Method for establishing a gene gun-mediated high-efficiency transgenic system of vaccinium

Abstract

The application provides a method for establishing a gene gun-mediated high-efficiency transgenic system of Vaccinium. The method comprises the following steps: taking sterile Vaccinium tender leaves as explants, and optimizing the treatment method of high-osmotic medium on the explants; a plasmid vector used for transfection comprises a first expression vector containing a transposase and a second expression vector used for integrating a target gene; after the target gene is integrated into the second expression plasmid, the first expression plasmid is mixed to prepare DNA-coated microprojectiles; the DNA-coated microprojectiles are bombarded on the explants under sterile conditions; the bombarded explants are placed on a regeneration medium to induce sprouting, and positive transgenic plants are obtained through pressure screening. The method can effectively improve the transgenic efficiency of Vaccinium, is not limited by species, can realize high-efficiency genetic transformation of various wild blueberries, greatly shortens the obtaining period of transgenic plants, and fills the key technical blank in the field.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to a method for establishing a highly efficient transgenic system of Vaccinium plants mediated by a gene gun. Background Technology

[0002] blueberry( Vaccinium (spp.) is a member of the Ericaceae family ( Ericaceae ) Vaccinium ( Vaccinium Blueberries are an important economic crop. As blueberries gain market influence, related species are also receiving increasing attention. Wild blueberries, such as the blueberry from Northeast my country, the European blueberry (unique to Europe), and the evergreen blueberry from the southern United States, are rich in anthocyanins, polyphenolic compounds, and vitamins, possessing significant antioxidant, anti-inflammatory, and neuroprotective physiological functions, and like blueberries, have immense development potential.

[0003] Transgenic technology is needed for functional gene research and gene editing breeding of both blueberries and wild blueberries. However, traditional Agrobacterium-mediated transgenic technology is developed for specific species, with significant differences in steps and formulations between species. Traditional blueberry transgenic technology is cumbersome, complex, and time-consuming, resulting in difficulties in mastering it and low conversion efficiency. There is an urgent need to develop a simple and universal method.

[0004] Gene gun bombardment technology, as a mature physical transformation method, delivers exogenous DNA directly to plant cells via high-speed micro-projectiles. It boasts significant advantages such as a wide host range, ease of operation, and the ability to transform multiple genes simultaneously. Currently, this technology has achieved remarkable results in important crops such as rice, wheat, and cotton, successfully cultivating several transgenic varieties with improved traits. Compared to Agrobacterium-mediated transformation, gene gun technology overcomes the limitations of host compatibility, simplifies the operation process, significantly shortens the transformation cycle, and typically maintains good regeneration capacity in transformed tissues. However, gene guns suffer from low transformation efficiency. This is because gene gun-mediated transformation lacks the gene integration-promoting protein factors found in Agrobacterium, resulting in extremely low transformation efficiency. The transgenic efficiency of gene gun-mediated transformation is approximately 0.04%, far below the experimental requirement of >3%. Therefore, increasing gene integration efficiency is crucial for the development of gene gun technology.

[0005] Transposases, typically encoded by transposons, recognize specific sequences at both ends of the transposon, dissociating the transposon from adjacent sequences and inserting it into a new DNA target site, without requiring homology. Therefore, transposases can serve as a means to facilitate DNA integration into the plant genome. Currently, the only known successful transposon transcription of plants using transposases is the ping-pong transposase from rice; transposases from other plants have been rarely reported.

[0006] Blueberry not only has unique flavor, but also is rich in anthocyanins and other health ingredients, which has broad prospects in the field of health food and medical raw material development. Although many excellent varieties have been bred by traditional hybridization, breakthrough varieties are difficult to emerge due to the limitation of gene pool.

[0007] Therefore, it is necessary to develop and utilize rich wild blueberry resources, and carry out gene editing breeding to accelerate the commercialization process of wild blueberry. The existing technology cannot realize efficient genetic transformation of blueberry and wild blueberry, which seriously restricts the development and utilization of the rare resource and the improvement process of the variety. Developing a high-efficiency gene gun transformation system suitable for various blueberries not only has important theoretical innovation value, but also can provide key technical support for the quality improvement and sustainable development of the blueberry industry. SUMMARY

[0008] The application provides a method for establishing a high-efficiency transgenic system of Vaccinium plants mediated by a gene gun, which can effectively improve the efficiency of establishing a transgenic system of Vaccinium plants, realize efficient genetic transformation of various wild blueberries, break through the species restriction, greatly shorten the obtaining period of transgenic plants, and solve the key technical blank in the field.

[0009] The specific technical scheme is as follows:

[0010] The application provides a method for establishing a high-efficiency transgenic system of Vaccinium plants mediated by a gene gun, which comprises the following steps:

[0011] (1) sterile Vaccinium plant tender leaves are used as explants and are placed in a high-osmotic medium for pretreatment;

[0012] (2) a DNA-coated microprojectile is prepared by using a gene gun technology to mix plasmids, and the DNA-coated microprojectile is used to bombard the pretreated explants under sterile conditions;

[0013] The mixed plasmid is obtained by mixing a first expression vector containing a transposase and a second expression vector containing a target gene;

[0014] (3) after the bombardment is completed, the bombarded explants are induced to sprout, and transgenic positive plants are obtained.

[0015] In the application, the efficiency of establishing a transgenic system of Vaccinium plants is effectively improved by “high-osmotic pretreatment + gene gun bombardment + subsequent tissue culture conditions”; the transformation rate is increased from 5 ‰ to 8.9% and 11.58%, the transformation period of Vaccinium uliginosum is shortened from 8 months to 3-4 months, and the transformation period of Vaccinium waxyleaf is shortened from 2-3 months to 1-2 months.

[0016] Further, in step (1), the Vaccinium plant is Vaccinium uliginosum or Vaccinium waxyleaf.

[0017] Further, in step (1), the formula of the high-osmotic 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.

[0018] Further, in step (1), the pretreatment method is:

[0019] Part of the leaf of the explant is laid flat on the high-osmotic medium with the abaxial surface facing up.

[0020] And / or, the area of the part of the leaf is > (0.4 cm x 0.4 cm).

[0021] And / or, the pretreatment time is 4~24 h; further preferably, the pretreatment time is 4~8 h or 6~8 h; more preferably, the pretreatment time is 8 h.

[0022] Further, 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.

[0023] The first original expression vector is selected from a plant overexpression vector: pCAMBIA1300, and other overexpression vectors can also be used, for example: pGreen, pAK320NEO and the like.

[0024] Further, the first original expression vector is selected from pCAMBIA1300, pGreen or pAK320NEO; further preferably, the first original expression vector is selected from pCAMBIA1300.

[0025] And / or, the nucleotide sequence of the transposase-encoding gene is as shown in SEQ ID NO. 1.

[0026] SEQ ID NO. 1:

[0027]

[0028] Further, 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.

[0029] The second original expression vector is selected from a plant overexpression vector: pCAMBIA1300, and other overexpression vectors can also be used, for example: including but not limited to pGreen, pAK320NEO, etc.

[0030] Further, the second original expression vector is selected from pCAMBIA1300, pGreen or pAK320NEO;

[0031] And / or, the molar ratio of the first expression vector to the second expression vector is 1:1~100; further preferably, the molar ratio of the first expression vector to the second expression vector is 1:1.

[0032] Further, in step (2), the DNA-coated microprojectile comprises: gold powder, spermidine, calcium chloride and mixed plasmid;

[0033] And / or, the molar ratio of the gold powder, spermidine and calcium chloride is 1:3600~4200:8000~12000;

[0034] And / or, the air pressure of the gene gun bombardment is 1100 -1200 psi;

[0035] And / or, the vacuum degree of the gene gun bombardment is 23-25 inch Hg;

[0036] And / or, the bombardment distance of the gene gun bombardment is 3-9 cm;

[0037] And / or, the bombardment frequency of the gene gun bombardment is 1~2 times.

[0038] The core use of spermidine and calcium chloride is to promote the combination of DNA and gold powder. Calcium chloride provides calcium ions (Ca²⁺) to neutralize the negative charge of DNA molecules, reduce the repulsive force between DNA and negatively charged microcarriers, and create conditions for their combination;

[0039] Spermidine, as a polyamine substance, further enhances the adsorption of DNA and microcarriers, helps DNA firmly adhere to the surface of microcarriers, ensures that foreign DNA can effectively enter target cells with microcarriers during bombardment, and improves the transformation efficiency.

[0040] Further, in step (3), the inducing budding comprises: after the bombarded explants are sequentially subjected to dark culture, photoperiod induction culture, regeneration culture, screening culture, germination culture and rooting culture, finally obtaining the transgenic shoots;

[0041] The dark culture comprises: first culturing the stably transformed cells in a high-osmotic medium, and then transferring the cells to a regeneration medium without mannitol for continuous culture;

[0042] And / or, in the dark culture, the formula of the high-osmotic 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;

[0043] And / or, in the dark culture, the culturing time in the high-osmotic medium is 16~18 h;

[0044] And / or, in the 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;

[0045] And / or, in the dark culture, the culturing time in the regeneration medium is 32~56 h.

[0046] Further, in step (3), the condition of the photoperiod induction culture is 16 h of light culture at 24℃, and 8 h of dark culture at 24℃;

[0047] And / or, the medium of the photoperiod induction 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 + 10 mg / L Hyg, pH = 5.20±0.02;

[0048] And / or, the condition of the regeneration culture is 14~18 h of light culture at 23~25℃;

[0049] And / or, the formula of the medium of 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.

[0050] Further, the condition of the step (3) is that,

[0051] And / or, the condition of the screening culture is that: 23~25℃ under light culture 14~18 h.

[0052] And / or, the medium formula of 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.

[0053] And / or, the condition of the germination culture is that: 23~25℃ under light culture 14~18 h.

[0054] And / or, the medium formula of the germination 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 + 15 mg / L Hyg, pH = 5.20±0.02.

[0055] And / or, the condition of the rooting culture is that: 23~25℃ under light culture 14~18 h.

[0056] And / or, the medium formula of 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.

[0057] Compared with the prior art, the present application has the following beneficial effects:

[0058] (1) The method successfully explores the gene gun bombardment technology as a general genetic transformation method for blueberry plants, provides a reliable experimental basis for future gene gun-mediated gene transformation of blueberries, fills the gap of key technologies, not only creates a new technology for functional gene transformation of blueberries, but also provides technical support for the study of the role of each functional gene in blueberries.

[0059] (2) By using transposase gene as an auxiliary plasmid, the efficiency of integrating the target gene into the genome is greatly improved, and the gene gun-mediated transgenic method fundamentally breaks through the limitation of low integration rate.

[0060] (3) The method can realize efficient transformation of the Vaccinium plant explant, and transgenic adventitious bud points can be obtained in 15-20 days, and the transgenic buds can be obtained through subsequent regeneration and screening culture, so that the breeding cycle is greatly shortened.

[0061] (4) The fresh leaves of the Vaccinium plant are used as the explant in the method, and the leaves are easy to obtain and simple to operate, so that the transformation process is simplified.

[0062] (5) The method can greatly reduce the operation threshold and time cost of the existing transgenic technology, save manpower and material resources for the transgenic company, and reduce the comprehensive cost by 1 / 3-1 / 2, and has good commercial promotion value. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 It is a bud picture of V. uliginosum under the bright field and dark field of the Zeiss macro variable body fluorescence microscope in Example 1.

[0064] Wherein, a is a positive bud point picture of successful transgenic under the macro variable body fluorescence microscope bright field after gene gun bombardment by using the method of the patent; b is a positive bud point picture of successful transgenic under the macro variable body fluorescence microscope GFP dark field after gene gun bombardment by using the method of the patent; c is a positive bud point picture of successful transgenic under the bright field by using other methods other than the method of the patent; d is a positive bud point picture of successful transgenic under the GFP dark field by using other methods other than the method of the patent; e is a positive bud picture of successful transgenic after gene gun bombardment by using the method of the example; f is a positive bud picture of successful transgenic after gene gun bombardment by using the method of the example under the dark field.

[0065] Figure 2 It is a bud picture of V. uliginosum under the bright field and dark field of the Zeiss macro variable body fluorescence microscope in Example 1.

[0066] Wherein, a is a positive bud point picture of successful transgenic under the macro variable body fluorescence microscope bright field; b is a positive bud point picture of successful transgenic under the GFP dark field;

[0067] c is a positive bud point picture of successful transgenic under the bright field of another plant; d is a positive bud point picture of successful transgenic under the dark field of another plant.

[0068] Figure 3 It is a gene gun-mediated transgenic transformation efficiency of the Vaccinium plant before and after plasmid modification in Example 5, treatment II of Example 4 and Example 6. DETAILED DESCRIPTION

[0069] In order to make the technical personnel in the art better understand the present application, the technical solutions of the present application are described clearly and completely below in combination with specific embodiments. It should be pointed out that the following detailed description is exemplary and only a part of the embodiments of the present application, but not all the embodiments.

[0070] Based on the embodiments in the present application, it should be pointed out that for those skilled in the art, without departing from the concept of the present application, several modifications and improvements can also be made, which should also be considered as the protection scope of the present application, which will not affect the effect and practicability of the present application.

[0071] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs. The experimental materials used in the embodiments of the present application are all conventional experimental materials in the art, which can be purchased through commercial channels. The experimental methods without detailed conditions are carried out according to the conventional experimental methods or according to the operation instructions recommended by the suppliers.

[0072] In the following embodiments of the present application, "WPM" refers to the 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 indole butyric acid; "CPPU" refers to forchlorfenuron; "Hyg" refers to hygromycin; "IAA" refers to indole acetic acid; "KT" refers to kinetin; "GA" refers to gibberellin; "Trytone" refers to tryptone; "Yeast Extract" refers to yeast extract.

[0073] The formula of the high-osmotic 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;

[0074] The formula of the 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;

[0075] The formula of the photoperiod induction medium is: 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;

[0076] The formula of the screening medium is: 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;

[0077] The formula of the germination medium is: 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;

[0078] The formula of the 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.

[0079] The formula of the LB liquid medium is: NaCl 10 g / L + Trytone 10 g / L + Yeast Extract 5 g / L.

[0080] The gene gun used in the application is a product of the American Bio-RAD company, which refers to a high-speed helium gene gun PDS-1000 / He (Bio-Rad).

[0081] The Buffer P1-P4 used in the application for extracting plasmids is a product of Yiside Biotechnology Co., Ltd. The original expression vector used in the application is purchased from Addgene, and is designed by the applicant.

[0082] The determination method of the transformation efficiency is: using a Zeiss macro variable body fluorescence microscope to observe, and under the irradiation of a laser, the blueberry regenerative bud with green fluorescence (GFP) is a transgenic bud.

[0083] The calculation method of the transformation efficiency is: the number of transgenic buds / the total number of explants. If more than one bud is a transgenic bud in the multiple buds grown on one explant material, it is still counted as one in counting.

[0084] Example 1

[0085] The embodiment provides a method for establishing a high-efficiency transgenic system of Vaccinium plants by using a gene gun, and the specific steps are as follows:

[0086] (1) sterile Vaccinium plant tender leaves are used as explants and are placed in a high-osmotic medium for pretreatment;

[0087] The specific steps are as follows:

[0088] (1-1) Obtaining and pre-culturing of explants. Fresh leaves of Vaccinium uliginosum (the wild blueberry with the highest market share in China, mainly grown in Northeast China) growing for three weeks were taken as explants in a sterile environment, the length of the leaves ranged from 0.5 cm to 0.8 cm, the width ranged from 0.4 cm to 0.6 cm, and the growth state was good; the leaves were laid flat on the regeneration medium plate for pre-culturing for 2 days.

[0089] (1-2) High-osmotic pretreatment. The leaves pre-cultured for 2 days were transferred to a high-osmotic medium plate containing 0.4 M mannitol for high-osmotic pretreatment for 8 hours.

[0090] (2) The mixed plasmid was made into DNA-coated microprojectiles by using a gene gun technique, and the pre-treated explants were bombarded with the DNA-coated microprojectiles under sterile conditions; the mixed plasmid was obtained by mixing a first expression vector containing a transposase and a second expression vector containing a target gene.

[0091] The specific steps are as follows:

[0092] (2-1) Extracting plasmid. The first expression vector is composed of a first original expression vector pCAMBIA1300 and a transposase-encoding gene (nucleotide sequence as 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) position between the CaMV 35S promoter and the NOS terminator of the first original expression vector pCAMBIA1300, and is cut by Sal I and Hind III; the second expression vector is composed of a second original expression vector pCAMBIA1300 and a target gene (i.e. a 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) position between the CaMV 35S promoter and the NOS terminator of the second original expression vector, and is cut by Sal I and BmHI; the above expression vectors are all obtained by amplification of Escherichia coli.

[0093] The specific method is as follows: a single colony of E. coli is picked from a culture medium plate and inoculated into 4 mL of LB liquid medium containing kanamycin, and cultured 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, the bacterial solution is transferred to a 1.5 mL or 2.0 mL centrifuge tube, centrifuged at 12000 rpm (≥13000 g) at room temperature for 1 min, and the supernatant is discarded; 200 μL of Buffer P1 is added to the centrifuge tube to resuspend the bacterial solution; 200 μL of Buffer P2 is added to the centrifuge tube for lysis, and gentle rolling is performed for 6-8 times, with a lysis time of not more than 5 min; the lysis solution becomes clear and viscous; 200 μL of Buffer P3 is further added to the centrifuge tube, and gentle rolling is performed for 6-8 times; after mixing well, the centrifuge tube is placed in a high-speed centrifuge and centrifuged at 12000 rpm (≥13000 g) at room temperature for 10 min; the supernatant after centrifugation is sucked into a 2 mL collection tube Hipure DNA adsorption column, and centrifuged at 12000 rpm (≥13000 g) at room temperature for 30 s; the adsorption column is taken out and the waste liquid is discarded; the adsorption column is placed back into the collection tube, 500 μL of Buffer P4 (containing anhydrous ethanol) is added, and centrifuged at 12000 rpm (≥13000 g) at room temperature for 30 s; the adsorption column is taken out and the waste liquid is discarded; the washing is repeated once; the adsorption column is placed back into the collection tube and centrifuged at 12000 rpm (≥13000 g) at room temperature for 1 min; the adsorption column is placed into a clean 1.5 mL centrifuge tube, and after standing for 1-2 min, 40 μL of elution solution is added to the center of the adsorption column membrane, and after standing at room temperature for 1-2 min, the adsorption column is centrifuged at 12000 rpm (≥13000 g) at room temperature for 1 min. 2 μL of the extracted plasmid is taken to measure the concentration; the rest of the plasmid is stored in a -20°C refrigerator.

[0094] (2-2) Gold powder required for biolistics is configured. The gold powder used in this embodiment has a diameter of 0.8-1.5 μm, and the amount of gold powder selected for biolistics is 1.5 milligrams per time, and 30 milligrams of gold powder is used for about 20 times of biolistics; the gold powder is placed into a 1.5 mL sterilized EP tube, 1000 microliters of 70% analytical pure ethanol is added to the EP tube, and the gold powder surface is washed uniformly by vigorous shaking on a vortex shaker for 3-5 min; the EP tube is placed at room temperature for 15 min, and the ethanol is discarded after high-speed centrifugation at 12000 rpm for 5 s; after the alcohol washing, 1000 microliters of sterile deionized water is added and left to stand for 1 min, and the supernatant is removed after centrifugation; the washing is performed for two to three times; 500 microliters of 50% glycerol is added to obtain the treated gold powder.

[0095] (2-3) Configuration of the microprojectile gun required to hit. 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) are mixed in a ratio of 1:1, then 25 μL of gold powder that has been pre-treated is taken, 10 μL of 0.10 M spermidine, 20 μL of plasmid DNA and 25 μL of 2.5 M calcium chloride are added to the gold powder suspension in turn; after each reagent is added, mix thoroughly and shake for 2 min, then immediately place on ice for 1 min; centrifuge at 12000 rpm for 10 s in a high-speed centrifuge, then discard the supernatant; add 140 μL of 70% ethanol, mix well with a gun head, then shake for 1 min; ultrasonic for 5 s, centrifuge at 12000 rpm for 10 s, discard the supernatant; add 140 μL of anhydrous ethanol, mix well with a gun head, then shake for 1 min, centrifuge at 12000 rpm for 10 s, discard the supernatant; finally add 5 μL of 100% ethanol to ensure complete resuspension of the gold powder, obtain DNA-coated microprojectiles with a concentration of 3000 ng / gun, and finally place on ice for standby.

[0096] (2-4) Gene gun treatment. In the clean bench, the surface of the gene gun and the vacuum chamber are disinfected with 75% ethanol and ultraviolet sterilized for 30 min in advance. The gene gun is hit under strict sterile conditions in the clean bench; before hitting, the blocking net and tray are sterilized in a high-pressure sterilization pot, and the membrane can be cracked and the carrier film is soaked in anhydrous ethanol for 15 min. In this embodiment, the PDS-1000 / He type gene gun produced by Bio-Rad Company is used for gene gun-mediated genetic transformation of the high-osmotic treated Vaccinium uliginosum leaves.

[0097] The specific bombardment process is as follows: after opening the clean bench, place the blocking net, fixing ring, rupturable membrane and object film on the sterile filter paper and dry in the clean bench; open the helium cylinder valve, adjust the pressure of the helium cylinder to be higher than the rupture pressure of the rupturable membrane 1100 psi, then open the transformer switch and the gene gun vacuum pump switch; install the rupturable membrane on the holder, then screw the holder clockwise to the gas accelerator and tighten with a wrench; install the object film on the fixing ring, fully resuspend the DNA-coated gold powder microbullets with the gun head, evenly coat the prepared microbullets on the center of the rupturable membrane, and then place it on the clean bench to dry; after drying, place the blocking net on the blocking net holder, place the fixing ring assembled with the object film on the upper end of the blocking net, and make the side with gold powder particles face the blocking net, then screw the cover tightly and insert it into the first layer of the bombardment chamber; place the medium plate containing explant material on the tray, at this time the explant material is concentrated and laid flat in the center of the medium plate, keep the bombardment distance at 3 cm, and close the door; first press the "VAC" key on the gene gun, when the vacuum degree reaches 25 inch Hg, quickly press the "HOLD" key, then press the "FIRE" key for a long time and keep it still, after hearing a "pop" sound, quickly release the key, the bombardment is completed, then press the "VENT" key, after the vacuum gauge returns to zero, take out the bombarded sample; the bombardment is 1 time.

[0098] (3) After the bombardment is completed, the bombarded explants are induced to sprout to obtain transgenic plants.

[0099] The specific steps are as follows:

[0100] (3-1) After the bombardment is completed, quickly cover the bombarded explant material; the bombarded material continues to be placed on the high-osmotic medium for 16 hours of dark culture to stabilize the transformed cells; then transfer to the regeneration medium without mannitol for 48 hours of dark culture; then perform photoperiod induction regeneration under light conditions, 16 h of light culture at 24°C, and 8 h of dark culture at 24°C; after 5-7 days of light culture, transfer the explant material to the regeneration medium containing hygromycin for regeneration culture, 16 h of light culture at 24°C;

[0101] (3-2) During the regeneration culture of explant leaf induction of sprout, after 15 to 20 days, transgenic sprout points will appear. At this time, the explant leaf with transgenic sprout points needs to be transferred to a new regeneration medium plate containing hygromycin to allow the sprout points to continue to grow and develop into transgenic sprouts. When a large number of adventitious sprouts grow and develop, transfer the explant material to the rooting medium containing hygromycin and continue to culture at 24°C under light for 16 hours. At the same time, replace the medium plate for the explant material every 15-20 days, and through this series of operations, positive plants can be finally obtained.

[0102] The conversion rate of the method of this example was determined, and the results are shown in Example 2.

[0103] Example 2

[0104] This example 2 is different from example 1 in that the high osmotic treatment time of the bilberry leaf (i.e. the explant material to be bombarded) is gradient designed (i.e. the treatment time of 8h in step (1-2) of example 1), and the rest of the steps are exactly the same as example 1.

[0105] The specific content is as follows:

[0106] The high osmotic treatment time is set to 0h, 2h, 4h, 6h, 8h, 10h, 12h, the air pressure is 1100 psi, the vacuum degree is kept at 25 inch Hg, the amount of plasmid is 3000 ng / gun, the bombardment distance is kept at 3 cm, and the bombardment times is 1 time. The bilberry leaf tissue explants after high osmotic treatment are bombarded by gene gun.

[0107] The results are shown in Table 1.

[0108] Table 1 Effect of high osmotic treatment time on GFP gene expression

[0109]

[0110] Through the detection of green fluorescent protein expression by Zeiss macro variable power fluorescence microscope, it is found that there is a direct correlation between high osmotic treatment time and green fluorescent protein expression.

[0111] As can be seen from Table 1, with the continuous increase of high osmotic treatment time, the expression efficiency of bilberry leaf tissue presents fluctuant increase. The transformation efficiency reaches the peak (9.10%-10.73%) in the range of 6-8 hours of high osmotic treatment time, which is significantly higher than that of other treatment times. When the high osmotic treatment time is less than 6h, the transformation efficiency gradually increases with the extension of the treatment time, which shows that a certain time of high osmotic treatment has a positive effect on cell state. But when the high osmotic treatment time exceeds 8h to 10h, the expression rate of GFP directly decreases to 2.44%, and to 12h, there is no GFP expression compared with other treatments at the same period, which shows that too long high osmotic treatment may cause cell damage or metabolic inhibition. By setting seven gradients of high osmotic treatment time, combined with the expression of green fluorescent protein of the final leaf tissue, it is analyzed that the preferred high osmotic treatment time for gene gun mediated bilberry leaf tissue genetic transformation is 8h.

[0112] Example 3

[0113] The difference between this embodiment 3 and embodiment 1 is that the amount of plasmid in the microprojectile configuration used for gene gun bombardment is gradient designed (i.e. the amount of plasmid in step (2-3) of embodiment 1 is 3000 ng / gun), and the rest of the steps are exactly the same as embodiment 1.

[0114] The specific content is as follows: under the premise that the high-osmotic treatment time is 8 h, the air pressure is 1100 psi, the vacuum degree is maintained at 25 inch Hg, the bombardment distance is 3 cm, and the bombardment times is 1 time, the amount of plasmid is set to 1500 ng / gun, 3000 ng / gun, and 4500 ng / gun three gradients, and the high-osmotic treated Vaccinium uliginosum leaf tissue explants are subjected to gene gun bombardment.

[0115] Table 2 Effect of plasmid amount on GFP gene expression

[0116]

[0117] Through the detection of green fluorescent protein expression by Zeiss macro variable magnification fluorescence microscope, it is found that there is a direct correlation between the amount of plasmid and the expression of green fluorescent protein.

[0118] It can be found from table 2 that under the condition of 3000 ng / gun of plasmid amount, the transformation efficiency reaches the highest (10.73%), which is significantly higher than 1500 ng / gun (6.67%) and 4500 ng / gun (5.91%).

[0119] Therefore, 3000 ng / gun can be used as the preferred parameter, and it is suggested that 1500-3000 ng / gun be used as the effective range. On the basis of 6-8 h high-osmotic treatment, the use of 3000 ng / gun of plasmid amount can synergistically improve the gene gun transformation efficiency.

[0120] Embodiment 4

[0121] The difference between this embodiment 4 and embodiment 1 is that the bombardment distance is gradient designed, and the rest of the steps are exactly the same as embodiment 1.

[0122] The specific content is as follows:

[0123] Under the premise that the amount of plasmid is 3000 ng / gun, the high-osmotic treatment time is 8 h, the air pressure is 1100 psi, the vacuum degree is maintained at 25 inch Hg, and the bombardment times is 1 time, the bombardment distance is set to 3 cm (treatment I), 6 cm (treatment II), and 9 cm (treatment III) three gradients, and 3 cm is the nearest distance that can be set by the device; the high-osmotic treated Vaccinium uliginosum leaf tissue explants are subjected to gene gun bombardment.

[0124] Table 3 Effect of bombardment distance on GFP gene expression

[0125]

[0126] The green fluorescence protein expression was detected by Zeiss macroscopic variable magnification body fluorescence microscope, and it was found that the three bombardment distances had obvious differences in the expression of green fluorescence protein in the leaf tissues of V. uliginosa after bombardment.

[0127] As shown in Table 3, the transformation efficiency of 6 cm was the highest (11.58%), which was better than that of 3 cm (10.73%) and 9 cm (4.82%). The transformation efficiency of 3 cm (10.73%) remained at a high level, but it was slightly lower than that of 6 cm. The transformation efficiency of 9 cm (4.82%) decreased sharply, which was only 37.36% of that of 6 cm, indicating that long-distance bombardment seriously affected the gene delivery efficiency. The preferred bombardment distance for the leaf tissue genetic transformation of V. uliginosa mediated by the gene gun was 6 cm. Based on the above results, it is recommended to perform gene gun bombardment under the synergistic conditions of 6-8 h high osmotic treatment, 3000 ng / shot plasmid dosage and 6 cm bombardment distance, so as to make the gene gun transformation efficiency as optimal as possible.

[0128] Example 5

[0129] The difference between this example 5 and example 1 is that the genetic transformation of V. uliginosa other than V. uliginosa, such as V. waxiygyllis, is performed by the gene gun mediation method, and the operation steps are the same as those of example 1.

[0130] Through the above method, the positive transgenic buds of V. waxiygyllis are obtained as shown in FIG. 5. Figure 2 As shown in FIG. 6, the transformation rate of the positive transgenic buds of V. waxiygyllis is 8.9%. Figure 3

[0131] Example 6

[0132] In this example 6, a plant transgenic recombinant plasmid before modification is used to perform genetic transformation of V. uliginosa by the gene gun mediation method. The only difference between this operation and example 1 is that the first expression vector is only composed of the first original expression vector pCAMBIA1300, and the remaining steps are consistent with those of example 1.

[0133] The experimental results show that the transformation efficiency of this method is about 5 ‰.

[0134] This data is in sharp contrast to the transformation rate obtained by the transgenic method mediated by the modified vector in V. waxiygyllis and V. uliginosa: the transformation rate of the modified vector in V. waxiygyllis (i.e. obtained in example 5) is 8.9%, and the transformation period is shortened from 2-3 months to 1-2 months; in V. uliginosa (i.e. obtained in treatment II of example 4), it is as high as 11.58%, and the transformation period is shortened from 8 months to 3-4 months.​

[0135] Thus, it can be seen that the transformation efficiency mediated by the recombinant plasmid before modification is far lower than that of the vector after modification. As shown in Figure 3 Table 2.

Claims

1. A method for establishing a gene gun-mediated high-efficiency transgenic system for Vaccinium species, characterized in that, include: (1) Use sterile young leaves of Vaccinium as explants, lay them flat in hypertonic medium with the abaxial surface facing up, and pretreat them; The formulation 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; The pretreatment time is 6-8 hours; (2) The mixed plasmid was made into DNA-encapsulated microbulbs using gene gun technology, and the DNA-encapsulated microbulbs were used to bombard the pretreated explants with gene gun under sterile conditions. The mixed plasmid is obtained by mixing a first expression vector containing a transposase-encoding gene and a second expression vector containing a target gene in a molar ratio of 1:1 to 100. The nucleotide sequence of the transposase encoding gene is shown in SEQ ID NO. 1; The gene gun bombardment parameters are: bombardment distance 3~9 cm; plasmid dosage 1500~3000 ng / gun; (3) After the bombardment is completed, the bombarded explants are induced to sprout to obtain transgenic positive plants; The induced bud formation process includes: sequentially subjecting the bombarded explants to dark culture, photoperiodic induced culture, regeneration culture, selection culture, budding culture and rooting culture, and finally obtaining transgenic buds; The dark culture includes: first stabilizing the transformed cells in a hypertonic medium, and then transferring them to a regeneration medium without mannitol for further culture; And / or, in dark culture, the hypertonic medium is formulated as follows: 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 16-18 h; And / or, in dark culture, the mannitol-free regeneration medium is formulated as follows: 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; And / or, the conditions for the rooting culture are: 23~25℃ under light for 14~18 h; And / or, the culture medium for the rooting culture is formulated as follows: 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.

2. The method for establishing as described in claim 1, characterized in that, In step (1), the preprocessing method is as follows: Take some leaves from the explant and lay them flat in a hypertonic culture medium with the abaxial surface facing up. And / or, the area of ​​the said partial leaf is > (0.4cm × 0.4cm).

3. The method for establishing as described in claim 1, characterized in that, 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 or pGreen.

4. The method for establishing as described in claim 1, characterized in that, In step (2), the second expression vector contains a second original expression vector and a target gene inserted into the second original expression vector; The second original expression vector was selected from pCAMBIA1300 or pGreen.

5. The method for establishing as described in claim 1, characterized in that, In step (2), the DNA-encapsulated microelastics include: gold powder, spermidine, calcium chloride, and a mixed plasmid; And / or, the molar ratio of the gold powder, spermidine, and calcium chloride is 1:3600~4200:8000~12000; And / or, the vacuum level of the gene gun bombardment is 23~25 inch Hg; And / or, the number of times the gene gun bombards is 1 to 2; And / or, the gene gun has a gas pressure of 1100~1200 psi.

6. The method for establishing as described in claim 1, characterized in that, In step (3), The photoperiod-induced culture conditions are: light culture at 24℃~25℃ for 16 h~18 h, and dark culture at 24℃~25℃ for 8 h~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 conditions for the regeneration culture are: light culture at 23~25℃ for 14~18 h; And / or, the culture medium formulation 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.

7. The method for establishing as described in claim 1, characterized in that, In step (3), And / or, the screening culture conditions are: light culture at 23~25℃ for 14~18 h; And / or, the culture medium formulation 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: light culture at 23~25℃ for 14~18 h; And / or, the germination culture medium formula 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.

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