CN112501211B - A method for genetic transformation of Eucommia by injection - Google Patents

Abstract

The invention relates to a method for transforming eucommia ulmoides by 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: preparation of genetic transformation receptor (1), culture of agrobacterium (2), preparation of agrobacterium resuspension (4), genetic transformation of eucommia (5), management of transgenic plant (6) and identification of transgenic plant. 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

A method for genetically transforming Eucommia by injection

technical field

The technical field of the invention is plant biotechnology, and specifically relates to a method for genetically transforming eucommia by injection.

Background technique

Eucommia ulmoides Oliver (Eucommia ulmoides Oliver) is a deciduous tree belonging to Eucommia ulmoides. In recent years, in the genetic transformation technology of Eucommia mediated by Agrobacterium, tissue culture technology is the main method, but there are factors such as low efficiency of callus induction differentiation, rooting of resistant shoots, and low survival rate of transgenic plants transplanted, resulting in Eucommia This type of research is relatively lagging behind, and it is difficult to obtain larger surviving plants.

The Agrobacterium injection method is a non-tissue culture genetic transformation method in which the genetically transformed recipient is injected with an Agrobacterium suspension in an open environment. The genetic transformation of the whole plant can be carried out directly on the plant, and there are no problems such as induction of differentiation, rooting of resistant shoots and later transplanting, and the survival of positive seedlings can be better guaranteed. In the present invention, the Eucommia ulmoides seedlings that have germinated for three days are genetically transformed by pulling out the first true leaf between two cotyledons. The Agrobacterium injection method has the advantages of simple operation, low cost, ability to transform large fragments of DNA, stable genetics, and high copy number. low merit. At present, the genetic transformation system of Agrobacterium injection has been widely used in dicotyledonous plants such as soybean and litchi, but the application in Eucommia has not been clearly reported, which restricts the research on Eucommia genomics and gene function.

Contents of the invention

The invention utilizes the genetic transformation technology of injecting eucommia seedlings with Agrobacterium to obtain transgenic plants, and provides technical support for the study of eucommia genomics and gene function.

A method for genetically transforming Eucommia by injection, comprising the following steps:

(1) Preparation of the genetic transformation receptor: select Eucommia ulmoides seedlings germinated for 2-4 days, and remove the first true leaf between the two cotyledons as the genetic transformation receptor for subsequent use;

(2) Cultivation of Agrobacterium: After transforming the vector with the target gene into Agrobacterium tumefaciens strain LBA4404, inoculate it on a solid YEP medium, and cultivate it in a 28°C incubator for 2 days; pick a single colony into 5ml liquid YEP medium, After 12 hours of shaking culture at 28°C and 180rpm, 100ul was added to 100ml liquid YEP medium, and cultured with shaking at 28°C and 180rpm until suitable for later use; the YEP medium contained 100mg/L kanamycin (Kan ) and 100mg/L rifampicin (Rif);

(3) Preparation of Agrobacterium resuspension: subpack the cultured Agrobacterium bacterium into 50ml centrifuge tubes, centrifuge at 5000rpm for 10min, discard the supernatant, and resuspend the thalline with the resuspension to be ready for use;

(4) Genetic transformation of Eucommia ulmoides: add acetosyringone (AS) and surfactant Silwet-L77 to the resuspension of Agrobacterium, inject 100 μL of the resuspension of Agrobacterium into between two cotyledons of Eucommia with a syringe, Incubate in the dark for 3 days at 28°C;

(5) Management of transgenic plants

The dark cultivation was cancelled, and the surviving plants were moved to the greenhouse of the transgenic plant demonstration base after one week, and the conventional fertilizer and water management was carried out.

The Eucommia seedlings are Eucommia seedlings germinated for 3 days.

The step (1) method for obtaining eucommia seedlings: select seeds with good growth vigor, wash the seeds with water three times, then soak the seeds in water and put them in a constant temperature incubator at 37°C overnight, then soak them with 400mg/L gibberellin for 5 -6 hours, take it out and wash it with water, sow it in moist nutrient soil, and germinate in 10-15 days.

The removal is extraction with surgical forceps.

The resuspension formula: MS 4.43g/L, sucrose 30g/L, 6-BA 3μM, 2-iP 3μM, adjust the pH to 5.8-6.0.

The YEP medium formula: yeast extract 10g/L, peptone 10g/L, sodium chloride 5g/L, pH 7.2, agar 15g/L added to the solid YEP medium.

Add 100 ul of acetosyringone with a concentration of 20 mg/ml to the Agrobacterium resuspension liquid in the step (4), and the concentration of the surfactant Silwet-L77 is 500 ul/L.

The concentration OD600 of the Agrobacterium suspension was 0.8.

The GUS gene is connected in the vector.

After the step (5), a transgenic plant identification step is included, and the identification includes GUS chemical histostaining detection of resistant plants and PCR detection of GUS gene.

GUS chemical tissue staining detection of resistant plants: After one month of management according to step (5), cut the newly grown leaves of Eucommia ulmoides seedlings and put them in a PCR tube, add GUS staining solution; put them in an incubator at 37°C Incubate, add 75% ethanol to elute until the chlorophyll is completely removed, and take pictures for observation.

In order to take transgenic leaves of Eucommia ulmoides and cut them into 1cm-2cm lengths, put them in PCR tubes, add 200ul of GUS staining solution to the PCR tubes, treat them in a vacuum at 15Kpa for 10min, and place them in a constant temperature incubator at 37°C for overnight reaction. Aspirate the dye solution and decolorize with 75% ethanol until the chlorophyll disappears.

PCR detection of transgenic plants: GUS staining solution was taken to detect positive leaves of plants, DNA was extracted by CTAB method, and further PCR detection was carried out.

PCR of the GUS gene: detection of 10 μL of the PCR system contains 1.0 μL of DNA, 0.2 μL of each primer, 5.0 ul of rTaq, and 3.6 μL of ddH2O. Reaction conditions: 94°C, 3min; 94°C, 30s; 54°C, 30s; 72°C, 1min; 35 cycles; extension at 72°C for 5min; storage at 12°C. After the PCR reaction is completed, 5 ul of the amplified product is electrophoresed in 1% agarose gel electrophoresis, observed and photographed under a gel imaging system.

In the present invention, the three-day-germinated Eucommia seedlings are used as genetic transformation recipients by pulling out the first true leaf between the two cotyledons, and the Agrobacterium suspension, acetosyringone and surface The active agent Silwet-L77 was dipped at different bacterial concentrations, and then cultured in the dark for 3 days. After new leaves were grown, GUS chemical tissue staining and PCR were used to identify the transgenic plants. The process studied the effect of different bacterial concentrations on Eucommia seedlings. Finally, the genetic transformation method and optimal transformation conditions of Agrobacterium-injected Eucommia seedlings were obtained. The method of the invention does not require a tissue culture process, and has high genetic transformation efficiency.

The invention utilizes the genetic transformation method of injecting eucommia seedlings with Agrobacterium to obtain transgenic plants, and provides technical support for the study of eucommia genomics and gene function.

Description of drawings

Figure 1 Preparation and injection of Eucommia seedling recipients.

Among them, A: Eucommia seedlings germinated for three days; B: Eucommia seedlings with the first true leaf pulled out; C: Eucommia seedlings injected with Agrobacterium suspension.

Fig. 2 Plants after genetic transformation.

Among them, A: the plant 15 days after genetic transformation; B: the transgenic plant one month after transplanting.

Figure 3 GUS staining of leaves of Eucommia ulmoides.

Among them, A: GUS staining does not appear blue wild-type Eucommia plant leaves; B, C, D: GUS staining blue transgenic Eucommia plant leaves.

Fig. 4 GUS gene PCR results.

Among them, M: DL2000 marker; WT: wild Eucommia plants; 1-7 are transgenic Eucommia plants

Detailed ways

The present invention will be described in further detail below in conjunction with embodiment.

The biological materials used in the following are all commercially available, and are preserved in the applicant's laboratory, and can be released to the public.

The resuspension formula: MS 4.43g/L, sucrose 30g/L, 6-BA 3μM, 2-iP 3μM, adjust the pH to 5.8-6.0.

The YEP medium formula: yeast extract 10g/L, peptone 10g/L, sodium chloride 5g/L, pH 7.2, agar 15g/L added to the solid YEP medium.

Embodiment 1 A kind of genetic transformation technology utilizing Agrobacterium to inject Eucommia seedlings

1.1 Genetic recipient preparation: The Eucommia ulmoides seedlings germinated three days ago, and the first true leaf between the two cotyledons was removed by surgical forceps as the genetic transformation recipient for future use. See Figure 1.

1.2 Cultivation of Agrobacterium: Agrobacterium tumefaciens strain LBA4404, the vector pCAMBIA1300 carrying the hygromycin resistance gene Hyg and GUS gene was inoculated into the culture medium containing 100 mg/mL kanamycin (Kan) and 100 mg/mL rifampicin (Rif) On the YEP solid medium, culture in 28℃ incubator for 2 days; pick a single colony and put it into 5ml YEP (containing 100mg/mL Kan and 100mg/mLRif) liquid medium, 28℃, 180rpm shaking culture for 12 hours, take 100ul Add to 100ml YEP (containing 100mg/mLKan and 100mg/mL Rif) liquid medium, shake at 28°C and 180rpm until suitable, and prepare for use.

1.3 Preparation of Agrobacterium resuspension: Divide the cultured Agrobacterium into 50ml centrifuge tubes, centrifuge at 5000rpm for 10min, discard the supernatant, resuspend the bacterial pellet with the resuspension, and prepare for use.

1.4 Genetic transformation of Eucommia ulmoides: Add acetosyringone (AS) and surfactant Silwet-L77 to the suspension of Agrobacterium ulmoides, inject 100 μL of Agrobacterium liquid into the two cotyledons of Eucommia ulmoides with a 1 mL syringe, 28 ℃, dark Culture 3d.

Table 1 Infection of Eucommia ulmoides seedlings under different bacterial concentration

1.5 Management of transgenic plants: cancel the dark cultivation for the dark cultivated plants, water every two days, and move the surviving plants to the greenhouse of the transgenic plant demonstration base after one week, and manage the conventional fertilizer and water. See Figure 2.

1.6 Identification of transgenic plants

1.6.1 GUS chemical tissue staining detection of resistant plants: take the transgenic leaves of Eucommia ulmoides and cut them into 1cm-2cm lengths, put them in a PCR tube, add 200ul of GUS staining solution to the PCR tube, and vacuum treatment at 15Kpa for 10min. Incubate overnight in a 37°C incubator. Aspirate the dye solution, add 1ml of 75% ethanol to decolorize until the chlorophyll disappears, and take pictures for observation. The results showed that in Table 1, a total of 2400 Eucommia plants were treated, and 140 positive plants were identified by GUS staining. Specifically: the OD ₆₀₀ values of the Agrobacterium bacterial liquid were 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, and 2.0; the survival rate and GUS staining rate were 77.33%, 77.67%, 66.00%, 58.33%, and 54.33% respectively , 67.33%, 53.00%, 62.00% and 1.33%, 10.00%, 2.67%, 3.33%, 7.00%, 1.70%, 11.00%, 9.60%; as can be seen from the table, when the optimal transformation condition OD ₆₀₀ value is 0.8, the plant The survival rate was 77.67%, and the GUS staining rate was 6.67%. See Figure 3.

1.6.2 PCR detection of transgenic plants

1.6.2.1 DNA extraction by CTAB method:

1. Add 1ml of 1.2% CTAB extraction buffer to a 2ml centrifuge tube, and add 20ul of β-mercaptoethanol, mix well and preheat at 65°C.

2. Take 0.1-0.2g of fresh leaves, grind until the cells are broken, and keep it in a water bath at 65°C for 1 hour, during which time it is inverted and mixed every 10 minutes.

3. Centrifuge at 13200rpm for 5min, pipette 800ul supernatant into a new centrifuge tube, add an equal volume of chloroform/phenol (1:1), and gently invert and mix 3-4 times.

4. Centrifuge at 13200rpm for 10min., transfer 700ul of the supernatant to a new centrifuge tube, add an equal volume of chloroform (pre-cooled), and mix gently by inversion.

5. Centrifuge at 13200rpm for 5min, transfer 600ul of the supernatant to a new centrifuge tube, add an equal volume of chloroform (pre-cooled), and gently invert to mix.

6. Centrifuge at 13200rpm for 5min, transfer 500ul of the supernatant to a new centrifuge tube, add 0.6-0.8 times isopropanol, mix gently by inversion, place at -20°C for 10min, centrifuge at 13200rpm for 10min, remove the supernatant .

7. Add 700ul of 75% alcohol, centrifuge at 13200rpm for 1min, repeat twice.

8. After removing the supernatant, dry it naturally and dissolve it in 30-50ul ddH ₂ O for later use.

1.6.2.2 PCR detection of GUS gene

The plants that were positive in the GUS staining solution were taken, and the GUS gene was detected by PCR. A 10 μL PCR system contains 1.0 μL of DNA, 0.2 μL of each primer, 5.0 ul of rTaq, and 3.6 μL of ddH2O. Reaction conditions: 94°C, 3min; 94°C, 30s; 54°C, 30s; 72°C, 1min; 35 cycles; extension at 72°C for 5min; storage at 12°C. After the PCR reaction is completed, 5 ul of the amplified product is electrophoresed in 1% agarose gel electrophoresis, observed and photographed under a gel imaging system. The results showed that 140 Eucommia ulmoides plants that were positive in the GUS staining solution were detected by PCR for the GUS gene. A total of 81 plants were detected as GUS gene integrated into the Eucommia genome, and the identified transgenic plants accounted for 57.86% of the positive plants. See Figure 4.

Claims (7)

1. A method utilizing injection genetic transformation Eucommia, comprising the following steps: (1) Preparation of the genetic transformation receptor: select Eucommia ulmoides seedlings germinated for 2-4 days, and remove the first true leaf between the two cotyledons as the genetic transformation receptor for subsequent use; (2) Cultivation of Agrobacterium: After transforming the vector with the target gene into Agrobacterium tumefaciens strain LBA4404, inoculate it on a solid YEP medium, and cultivate it in a 28°C incubator for 2 days; pick a single colony into 5ml liquid YEP medium, After 12 hours of shaking culture at 28°C and 180rpm, 100ul was added to 100ml liquid YEP medium, and cultured with shaking at 28°C and 180rpm until suitable for later use; the YEP medium contained 100mg/L kanamycin (Kan ) and 100mg/L rifampicin (Rif); (3) Preparation of Agrobacterium resuspension: subpack the cultured Agrobacterium bacterium into 50ml centrifuge tubes, centrifuge at 5000rpm for 10min, discard the supernatant, and resuspend the thalline with the resuspension to be ready for use; (4) Genetic transformation of Eucommia ulmoides: add acetosyringone (AS) and surfactant Silwet-L77 to the resuspension of Agrobacterium, inject 100 μL of the resuspension of Agrobacterium into between two cotyledons of Eucommia with a syringe, Incubate in the dark for 3 days at 28°C; (5) Management of transgenic plants For the dark cultured plants, cancel the dark culture, and move the surviving plants to the greenhouse of the transgenic plant demonstration base one week later, and manage the conventional fertilizer and water; The resuspension formula: MS 4.43g/L, sucrose 30g/L, 6-BA 3μM, 2-iP 3μM, adjust the pH to 5.8-6.0; Acetosyringone 100ul with a concentration of 20mg/ml was added to the Agrobacterium resuspension liquid in the step (4), and the concentration of the surfactant Silwet-L77 was 500ul/L; The concentration OD600 of the Agrobacterium suspension was 0.8. 2. The method according to claim 1, said eucommia seedlings are eucommia ulmoides seedlings germinated for 3 days. 3. the method according to claim 2, described step (1) eucommia seedling obtaining method: choose the good seed of growth vigor, wash the seed three times with water, then soak the seed in water and put into 37 ℃ constant temperature incubator overnight, Then soak with 400mg/L gibberellin for 5-6 hours, take it out, wash it with water, sow it in moist nutrient soil, and germinate in 10-15 days. 4. The method of claim 1, wherein the removal is extraction with surgical forceps. 5. The method according to claim 1, wherein the GUS gene is linked in the vector. 6. The method according to claim 5, after said step (5), comprising a transgenic plant identification step, said identification comprising GUS chemical histostaining detection of resistant plants and PCR detection of GUS gene. 7. The method according to claim 6, the GUS chemical histostaining detection of the resistant plant: after one month of management by step (5), the newly grown blades of Eucommia ulmoides seedlings are clipped and put into the PCR tube, and GUS is added Staining solution; incubate it in an incubator at 37°C, add 75% ethanol to elute until the chlorophyll is completely removed, and take pictures for observation; The PCR detection of the transgenic plants: take the plants whose leaves are positive with the GUS staining solution, and use the CTAB method to extract DNA for further PCR detection.

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