CN112501211A - Method for genetic transformation of eucommia ulmoides by injection - Google Patents
Method for genetic transformation of eucommia ulmoides by injection Download PDFInfo
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- CN112501211A CN112501211A CN202011516536.0A CN202011516536A CN112501211A CN 112501211 A CN112501211 A CN 112501211A CN 202011516536 A CN202011516536 A CN 202011516536A CN 112501211 A CN112501211 A CN 112501211A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
Abstract
The invention relates to a method for transforming eucommia ulmoides by utilizing injection genetic transformation. The method comprises the following specific operation steps of taking eucommia seedlings germinating for three days, pulling out a first true leaf between two cotyledons to serve as a genetic transformation receptor, and injecting agrobacterium tumefaciens resuspension with different concentrations between the two cotyledons of the eucommia seedlings: (1) preparation of genetic transformation receptor (2) culture of agrobacterium (3) preparation of agrobacterium resuspension (4) genetic transformation of eucommia (5) management of transgenic plant (6) transgenic plant identification. The invention does not need tissue culture technology, does not need aseptic operation except for culturing agrobacterium, has the advantages of simple operation, lower cost, capability of transforming large-fragment DNA, stable heredity, low copy number and the like, and becomes the most widely applied genetic transformation method at present.
Description
Technical Field
The invention belongs to the technical field of plant biotechnology, and particularly relates to a method for transforming eucommia ulmoides by injection genetic transformation.
Background
Eucommia ulmoides (Eucommia ulmoides Oliver) is a deciduous tree in Eucommia of Eucommiaceae, is a rare Chinese medicinal material in China, and has important medicinal value and economic value. In recent years, in the genetic transformation technology of agrobacterium-mediated eucommia, a tissue culture technology is mainly used, but factors such as low callus induced differentiation efficiency, rooting of resistant buds, low transplanting survival rate of transgenic plants and the like exist, so that the research of eucommia is relatively lagged, and a large survival plant is difficult to obtain.
The agrobacterium injection method is a non-tissue culture genetic transformation method for injecting agrobacterium resuspension to a genetic transformation receptor in an open environment. The whole plant genetic transformation can be directly carried out on the plant, the problems of induced differentiation, rooting of resistant buds, late transplanting and the like do not exist, and the survival of the positive seedlings can be better ensured. According to the invention, the eucommia ulmoides seedlings germinating for three days are subjected to genetic transformation by taking the first true leaf between two cotyledons as a material, and the agrobacterium injection method has the advantages of simple operation, low cost, capability of transforming large-fragment DNA, stable heredity, low copy number and the like. At present, genetic transformation systems of agrobacterium injection methods are widely applied to dicotyledonous plants such as soybean, litchi and the like, but the application of eucommia is not reported clearly, and research on eucommia genomics and gene functions is restricted.
Disclosure of Invention
The invention obtains transgenic plants by utilizing the genetic transformation technology of injecting eucommia seedlings by agrobacterium to provide technical support for research of eucommia genomics and gene functions.
A method for genetic transformation of eucommia ulmoides by injection comprises the following steps:
(1) preparation of genetic transformation receptors: selecting eucommia seedlings germinating for 2-4 days, and removing a first true leaf between two cotyledons to serve as a genetic transformation receptor for later use;
(2) and (3) culturing agrobacterium: transforming the agrobacterium tumefaciens strain LBA4404 by the vector with the target gene, inoculating the agrobacterium tumefaciens strain LBA4404 to a solid YEP culture medium, and culturing for 2d in an incubator at 28 ℃; picking single colony to 5ml liquid YEP culture medium, shaking and culturing at 28 deg.C and 180rpm for 12 hr, adding 100ul into 100ml liquid YEP culture medium, shaking and culturing at 28 deg.C and 180rpm to suitable value for use; 100mg/L kanamycin (Kan) and 100mg/L rifampicin (Rif) on the YEP medium;
(3) preparation of agrobacterium tumefaciens resuspension: subpackaging the cultured agrobacterium liquid into a 50ml centrifugal tube, centrifuging at 5000rpm for 10min, discarding the supernatant, and resuspending the thallus precipitate with a resuspension solution for later use;
(4) genetic transformation of eucommia ulmoides: adding Acetosyringone (AS) and a surfactant Silwet-L77 into the agrobacterium tumefaciens heavy suspension liquid, injecting 100 mu L of the agrobacterium tumefaciens heavy suspension liquid into a space between two eucommia ulmoides cotyledons by using an injector, and culturing in the dark at 28 ℃ for 3 d;
(5) management of transgenic plants
And (4) canceling dark culture of the dark cultured plants, transferring the survived plants to a greenhouse of a transgenic plant demonstration base after one week, and performing conventional fertilizer and water management.
The eucommia ulmoides seedling is a eucommia ulmoides seedling which germinates for 3 days.
The method for obtaining the eucommia ulmoides seedlings in the step (1) comprises the following steps: selecting seeds with good growth vigor, washing the seeds with water for three times, soaking the seeds in the water, putting the seeds into a constant-temperature incubator at 37 ℃ for overnight, soaking the seeds in 400mg/L gibberellin for 5 to 6 hours, taking the seeds out, washing the seeds with clear water, sowing the seeds in humid nutrient soil, and germinating and sprouting the seeds for 10 to 15 days.
The removal is performed by pulling out surgical forceps.
The formula of the heavy suspension comprises: MS 4.43g/L, sucrose 30g/L, 6-BA 3. mu.M, 2-iP 3. mu.M, and adjusting pH to 5.8-6.0.
The YEP culture medium formula comprises: 10g/L of yeast extract, 10g/L of peptone, 5g/L of sodium chloride, pH 7.2 and 15g/L of agar added into a solid YEP culture medium.
100ul of 20mg/ml acetosyringone is added into the agrobacterium tumefaciens heavy suspension liquid in the step (4), and the concentration of the surfactant Silwet-L77 is 500 ul/L.
The concentration OD600 of the agrobacterium tumefaciens resuspension is 0.8.
The vector is connected with GUS gene.
And (5) a transgenic plant identification step is included after the step, and the identification comprises GUS chemical tissue staining detection of the resistant plant and PCR detection of a GUS gene.
GUS chemical tissue staining detection of resistant plants: after one month of management according to the step (5), cutting leaves newly grown from eucommia ulmoides seedlings, putting the leaves into a PCR tube, and adding GUS staining solution; incubating the culture medium in an incubator at 37 ℃, adding 75% ethanol for elution until chlorophyll is completely removed, and photographing for observation.
The transgenic eucommia leaves are cut into about 1cm-2cm long and then placed in a PCR tube, 200ul of GUS dye solution is added into the PCR tube, vacuum treatment is carried out for 10min at 15Kpa, and the PCR tube is placed in a constant-temperature incubator at 37 ℃ for overnight reaction. And (5) sucking the dye liquor, and decoloring with 75% ethanol until chlorophyll disappears.
PCR detection of transgenic plants: and (3) detecting plants with positive leaves by using GUS dye solution, extracting DNA by using a CTAB method, and carrying out further PCR detection.
PCR of the GUS gene: the PCR system for detecting 10. mu.L contains 1.0. mu.L of DNA, 0.2. mu.L of each primer, 5.0ul of rTaq and 3.6. mu.L of ddH2O 3.6. Reaction conditions are as follows: 94 ℃ for 3 min; 30s at 94 ℃; at 54 ℃ for 30 s; 72 ℃ for 1 min; 35 cycles; extending for 5min at 72 ℃; storing at 12 deg.C. After the PCR reaction, 5ul of the amplified product was electrophoresed in 1% agarose gel, observed under a gel imaging system and photographed.
According to the invention, eucommia seedlings germinating for three days are taken, a first true leaf between two cotyledons is removed to serve as a genetic transformation receptor, agrobacterium tumefaciens heavy suspension liquid, acetosyringone and a surfactant Silwet-L77 are injected between the two cotyledons of the eucommia seedlings, after dip-dyeing is carried out under different liquid concentration, dark culture is carried out for 3d, GUS chemical tissue dyeing and PCR identification of transgenic plants are carried out after new leaves grow out, the influence of different liquid concentrations on the eucommia seedlings is researched in the process, and finally, the genetic transformation method and the optimal transformation condition of the eucommia seedlings injected with agrobacterium tumefaciens are obtained. The method of the invention does not need tissue culture process and has high genetic transformation efficiency.
The invention obtains the transgenic plant by utilizing the genetic transformation method of injecting eucommia ulmoides seedlings by agrobacterium to provide technical support for research of the genomics and the gene function of eucommia ulmoides.
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FIG. 1 preparation and injection of eucommia ulmoides seedling recipients.
Wherein, A: germinating three-day eucommia ulmoides seedlings; b: removing the first leaf of eucommia ulmoides seedling; c: injecting agrobacterium tumefaciens resuspension to the eucommia ulmoides seedlings.
FIG. 2 genetically transformed plants.
Wherein, A: plants 15 days after genetic transformation; b: transplanting the transgenic plants one month later.
FIG. 3GUS staining of eucommia leaves.
Wherein, A: the leaves of the eucommia ulmoides plants of blue wild type are not shown in GUS staining; B. c, D: GUS stains blue leaves of transgenic eucommia ulmoides plants.
FIG. 4GUS gene PCR results.
Wherein, M: DL2000 marker; WT is wild eucommia ulmoides plant; 1-7 is transgenic eucommia plant
Detailed Description
The present invention will be described in further detail with reference to examples.
All the following biomaterials were commercially available and stored in the laboratory of the applicant, and were publicly distributed.
The formula of the heavy suspension comprises: MS 4.43g/L, sucrose 30g/L, 6-BA 3. mu.M, 2-iP 3. mu.M, and adjusting pH to 5.8-6.0.
The YEP culture medium formula comprises: 10g/L of yeast extract, 10g/L of peptone, 5g/L of sodium chloride, pH 7.2 and 15g/L of agar added into a solid YEP culture medium.
Example 1A genetic transformation technique for injecting eucommia ulmoides seedlings by Agrobacterium
1.1 genetic receptor preparation: the method comprises the steps of taking eucommia seedlings germinating for three days, pulling out a first true leaf between two cotyledons through surgical forceps to serve as a genetic transformation receptor for later use. As shown in fig. 1.
1.2 culture of Agrobacterium: agrobacterium tumefaciens strain LBA4404, vector pCAMBIA1300 vector carrying hygromycin resistance gene Hyg and GUS gene, was inoculated on YEP solid medium containing 100mg/mL kanamycin (Kan) and 100mg/mL rifampicin (Rif), and cultured in an incubator at 28 ℃ for 2 days; single colonies were picked up into 5mL YEP (containing 100mg/mL Kan and 100mg/mL Rif) liquid medium, and after 12 hours of shaking culture at 28 ℃ and 180rpm, 100ul was added to 100mL YEP (containing 100mg/mL Kan and 100mg/mL Rif) liquid medium, and shaking culture at 28 ℃ and 180rpm was carried out until appropriate, to prepare it for use.
1.3 preparation of Agrobacterium resuspension: and (3) subpackaging the cultured agrobacterium tumefaciens bacterial liquid in a 50ml centrifugal tube, centrifuging at 5000rpm for 10min, discarding the supernatant, and resuspending the bacterial precipitate with a resuspension solution for later use.
1.4 genetic transformation of eucommia ulmoides: adding Acetosyringone (AS) and a surfactant Silwet-L77 into the agrobacterium tumefaciens heavy suspension, injecting 100 mu L of agrobacterium tumefaciens liquid into the space between two eucommia ulmoides cotyledons by using a 1mL injector, and culturing for 3d in the dark at 28 ℃.
TABLE 1 Dip-dyeing of eucommia ulmoides seedlings under different bacterial liquid concentrations
1.5 management of transgenic plants: and (4) canceling dark culture of the dark-cultured plants, watering once every two days, transferring the survived plants to a greenhouse of a transgenic plant demonstration base after one week, and performing conventional fertilizer and water management. As shown in fig. 2.
1.6 identification of transgenic plants
1.6.1 GUS chemical tissue staining detection of resistant plants: the transgenic eucommia leaves are cut into about 1cm-2cm long and then placed in a PCR tube, 200ul of GUS dye solution is added into the PCR tube, vacuum treatment is carried out for 10min under 15Kpa, and then the PCR tube is placed in a constant temperature incubator at 37 ℃ for incubation overnight. Sucking the dye solution, adding 1ml of 75% ethanol for decolorization until chlorophyll disappears, and photographing for observation. The results showed that 2400 eucommia ulmoides were co-treated in Table 1, and that 140 positive plants were identified by GUS staining. The method specifically comprises the following steps: agrobacterium liquid OD600Values of 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, respectively; the survival rate and GUS staining rate are 77.33%, 77.67%, 66.00%, 58.33%, 54.33%, 67.33%, 53.00%, 62.00% and 1.33%, 10.00%, 2.67%, 3.33%, 7.00%, 1.70%, 11.00%, 9.60%, respectively; as can be seen from the table, the optimum conversion conditions OD600At a value of 0.8, the plant survival rate was 77.67%, and the GUS staining rate was 6.67%. As shown in fig. 3.
1.6.2 PCR detection of transgenic plants
1.6.2.1 extraction of DNA by CTAB method:
1. adding 1ml of 1.2% CTAB extraction buffer solution into a 2ml centrifuge tube, adding 20ul of beta-mercaptoethanol, uniformly mixing, and preheating at 65 ℃.
2. Taking 0.1-0.2g of fresh leaves, grinding until the cells are broken, preserving the heat in a water bath at 65 ℃ for 1h, and reversing and mixing the mixture once every 10min.
3. 13200rpm, centrifuge for 5min, absorb 800ul of supernatant into a new centrifuge tube, add equal volume of chloroform/phenol (1:1), mix by gently inverting 3-4 times.
4. 13200rpm, centrifuge for 10min, transfer supernatant 700ul into a new centrifuge tube, add equal volume of chloroform (precool), mix by gentle inversion.
5. 13200rpm, centrifuge for 5min, transfer the supernatant 600ul into a new centrifuge tube, add equal volume of chloroform (precool), mix by gentle inversion.
6. 13200rpm, centrifuging for 5min, transferring supernatant 500ul into a new centrifuge tube, adding 0.6-0.8 times of isopropanol, slightly inverting and mixing, standing at-20 deg.C for 10min, centrifuging at 13200rpm for 10min, and removing supernatant.
7. 700ul of 75% ethanol was added, 13200rpm was applied, and centrifugation was performed for 1min, and repeated 2 times.
8. Removing supernatant, naturally drying, and dissolving in 30-50ul ddH2And O for later use.
1.6.2.2 GUS gene PCR detection
And (4) taking a GUS dye solution to detect positive plants, and carrying out PCR detection on GUS genes. 10 uL of PCR system contained 1.0 uL of DNA, 0.2 uL of each primer, 5.0ul of rTaq, and 3.6 uL of ddH2O3. Reaction conditions are as follows: 94 ℃ for 3 min; 30s at 94 ℃; at 54 ℃ for 30 s; 72 ℃ for 1 min; 35 cycles; extending for 5min at 72 ℃; storing at 12 deg.C. After the PCR reaction, 5ul of the amplified product was electrophoresed in 1% agarose gel, observed under a gel imaging system and photographed. The results show that PCR detection of GUS gene is carried out on 140 eucommia ulmoides plants which are positive to GUS dye liquor detection, 81 eucommia ulmoides plants are detected to be integrated into the eucommia ulmoides genome, and the transgenic plants are identified to account for 57.86% of the positive plants. As shown in fig. 4.
Claims (10)
1. A method for genetic transformation of eucommia ulmoides by injection comprises the following steps:
(1) preparation of genetic transformation receptors: selecting eucommia seedlings germinating for 2-4 days, and removing a first true leaf between two cotyledons to serve as a genetic transformation receptor for later use;
(2) and (3) culturing agrobacterium: transforming the agrobacterium tumefaciens strain LBA4404 by the vector with the target gene, inoculating the agrobacterium tumefaciens strain LBA4404 to a solid YEP culture medium, and culturing for 2d in an incubator at 28 ℃; picking single colony to 5ml liquid YEP culture medium, shaking and culturing at 28 deg.C and 180rpm for 12 hr, adding 100ul into 100ml liquid YEP culture medium, shaking and culturing at 28 deg.C and 180rpm to suitable value for use; 100mg/L kanamycin (Kan) and 100mg/L rifampicin (Rif) on the YEP medium;
(3) preparation of agrobacterium tumefaciens resuspension: subpackaging the cultured agrobacterium liquid into a 50ml centrifugal tube, centrifuging at 5000rpm for 10min, discarding the supernatant, and resuspending the thallus precipitate with a resuspension solution for later use;
(4) genetic transformation of eucommia ulmoides: adding Acetosyringone (AS) and a surfactant Silwet-L77 into the agrobacterium tumefaciens heavy suspension liquid, injecting 100 mu L of the agrobacterium tumefaciens heavy suspension liquid into a space between two eucommia ulmoides cotyledons by using an injector, and culturing in the dark at 28 ℃ for 3 d;
(5) management of transgenic plants
And (4) canceling dark culture of the dark cultured plants, transferring the survived plants to a greenhouse of a transgenic plant demonstration base after one week, and performing conventional fertilizer and water management.
2. The method of claim 1, wherein the eucommia ulmoides seedling is a eucommia ulmoides seedling that germinates for 3 days.
3. The method according to claim 2, wherein the step (1) is a method for obtaining eucommia ulmoides seedling: selecting seeds with good growth vigor, washing the seeds with water for three times, soaking the seeds in the water, putting the seeds into a constant-temperature incubator at 37 ℃ for overnight, soaking the seeds in 400mg/L gibberellin for 5 to 6 hours, taking the seeds out, washing the seeds with clear water, sowing the seeds in humid nutrient soil, and germinating and sprouting the seeds for 10 to 15 days.
4. The method of claim 1, wherein the removing is with forceps removal.
5. The method of claim 1, the resuspension fluid formulation: MS 4.43g/L, sucrose 30g/L, 6-BA 3. mu.M, 2-iP 3. mu.M, and adjusting pH to 5.8-6.0.
6. The method of claim 5, wherein 100ul acetosyringone with a concentration of 20mg/ml and 500ul surfactant Silwet-L77 are added to the Agrobacterium re-suspension liquid of step (4).
7. The method of claim 1, wherein the agrobacterium resuspension has a concentration OD600 of 0.8.
8. The method of claim 1, wherein the vector comprises a GUS gene linked thereto.
9. The method according to claim 8, comprising a transgenic plant identification step after step (5), said identification comprising GUS chemical tissue staining detection of resistant plants and PCR detection of GUS gene.
10. The method of claim 9, wherein the GUS chemical tissue staining of the resistant plant is detected by: after one month of management according to the step (5), cutting leaves newly grown from eucommia ulmoides seedlings, putting the leaves into a PCR tube, and adding GUS staining solution; incubating the strain in an incubator at 37 ℃, adding 75% ethanol for elution until chlorophyll is completely removed, and photographing for observation;
PCR detection of the transgenic plant: and (3) detecting plants with positive leaves by using GUS dye solution, extracting DNA by using a CTAB method, and carrying out further PCR detection.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112931227A (en) * | 2021-04-26 | 2021-06-11 | 北京林业大学 | Method for whole plant induction plant regeneration of each part of eucommia ulmoides and construction of transgenic plant regeneration system |
| CN113122569A (en) * | 2021-04-26 | 2021-07-16 | 北京林业大学 | Construction method of eucommia transgenic plant regeneration system |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070033678A1 (en) * | 2005-07-27 | 2007-02-08 | J.R. Simplot Company | Precise cutting |
| CN1948480A (en) * | 2006-11-15 | 2007-04-18 | 贵州大学 | Agricultural bacillus introduced eucommia trangenic method |
| CN104561089A (en) * | 2014-12-26 | 2015-04-29 | 淮北师范大学 | Culture method of genetically modified muskmelon tissue culture seedlings and application |
| CN105132457A (en) * | 2015-10-19 | 2015-12-09 | 宁夏农林科学院 | Method for conducting rapid genetic transformation of alfalfa |
| CN110656129A (en) * | 2019-11-05 | 2020-01-07 | 贵州大学 | Genetic transformation method of agrobacterium-mediated coix lacryma-jobi |
-
2020
- 2020-12-21 CN CN202011516536.0A patent/CN112501211B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070033678A1 (en) * | 2005-07-27 | 2007-02-08 | J.R. Simplot Company | Precise cutting |
| CN1948480A (en) * | 2006-11-15 | 2007-04-18 | 贵州大学 | Agricultural bacillus introduced eucommia trangenic method |
| CN104561089A (en) * | 2014-12-26 | 2015-04-29 | 淮北师范大学 | Culture method of genetically modified muskmelon tissue culture seedlings and application |
| CN105132457A (en) * | 2015-10-19 | 2015-12-09 | 宁夏农林科学院 | Method for conducting rapid genetic transformation of alfalfa |
| CN110656129A (en) * | 2019-11-05 | 2020-01-07 | 贵州大学 | Genetic transformation method of agrobacterium-mediated coix lacryma-jobi |
Non-Patent Citations (1)
| Title |
|---|
| 王超: "杜仲直接转化法研究", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112931227A (en) * | 2021-04-26 | 2021-06-11 | 北京林业大学 | Method for whole plant induction plant regeneration of each part of eucommia ulmoides and construction of transgenic plant regeneration system |
| CN113122569A (en) * | 2021-04-26 | 2021-07-16 | 北京林业大学 | Construction method of eucommia transgenic plant regeneration system |
| CN113122569B (en) * | 2021-04-26 | 2022-07-08 | 北京林业大学 | Construction method of eucommia transgenic plant regeneration system |
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