Agrobacterium rhizogenes-mediated leek genetic transformation method

Abstract

The invention discloses an agrobacterium rhizogenes-mediated rapid genetic transformation method for Chinese chives. The method mainly comprises the following steps: (1) culturing Chinese chives seedlings; (2) preparing a recombinant agrobacterium rhizogenes bacterial suspension with a target gene; (3) cutting roots, soaking and infecting; (4) inducing transgenic hairy roots; and (5) identifying and obtaining the compound transgenic leek plant. According to the method, a stable and efficient leek genetic transformation system is established through a direct soaking method, leek seedlings are adopted as explants, a composite transgenic line containing transgenic roots can be rapidly obtained without culture of calluses, the transformation period is greatly shortened, and the problems existing in an existing agrobacterium tumefaciens mediation method are solved; the method is simple to operate, does not need sterile environment operation, is low in cost and short in time, can quickly form a stable genetic Chinese chive compound transgenic plant, and provides a low-cost and easily available compound gene plant model and a construction method for Chinese chive functional gene verification and Chinese chive root development research.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering and genetic breeding, and more specifically relates to a method for genetic transformation of leek mediated by Agrobacterium rhizogenes. Background Art

[0002] Leek (Allium tuberosum Rottler ex Sprengle), also known as grass stalactite and yang grass, is a perennial root plant of the genus Allium (Allium L.) of the Amaryllis family native to my country. It is mainly edible with leaves, pseudostems and flower stalks. Leek contains protein, fat, cellulose, carotene, vitamin C, trace elements and various organic sulfur compounds. Modern medical research shows that leek has the effects of antibacterial and anti-inflammatory, lowering blood lipids, preventing coronary heart disease and hypertension, and improving the body's immune ability, and has high medical and health care value. However, the lack of effective stress resistance, disease resistance and insect resistance genes in the natural population of leek seriously restricts the improvement of leek yield and quality. The mining and functional verification of important leek genes are crucial to leek biological breeding, but the lack of a fast and effective genetic transformation system has greatly limited the relevant research on important functional genes of leek. Therefore, it is crucial to establish a stable, efficient and easy-to-operate leek genetic transformation system for the study of gene function and biological breeding of leek.

[0003] At present, most methods for genetic transformation of plants mainly include: (1) Gene gun method, which uses gene gun to directly introduce foreign DNA into plant cells. (2) Agrobacterium-mediated method, which uses Agrobacterium tumefaciens to carry foreign genes and infect leek callus to achieve genetic transformation. (3) Microinjection method, which directly injects foreign DNA into plant cells. (3) Electroporation method, which promotes the introduction of foreign DNA by treating plant cells with electric shock. Among them, the Agrobacterium-mediated method is relatively simple to operate and suitable for large-scale transformation, but it may be limited by the plant genotype; while the microinjection method and electroporation method have higher transformation efficiency, but the operation is complicated and the cost is high.

[0004] So far, the only methods that have successfully achieved genetic transformation in leek and obtained transformed plants are the gene gun method and the Agrobacterium-mediated method. Due to the limitations of the leek genotype, the leek's weak regeneration ability, unstable genetic transformation, and extremely low transformation efficiency, the genetic transformation of leek has always been a difficult problem that needs to be overcome.

[0005] The Agrobacterium tumefaciens-mediated leek genetic transformation system established in the prior art (Establishment of the leek genetic transformation system mediated by Agrobacterium tumefaciens, Zhang Song, 2003) uses the GUS gene as a reporter gene and cultivates regenerated plants using the callus tissue of sterile seedlings. Its transformation cycle is long and must be carried out under sterile conditions, which is time-consuming and labor-intensive, difficult to operate, and costly. The use of this method for leek genetic transformation has the problems of low transformation efficiency and weak repeatability. At the same time, existing studies have basically carried out genetic transformation through leek callus tissue, and all have the problems of unstable transformation efficiency, high contamination rate and long cycle. At present, there are still few methods for establishing new leek genetic transformation systems. In order to obtain more leek genetic transformation systems, solve the defects of the existing Agrobacterium tumefaciens-mediated leek genetic transformation methods, and overcome the problem of using callus tissue for genetic transformation, it is necessary to provide more low-cost, easy-to-obtain, high-transformation efficiency, short-time leek genetic transformation methods and composite gene plant models. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the defects of the existing genetic transformation method of leek using Agrobacterium tumefaciens-mediated callus tissue, such as harsh conditions, too long transformation cycle, low efficiency, great operation difficulty, high cost, etc., as well as the shortcomings of the leek genetic transformation method, and to provide a genetic transformation method of leek mediated by Agrobacterium tumefaciens.

[0007] The invention aims to provide a method for genetic transformation of leek mediated by Agrobacterium rhizogenes.

[0008] Another object of the present invention is to provide an application of a method for genetic transformation of leek mediated by Agrobacterium rhizogenes.

[0009] Another object of the present invention is to provide a composite transgenic plant.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] The present invention provides a method for genetic transformation of leek mediated by Agrobacterium rhizogenes, comprising the following steps:

[0012] (1) The explants are leek seedlings;

[0013] (2) Transform the target gene into Agrobacterium rhizogenes to prepare a suspension of Agrobacterium rhizogenes. The OD of the suspension is 600 The value is 0.8~1.2;

[0014] (3) Cut the roots of leek seedlings and soak them in a suspension of Agrobacterium rhizogenes for 10 to 20 minutes;

[0015] (4) dark culturing the infected leek seedlings for 1 to 2 days, and then continuing to cultivate them after transplanting;

[0016] (5) After identifying the new roots of the leek seedlings, positive leek plants can be obtained.

[0017] The present invention adopts a recombinant Agrobacterium rhizogenes suspension, establishes a stable and efficient leek genetic transformation system by a direct immersion method, and obtains a composite transgenic strain containing transgenic roots. The method provided by the present invention adopts leek seedlings as explants, does not require callus tissue for cultivation, does not require operation under a sterile environment, and directly cuts the roots of leek seedlings for immersion, greatly shortens the transformation time, and can efficiently induce transgenic hairy roots. It overcomes the defects of harsh conditions, too long transformation cycle, low efficiency, high operation difficulty, and high cost in the genetic transformation method using Agrobacterium tumefaciens-mediated callus tissue. The method provided by the present invention is simple to operate, low in cost, short in cycle, can quickly form a stable and inherited leek composite transgenic plant, greatly reduces the operating cost and difficulty of the leek genetic system, can be widely promoted and applied, and provides more new plant models for leek genetic improvement and gene function research.

[0018] Furthermore, in step (1), the leek seeds are sterilized and then germinated to obtain leek seedlings. Germination conditions: 25-28°C, 12-16h light / 8-12h dark.

[0019] Preferably, the germination conditions are 25°C, 16h light / 8h dark.

[0020] In particular, based on the method provided by the present invention, other Agrobacterium rhizogenes strains commonly used in the art can also be used to carry out genetic transformation of leek seedlings.

[0021] Preferably, the Agrobacterium rhizogenes strain in step (2) is the cucumopine type Agrobacterium rhizogenes K599, which contains a cucumopine type Ri plasmid and has streptomycin resistance.

[0022] Preferably, the OD of the Agrobacterium rhizogenes suspension in step (2) is 600 The value is 1.0.

[0023] Furthermore, in step (2), the Agrobacterium rhizogenes is cultured in LB liquid culture medium, and the culture medium also contains 50-100 mg / L kanamycin and 50-100 mg / L streptomycin.

[0024] Preferably, the LB liquid culture medium also contains 50 mg / L kanamycin and 50 mg / L streptomycin.

[0025] Furthermore, in step (2), an MS solution containing 50-100 μM acetosyringone and 3-5% sucrose is used to prepare the Agrobacterium rhizogenes suspension.

[0026] Preferably, the Agrobacterium rhizogenes suspension is prepared using a MS solution containing 100 μM acetosyringone and 3% sucrose.

[0027] Preferably, the leek seedlings are 10 days old.

[0028] Furthermore, the conditions for continuing the culture in step (4) are: 24-28° C., 16 h light / 8 h dark environment.

[0029] Furthermore, in step (5), the hairy roots are stained and identified by PCR.

[0030] As a more preferred embodiment, the present invention provides a specific method for genetic transformation of leek mediated by Agrobacterium rhizogenes:

[0031] (1) Culture of leek seedlings: Select plump leek seeds, soak them in 75% ethanol for 1 min, and then sterilize them with 5% sodium hypochlorite solution for 1 min; place the seeds in a culture dish covered with two layers of moist gauze or filter paper for culture, and culture them at 25° C., 16 h light / 8 h dark conditions for 10 days to obtain leek seedlings;

[0032] (2) Preparation of Agrobacterium rhizogenes suspension: The target gene plasmid is 1304 vector, which has kanamycin resistance and contains GUS reporter gene. The Agrobacterium strain is cucumber alkaline type Agrobacterium rhizogenes K599, which has streptomycin resistance; the target gene plasmid is transferred into K599 competent cells by freeze-thaw method; the Agrobacterium rhizogenes strain K599 containing the target gene (such as containing 1304 vector) is inoculated into 3mL LB liquid culture medium (containing 50mg / L kanamycin and 50mg / L streptomycin), and cultured at 28℃ and 200rpm for 24h; 1mL of the cultured bacterial solution is centrifuged at 10000rpm for 10min at room temperature, and resuspended in MS solution (containing 100μM acetosyringone and 3% sucrose), and the OD of the bacterial suspension is 600 The value is adjusted to 1.0;

[0033] (3) Select 10-day-old leek seedlings with good growth status as explants, remove the old roots of leek, and soak the explants in K599 bacterial suspension for 10 min;

[0034] (4) The infected leek explants were placed in a culture dish covered with two layers of moist gauze or filter paper (humidity controlled at 60-80%) and cultured in the dark for 2 days, and then cultured under the conditions of 25°C, 16 h light / 8 h dark; the middle leek hairy roots were selected within 14-21 days to detect the expression of the GUS gene; at the same time, the GUS gene was identified by PCR to obtain positive leek plants.

[0035] The present invention provides application of the method in constructing a composite transgenic leek strain.

[0036] The present invention also provides a composite transgenic leek strain, which is constructed by adopting the above method.

[0037] The present invention has the following beneficial effects:

[0038] The invention provides a method for establishing a genetic transformation system of leek mediated by Agrobacterium rhizogenes, wherein the root-cut leek seedlings are soaked in a bacterial solution of Agrobacterium rhizogenes K599 (containing a target gene) to obtain a composite transgenic plant containing transgenic roots. First, the soaking method is simple and convenient to operate, and multiple leek plants can be infected simultaneously; second, the experimental operation does not require aseptic conditions, which is more time-saving and labor-saving than traditional tissue culture, and the required equipment is simple, cost-saving, and easy to promote in production practice; and the experimental cycle is short, and the genetic transformation system cycle is 26-33d, which effectively shortens the cycle of obtaining transgenic leek. The method greatly reduces the operating cost and difficulty of the leek genetic system, and provides a low-cost, easily obtainable composite gene plant model for the verification of leek functional genes and the study of leek root system development. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a flow chart of genetic transformation of leek mediated by Agrobacterium rhizogenes (Figure A is a schematic diagram of the growth of leek seedlings in a culture dish; Figure B is a schematic diagram of root cutting of leek explants; Figure C is a schematic diagram of immersion infection of leek by Agrobacterium rhizogenes; Figure D is a schematic diagram of the growth of leek with hairy roots obtained 14 days after infection. The hairy roots are in the red box).

[0040] Figure 2 This is the result of GUS staining of leek hairy roots (the blue roots in the red frame in the figure are transgenic roots).

[0041] Figure 3 This is a PCR validation diagram of the GUS gene in leek hairy roots (in the figure, "+" is the 1304 plasmid positive control; "-" is the leek root genomic DNA negative control; M is the DNA 2000 marker; 1-8 are the PCR results of the hairy root genomic DNA). DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0043] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0044] Example 1 A method for genetic transformation of leek mediated by Agrobacterium rhizogenes

[0045] 1. Leek seedling cultivation

[0046] Select plump leek seeds, soak them in 75% ethanol for 1 minute, and then sterilize them with 5% sodium hypochlorite solution for 1 minute. Place the seeds in a culture dish covered with two layers of moist gauze or filter paper for cultivation at 25°C, 16h light / 8h dark conditions for 10 days. During this period, add water to keep them moist.

[0047] 2. Preparation of Agrobacterium rhizogenes suspension

[0048] (1) Transformation of Agrobacterium rhizogenes: The 1304 vector plasmid with the GUS marker was transformed into K599 competent cells by the freeze-thaw method. Take 10 μL of the 1304 vector plasmid and add it to 100 μL of Agrobacterium rhizogenes K599 competent cells. After mixing, immediately place it on ice for 30 minutes, treat it with liquid nitrogen for 5 minutes, heat shock it in a 37°C water bath for 5 minutes, add 1 mL of LB liquid culture medium (containing 50 mg / L streptomycin), and culture it at 28°C and 200 rpm for 4 hours. Centrifuge the bacterial solution at 10,000 rpm for 2 minutes at room temperature, discard most of the supernatant, and retain 200 μL of the supernatant. Suspend the bacterial cells, spread them on LB solid culture medium (containing 50 mg / L kanamycin and 50 mg / L streptomycin), and culture them at 28°C for 2-3 days until a single colony grows.

[0049] (2) Identification of positive rhizogenes: Pick a single colony and culture it in 3 mL of LB liquid medium (containing 50 mg / L kanamycin and 50 mg / L streptomycin) for 24 h, and culture it in a shaking incubator at 28°C and 200 rpm for 24 h. Perform PCR identification on the bacterial solution, and the primer sequences used are as follows:

[0050] GUS-F: 5'-GAATTGATCAGCGTTGGTGGG-3';

[0051] GUS-R: 5'-CGATGGATTCCGGCATAGTTA-3';

[0052] The PCR reaction system was: 6.5 μL 2×M5 HiPer plus Taq HiFi PCR Mix (Beijing Polymer Biotechnology Co., Ltd.), 1 μL forward primer, 1 μL reverse primer, 1 μL bacterial solution, 3 μL ddH2O; the reaction program was 95°C for 3 min, 95°C for 30 s, 50°C for 30 s, 72°C for 40 s, 33 cycles, 72°C for 10 min. The size of the PCR amplified fragment was 430 bp, and the obtained K599-1304 positive bacterial solution was stored in a -80°C refrigerator.

[0053] (3) Preparation of bacterial suspension: Take 30 μL of K599-1304 positive bacterial solution and inoculate it into 3 mL of LB liquid culture medium (containing 50 mg / L kanamycin and 50 mg / L streptomycin), and culture it at 28°C, 200 rpm and shaking for 24 h. Take 1 mL of K599-1304 positive bacterial solution and inoculate it into 100 mL of LB liquid culture medium (containing 50 mg / L kanamycin and 50 mg / L streptomycin), and culture it at 28°C, 200 rpm and shaking for 24 h. Centrifuge the bacterial solution at 10,000 rpm for 10 min at room temperature, discard the supernatant, resuspend the bacterial solution in 50 mL of MS solution (containing 100 μM acetosyringone and 3% sucrose), and use a UV spectrophotometer to measure the OD value of the bacterial suspension. 600 Adjust the value to 1.0.

[0054] 3. Infection of leek seedling explants

[0055] Select 10-day-old leek seedlings that are in good growth condition, such as Figure 1 As shown in A, use a scalpel to remove the old roots of the leek. Figure 1 As shown in B, leek explants were immersed in a suspension of Agrobacterium rhizogenes (OD 600 =1.0), the soaking time is 10min, such as Figure 1 C. After soaking, carefully transfer the leek explants to a culture dish covered with two layers of moist gauze or filter paper and culture them in the dark for 2 days. Then transfer them to a plant culture room and culture them at 25°C, 16h light / 8h dark for 14-21 days. The newly grown underground roots are the transformed leek hairy roots, as shown in Figure 1 As shown in D.

[0056] Example 2 Identification of transgenic hairy roots

[0057] 1. GUS staining

[0058] Soak the leek with hairy roots in GUS staining solution and incubate overnight at 28°C. The formula of GUS staining solution is shown in Table 1, which needs to be prepared before use, and take photos of the GUS staining of the hairy roots.

[0059] Table 1 GUS staining solution formula

[0060]

[0061] The staining results are as follows Figure 2 As shown, the leek hairy roots are dyed blue, indicating that the exogenous GUS gene is integrated into the genome of the leek hairy roots, indicating that it is feasible to transfer the target gene into the leek root system using this genetic system.

[0062] 2. PCR amplification of GUS gene

[0063] The genomic DNA of leek hairy roots was extracted and PCR amplified for GUS gene. The GUS primers used were: GUS-F: 5'-GAATTGATCAGCGTTGGTGGG-3', GUS-R: 5'-CGATGGATTCCGGCATAGTTA-3'. The PCR reaction system was: 6.5μL 2×M5 HiPer plus Taq HiFi PCR Mix (Beijing Polymer Biotechnology Co., Ltd.), 1μL forward primer, 1μL reverse primer, 1μL bacterial solution, 3μL ddH2O; the reaction program was 95℃3min, 95℃30s, 50℃30s, 72℃40s, 33 cycles, 72℃10min, followed by electrophoresis detection.

[0064] The electrophoresis results are as follows Figure 3 As shown, the target bands were detected in 2 of the 8 hairy root materials, indicating that the GUS gene was successfully transformed into the hairy roots.

[0065] Example 3 Condition Optimization

[0066] 1. Agrobacterium rhizogenes concentration

[0067] In order to further improve the positive rate of hairy roots, the bacterial concentration (OD 600 The values ​​were set to 0.4, 0.6, 0.8, 1.0 and 1.2 respectively, and the remaining steps were exactly the same as in Example 1, and transformation and staining were performed respectively, and the number of induced hairy root plants, the number of positive plants, the hairy root induction rate and the positive rate were counted.

[0068] The specific results are shown in Table 2, which show that the concentration of Agrobacterium rhizogenes affects the induction of hairy roots. 600 =0.8~1.2, there are more positive strains after induction and the positive rate is higher.

[0069] Table 2 Effect of bacterial solution concentration on hairy root induction rate and positive rate

[0070] <![CDATA[Bacterial liquid concentration (OD 600 )]]> Total number of infected plants Number of induced hairy roots Number of positive strains Hairy root induction rate Positive rate 0.4 110 15 3 13.64％ 2.73％ 0.6 110 18 6 16.36％ 5.45％ 0.8 106 26 13 24.53％ 12.26％ 1.0 90 39 12 43.33％ 13.33％ 1.2 130 30 12 25.00％ 10.00％

[0071] 2. Explant infection time

[0072] The immersion infection time of the explants in Example 1 was set to 5 min, 10 min, 15 min and 20 min, respectively. The remaining steps were exactly the same as in Example 1. Transformation and staining were performed respectively, and the number of induced hairy root plants, the number of positive plants, the hairy root induction rate and the positive rate were counted.

[0073] The specific results are shown in Table 3. It can be seen from Table 3 that when the explant immersion time is 10 to 20 minutes, the positive rate of hairy roots is higher.

[0074] Table 3 Effect of immersion time on hairy root induction rate and positive rate

[0075] Soaking time (min) Total number of plants soaked Number of induced hairy roots Number of positive strains Hairy root induction rate Positive rate 5 112 20 7 17.86％ 6.25％ 10 104 28 13 26.92％ 12.50％ 15 102 24 12 23.53％ 11.76％ 20 100 18 10 18.00％ 10.00％

[0076] Comparative Example 1: Method for genetic transformation of leek mediated by Agrobacterium tumefaciens

[0077] The method of using Agrobacterium tumefaciens-mediated transformation of leek genes was carried out according to the existing technology: Zhang Song, Da Kedong, Jiang Luyan, et al. Establishment of the leek gene transformation system mediated by Agrobacterium tumefaciens [J]. Acta Horticulturae Sinica, 2003, 30(1): 4. DOI: 10.3321 / j.issn:0513-353X.2003.01.009.

[0078] Referring to the method of comparative example 1, leek gene transformation was carried out. The results showed that the success rate of transformation using Agrobacterium tumefaciens was extremely low and the transformation cycle was too long. The present invention spent nearly three months on the transformation according to the method of comparative example 1, but the transformation was not successful. The transformation efficiency of this method was very low and the repeatability was not strong.

[0079] Based on the data of successful transformation in the prior art, the transformation method mediated by Agrobacterium tumefaciens of Example 1 and Comparative Example 1 is further compared from the perspective of experimental operation difficulty and experimental cycle. As shown in Table 4, it can be seen that the method for genetic transformation of leek mediated by Agrobacterium rhizogenes of the present invention has low requirements for the operating environment, can directly use leek seedlings as explants for genetic transformation, can greatly shorten the transformation cycle, and the genetic transformation system cycle is 26-33d, which is at least half the time compared to Agrobacterium tumefaciens mediation. In addition, this method has the advantages of convenient operation, simple equipment requirements, and low environmental requirements. The experimental operation does not need to be carried out under sterile conditions. Compared with traditional tissue culture, it is more time-saving and labor-saving, and is easy to promote in production practice, providing a low-cost and easily accessible method for genetic transformation of leek for the verification of leek functional genes and the study of leek root system development.

[0080] Table 4 Comparison of operation difficulty and cycle of different genetic transformation methods

[0081] Method Type Operating Environment explant Experimental cycle Genetic transformation of Chinese chives mediated by Agrobacterium rhizogenes Non-sterile seedling 26-33d Genetic transformation of Chinese chives mediated by Agrobacterium tumefaciens Sterile Callus 73-78d

[0082] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for genetic transformation of leek mediated by Agrobacterium rhizogenes, characterized in that: The following steps are involved: (1) The explants are leek seedlings; (2) Transform the target gene into Agrobacterium rhizogenes to prepare a suspension of Agrobacterium rhizogenes. The OD of the suspension is 600 The value is 0.8~1.2; (3) Cut the roots of leek seedlings and soak them in a suspension of Agrobacterium rhizogenes for 10 to 20 minutes; (4) dark culturing the infected leek seedlings for 1 to 2 days, and then continuing to cultivate them after transplanting; (5) After identifying the new roots of the leek seedlings, positive leek plants can be obtained.

2. The method according to claim 1, characterized in that The leek seedlings in step (1) are 7-14 days old.

3. The method according to claim 1, characterized in that The Agrobacterium rhizogenes strain in step (2) is K599 strain.

4. The method according to claim 3, characterized in that In step (2), Agrobacterium rhizogenes is cultured in LB liquid culture medium containing 50-100 mg / L kanamycin and 50-100 mg / L streptomycin.

5. The method according to claim 4, characterized in that In step (2), an MS solution containing 50-100 μM acetosyringone and 3-5% sucrose is used to prepare the Agrobacterium rhizogenes suspension.

6. The method according to claim 1, characterized in that The humidity of the dark culture in step (3) is 60-80%.

7. The method according to claim 1, characterized in that The conditions for continuing the culture in step (4) are: 25-28° C., 12-16 h light / 8-12 h dark environment.

8. The method according to claim 1, characterized in that In step (5), the newly grown roots are stained and identified by PCR.

9. Use of the method according to any one of claims 1 to 8 in constructing a composite transgenic leek strain.

10. A composite transgenic leek strain, characterized in that: The method is constructed by any one of claims 1 to 8.

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