Agrobacterium tumefaciens-mediated blumea balsamifera callus genetic transformation method and application
Through the Agrobacterium tumefaciens transformation method mediated by Agrobacterium tumefaciens, the beet-elucin synthesis gene was successfully transferred to the callus callus, which solved the problem of difficulty in transforming callus callus, and achieved efficient transformation and functional genome of the callus Technical support was provided by academic research.
Patent Information
- Application Number
- CN202411212347.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-30
- Publication Date
- 2024-12-10
AI Technical Summary
The difficulty in stable transformation of callus in Aiba callus is the main bottleneck restricting the genomics research of function of Aiba callus, and the existing technology is difficult to efficiently realize the genetic transformation of callus in Aiba callus.
Agrobacterium tumefa callus transformation method mediated by Agrobacterium tumefa callus, the stem segments or leaves of Agrobacterium tumefa were precultured, and the infection solution was prepared, and the infection and co-culture were carried out, combined with antibiotic screening and liquid culture. The beet-elutin synthesis gene p35S-RUBY was successfully transformed into the worm callus tissue.
The efficient transformation of callus in Ai Callus was achieved, with a total conversion rate of 53%, breaking through the technical obstacles of the difficulty of transforming callus in Ai Callus and providing technical support for the functional genomics research of Ai.
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Figure CN119101702A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of plant tissue culture technology and plant transgenic technology, and specifically relates to a method and application of genetic transformation of mugwort callus mediated by Agrobacterium tumefaciens. Background Art
[0002] Artemisia argyi Lévl.et Van is a perennial herb belonging to the genus Artemisia of the Asteraceae family. It is a widely used Chinese herbal medicine. Its leaves are used as medicine, and have many functions such as warming the meridians and dispelling cold, removing dampness and relieving itching. In production, Artemisia argyi is mainly propagated asexually by rhizome division, but due to the limitation of division time, it can only be divided within a short time before the perennial root sprouts. Although Artemisia argyi can also be propagated by seeds, because Artemisia argyi is a short-day plant, it blooms and bears fruit late, and it is not easy to obtain mature seeds. At the same time, the quality of sowing seedlings is uneven. Therefore, there are problems in production at present such as low seedling reproduction rate and uneven seedling quality.
[0003] With the analysis of the mugwort genome, the construction of a genetic transformation system for mugwort is the technical basis for in-depth exploration of key functional genes in mugwort leaves and studying their functions. At present, the commonly used genetic transformation methods include Agrobacterium-mediated transformation, gene gun-mediated transformation, PEG-mediated protoplast transformation, and plant virus vector-mediated transformation. Among them, the Agrobacterium-mediated transformation method is relatively simple to operate and is a commonly used transformation method. However, the difficulty in stable transformation of mugwort callus is the main bottleneck restricting the research on the functional genomics of mugwort. How to optimize the genetic transformation system of mugwort callus and improve the efficient transformation of mugwort callus has become an urgent problem to be overcome in the research on the functional genomics of mugwort. Summary of the invention
[0004] The purpose of the present invention is to provide a method and application of genetic transformation of mugwort callus mediated by Agrobacterium tumefaciens to solve the problems existing in the above-mentioned prior art. By using Agrobacterium tumefaciens to mediate the transformation of mugwort callus, the betalain synthesis gene p35S-RUBY is successfully transformed into mugwort callus, and a large amount of transgenic callus that accumulates betalain and becomes red is obtained. The technical barrier of difficult transformation of mugwort callus is broken through, and technical support is provided for the research of functional genomics of mugwort.
[0005] In order to achieve the above object, the present invention is implemented through the following technical solutions:
[0006] The first aspect of the present invention provides a method for genetic transformation of mugwort callus mediated by Agrobacterium tumefaciens, comprising the following steps:
[0007] S1. Take the stem segments or leaves of sterile mugwort seedlings as explants for pre-culture;
[0008] S2, activating the Agrobacterium tumefaciens to prepare an Agrobacterium tumefaciens infection solution;
[0009] S3, placing the pre-cultured explants in the infection solution prepared in step S2 for infection, so that the explants are in full contact with the Agrobacterium tumefaciens;
[0010] S4. After the infection is completed, the bacterial liquid on the surface of the explant is sucked dry and then transferred to the culture medium for co-cultivation;
[0011] S5, transferring the explants after the co-cultivation is completed to a culture medium containing antibiotics for sterilization culture to obtain explants containing callus tissue;
[0012] S6. After the callus tissue is removed from the explant, it is placed in a liquid culture medium for subculture.
[0013] The second aspect of the present invention provides the use of the method for genetic transformation of mugwort callus mediated by Agrobacterium tumefaciens described in the first aspect in genetic transformation of mugwort callus.
[0014] The beneficial effects of the above embodiments of the present invention are as follows:
[0015] The invention is easy to obtain materials, simple and easy to operate, and has a short cycle. The betalain synthesis gene p35s-RUBY is successfully transferred into the mugwort callus tissue by using Agrobacterium tumefaciens-mediated mugwort callus transformation, and transgenic callus tissue that accumulates betalain and appears red is obtained. The total transformation rate is as high as 53%, and it is successfully subcultured. This method breaks through the technical obstacles of mugwort callus tissue transformation and provides technical support for the research of mugwort functional genomics. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.
[0017] Figure 1 This is the process of callus induction of mugwort stem segments and leaves in MS+1.5 mg / L 2,4-D medium in Example 1 of the present invention (Ⅰ and Ⅳ are respectively the initial stage of inoculation of mugwort leaves and stem segments; Ⅱ and Ⅴ are respectively the inoculation and culture of mugwort leaves and stem segments for 15 days; Ⅲ and Ⅵ are respectively the subculture of mugwort leaves and stem segments for 15 days. The scale in the figure is 1 cm);
[0018] Figure 2 The effects of different concentrations of hygromycin on callus induction of stem segments and leaves in Example 2 of the present invention (effects of hygromycin I-IV on stem segments; a: 1 mg / L; II: 2 mg / L; III: 3 mg / L; IV: 4 mg / L; effects of hygromycin V-VIII on leaves; V: 1 mg / L; VI: 2 mg / L; VII: 3 mg / L; VIII: 4 mg / L, scale bar 1 cm in the figure);
[0019] Figure 3 The genetic transformation process of the leaves and stems of Artemisia argyi in Example 1 of the present invention (A: genetic transformation process of leaves of Artemisia argyi; B: genetic transformation process of stems of Artemisia argyi; I: pre-culture of explants; II: infection of explants with Agrobacterium; III: co-culture of explants; IV-VI: degerming and screening culture of explants);
[0020] The diagrams are for illustrative purposes only. DETAILED DESCRIPTION
[0021] The present invention is achieved by the following steps:
[0022] S1. Take the stem segments or leaves of sterile mugwort seedlings as explants for pre-culture;
[0023] S2, activating the Agrobacterium tumefaciens to prepare an Agrobacterium tumefaciens infection solution;
[0024] S3, placing the pre-cultured explants in the infection solution prepared in step S2 for infection, so that the explants are in full contact with the Agrobacterium tumefaciens;
[0025] S4. After the infection is completed, the bacterial liquid on the surface of the explant is sucked dry and then transferred to the culture medium for co-cultivation;
[0026] S5, transferring the explants after the co-cultivation is completed to a culture medium containing antibiotics for sterilization culture to obtain explants containing callus tissue;
[0027] S6. After the callus tissue is removed from the explant, it is placed in a liquid culture medium for subculture.
[0028] Among them, in S1, the pre-culture is to cut the leaves of sterile wormwood seedlings into 1-2 cm 2 Or cut the stem segments into 1-2 cm pieces and perform pre-cultivation;
[0029] The pre-culture is: MS+1.5 mg / L 2,4-D culture medium, cultured at 22-28° C. for 2-6 days.
[0030] In S2, the activation treatment is to inoculate the Agrobacterium tumefaciens cells in LB liquid culture medium and culture them at 25-30° C. for 48 hours;
[0031] Select a single colony and transfer it to LB liquid medium, and culture it with shaking at 22-28°C overnight;
[0032] Centrifuge to enrich Agrobacterium tumefaciens cells, and use MGL liquid medium to resuspend the cells to prepare infection solution until OD600 is 0.3-1.2;
[0033] The formula of the MGL medium is 5 g tryptone, 5 g NaCl, MgSO4 7H 2 O 0.1g, KH 2 PO 4 0.25g, mannitol 5g, glycine 1g;
[0034] The Agrobacterium tumefaciens cell is the Agrobacterium EHA105 strain containing the p35S-RUBY plasmid;
[0035] The LB medium contained 50 μg / mL spectinomycin and 20 μg / mL rifampicin.
[0036] In order to enhance the transformation effect, acetosyringone (AS) is added to the infection solution, and its concentration is controlled at 50-100 μM.
[0037] In S3, the infection time is 5 to 45 minutes.
[0038] In S4, the co-culture medium is MS solid culture medium, and the co-culture is carried out at 22-28° C. for 2 to 4 days under dark conditions.
[0039] In S5, the medium used in the degerming culture was MS + 1.5 mg / L 2,4-D;
[0040] The antibacterial antibiotic is one or more of timentin, sporemycin or carbenicillin; the concentration of the antibacterial antibiotic is 30-400 mg / L.
[0041] Preferably, the antibacterial antibiotic is timentin, and the concentration of timentin is 100-200 mg / L;
[0042] Alternatively, the screening antibiotic is hygromycin, and the concentration of hygromycin is 1-4 mg / L.
[0043] In S6, the subculture is a medium of MS+1.5 mg / L 2,4-D+0.6% agar+3% sucrose, and is cultured under light at 25-30° C. for 15-20 days.
[0044] The present invention is further described in detail below with reference to the accompanying drawings and examples.
[0045] The MS medium and LB medium used in the present invention are both commercially available basic medium. The MGL medium is a self-prepared medium. The formula of the MGL medium (1L) is 5g of tryptone, 5g of NaCl, and MgSO 4 7H 2 O 0.1g, KH 2 PO 4 0.25g, mannitol 5g, glycine 1g.
[0046] Example 1
[0047] S1. Take the stem segments or leaves of sterile mugwort seedlings as explants for pre-culture
[0048] Cut the leaves of sterile mugwort seedlings into 1-2 cm 2 , stem segments were cut into 1-2 cm segments and inoculated into MS + 1.5 mg / L 2,4-D callus induction medium for pre-culture ( Figure 3 A, BⅠ).
[0049] S2, activating Agrobacterium tumefaciens to prepare Agrobacterium tumefaciens infection solution
[0050] The Agrobacterium EHA105 strain containing the p35S-RUBY plasmid was activated on LB medium containing 50 μg / mL spectinomycin and 20 μg / mL rifampicin, and then cultured in an incubator at 28°C for 48 hours; a single colony after activation culture was selected and transferred to LB liquid culture medium, and cultured overnight at 28°C with shaking; the bacteria were collected by centrifugation at 8000rpm for 5min, the bacterial solution was washed once with sterile water to remove the residual LB culture medium, and the bacteria were resuspended in MGL liquid culture medium to an absorbance of 0.6 (OD600 nm) for use, and 50 μM acetosyringone was added before use.
[0051] S3, infection and transformation and S4, co-cultivation
[0052] Place the mugwort explants in the infection solution of Agrobacterium activated by acetosyringone and infect for 15 min ( Figure 3 A, BⅡ); after infection, remove the explants, dry the excess bacterial solution on sterile filter paper, and then place them on the culture medium covered with sterile filter paper; co-culture in the dark for 2 days ( Figure 3 A, BⅢ).
[0053] S5, Induction of transgenic callus
[0054] After the co-culture, the explants were transferred to MS + 1.5 mg / L 2,4-D callus induction medium containing timentin antibacterial antibiotics and hygromycin selection antibiotics for primary culture ( Figure 3 A, BⅣ).
[0055] S6, Obtaining transgenic callus and subculture
[0056] The transgenic callus was separated from the explant and inoculated into a callus medium containing MS + 1.5 mg / L 2,4-D + 0.6% agar + 3% sucrose, and cultured at 25°C under light to obtain a large number of subcultured transgenic callus ( Figure 3 A, BⅤ-Ⅵ).
[0057] The process of callus induction and subculture proliferation of leaves and stems of Artemisia selengensis Figure 1 As shown in Figure 1, about 15 days after inoculation, callus tissue can be observed on the edge of the leaves of Artemisia argyi, and the stem segments swell to form callus tissue. The callus tissue was cut off and subcultured for 15 days, and the following can be observed: Figure 1 A large amount of callus tissue of III and VI.
[0058] Example 2: Effects of different concentrations of hygromycin on callus induction in stem segments and leaves
[0059] The plant overexpression vector plasmid used in the present invention has a hygromycin resistance marker gene, so hygromycin is selected as the screening antibiotic. The sensitivity results of the stem segments and leaves of Artemisia selengensis to hygromycin are shown in the attached figure. Figure 2 As shown, hygromycin had a significant inhibitory effect on the callus induction of the two explants of Artemisia argyi, and both showed a trend of increasing inhibition with increasing hygromycin concentration.
[0060] When the hygromycin concentration was 3 mg / L, the stem segments darkened and dried up, and the callus induction of the stem segments was completely inhibited. Therefore, when using stem segments as transformation materials, the hygromycin screening concentration was selected to be 3 mg / L when screening resistant callus and buds.
[0061] When the concentration of hygromycin was 2 mg / L, the leaves turned green, yellow and brown, and the leaf edges did not swell, and the ability to induce callus was completely inhibited. When leaves were used as transformation materials, the selective concentration of hygromycin was 2 mg / L, which could ensure the growth of resistant callus and adventitious buds and reduce the generation of false positives.
[0062] Example 3: Effects of different antibacterial antibiotics on callus induction of Artemisia argyi
[0063] In order to inhibit the excessive growth of Agrobacterium and affect the callus induction of Artemisia argyi explants, the stem segments and leaves of Artemisia argyi were inoculated on callus induction medium containing antibacterial antibiotics such as timentin, cephalosporin, carbenicillin and other antibacterial antibiotics at different mass concentrations to observe the effects of different antibacterial antibiotics on the callus induction of Artemisia argyi.
[0064] After 20 days of culture, the antibacterial rate and callus induction rate were statistically analyzed, as shown in Table 1. It can be seen that timentin has the strongest inhibitory effect on Agrobacterium EHA105, and can significantly inhibit the growth of Agrobacterium at a concentration of 100 mg / L; Agrobacterium can still grow vigorously at a concentration of 400 mg / L of cephalosporin; 30-60 mg / L carbenicillin cannot inhibit the growth of Agrobacterium. The antibacterial effects of the three antibiotics on Agrobacterium EHA105 are timentin>cephalosporin>carbenicillin.
[0065] Table 1 Effects of different antibacterial antibiotics on callus induction of Artemisia argyi
[0066]
[0067]
[0068] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.
Claims
1. A method for transformation of mugwort callus mediated by Agrobacterium tumefaciens, characterized in that: The following steps are involved: S1. Take the stem segments or leaves of sterile mugwort seedlings as explants for pre-culture; S2, activating the Agrobacterium tumefaciens to prepare an Agrobacterium tumefaciens infection solution; S3, placing the pre-cultured explants in the infection solution prepared in step S2 for infection, so that the explants are in full contact with the Agrobacterium tumefaciens; S4. After the infection is completed, the bacterial liquid on the surface of the explant is sucked dry and then transferred to the culture medium for co-cultivation; S5, transferring the explants after the co-cultivation is completed to a culture medium containing antibiotics for sterilization culture to obtain explants containing callus tissue; S6. After the callus tissue is removed from the explant, it is placed in a liquid culture medium for subculture.
2. The method for transforming mugwort callus according to claim 1, characterized in that: In S1, the pre-culture is to cut the leaves of sterile mugwort seedlings into 1-2 cm 2 Or cut the stem segments into 1-2 cm pieces and perform pre-cultivation; The pre-culture is: MS+1.5 mg / L 2,4-D culture medium, cultured at 22-28° C. for 2-6 days.
3. The method for transforming mugwort callus according to claim 1, characterized in that: In S2, the activation treatment is to inoculate the Agrobacterium tumefaciens cells in LB liquid culture medium and culture them at 25-30° C. for 48 hours; Select a single colony and transfer it to LB liquid medium, and culture it with shaking at 22-28°C overnight; Centrifuge to enrich Agrobacterium tumefaciens cells, and use MGL liquid medium to resuspend the cells to prepare infection solution to OD600 of 0.3-1.2; The formula of the MGL medium is 5 g of tryptone, 5 g of NaCl, 0.1 g of MgSO4·7H2O, 0.25 g of KH2PO4, 5 g of mannitol, and 1 g of glycine; The Agrobacterium tumefaciens cell is the Agrobacterium EHA105 strain containing the p35S-RUBY plasmid; The LB medium contained 50 μg / mL spectinomycin and 20 μg / mL rifampicin.
4. The method for transforming mugwort callus according to claim 1, characterized in that: In S2, acetosyringone is added to the infection solution, and its concentration is controlled to be 50-100 μM.
5. The method for transforming mugwort callus according to claim 1, characterized in that: In S3, the infection time is 5 to 45 minutes.
6. The method for transforming mugwort callus according to claim 1, characterized in that: In S4, the co-culture medium is MS solid culture medium, and the co-culture is carried out at 22-28° C. for 2 to 4 days under dark conditions.
7. The method for transforming mugwort callus according to claim 1, characterized in that: In S5, the medium used in the degerming culture was MS + 1.5 mg / L 2,4-D; The antibacterial antibiotic is one or more of timentin, sporemycin or carbenicillin; the concentration of the antibacterial antibiotic is 30-400 mg / L.
8. The method for transforming mugwort callus according to claim 7, characterized in that: In S5, the antibacterial antibiotic is timentin, and the concentration of timentin is 100-200 mg / L; Alternatively, the screening antibiotic is hygromycin, and the concentration of hygromycin is 1-4 mg / L.
9. The method for transforming mugwort callus according to claim 1, characterized in that: In S6, the subculture is a medium of MS+1.5 mg / L 2,4-D+0.6% agar+3% sucrose, and is cultured under light at 25-30° C. for 15-20 days.
10. Use of the method for transforming mugwort callus according to any one of claims 1 to 9 in transforming mugwort callus.