 14-18 h; And / or, the culture medium formula for the rooting culture is: WPM + 2%~4% sucrose + 7.5 g / L~9.0 g / L agar + 0.2 mg / L~0.4 mg / L IAA + 0.5 mg / L~2.0 mg / L KT + 12 mg / L~18 mg / L Hyg, pH = 5.20±0.02.

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