Construction method of agrobacterium rhizogenes mediated blueberry genetic transformation system

Abstract

The invention discloses a construction method of an agrobacterium rhizogenes mediated blueberry genetic transformation system, and belongs to the technical field of genetic engineering. The construction method provided by the invention comprises the following steps: taking semi-lignified green branches of blueberries, topping, cutting into 6-8cm cuttings with more than two bud points and leaf removal, injecting an infection solution consisting of agrobacterium rhizogenes K599 bacterial liquid with OD600 of 1.0, 200 [mu] mol / L acetosyringone and 0.05% SL-77 into two ends of the cuttings and axillary bud points for infection, and carrying out seedling culture. And then carrying out culture, mildew prevention and transgenic line verification. By using the construction method provided by the invention, a blueberry genetic transformation system can be efficiently constructed, and the highest transformation rate is 29.75%. Technical support is provided for achieving the purposes of simple, rapid and efficient blueberry genetic transformation and breeding, and wide application prospects are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a method for constructing a blueberry genetic transformation system mediated by Agrobacterium rhizogenes. Background Art

[0002] Blueberry is a plant of the genus Vaccinium L. in the family Ericaceae, native to North America. Blueberry has high health value, and the breeding of blueberry varieties with strong market competitiveness is of great significance to the development of the blueberry industry. With the development of modern molecular biology, molecular breeding technologies such as gene editing can be used to promote the improvement and innovation of blueberry germplasm and accelerate the breeding process. Conventional hybrid breeding combined with molecular breeding has gradually become a new breeding trend.

[0003] An efficient and stable blueberry genetic transformation system is an important research method for discovering key genes of blueberries, studying excellent traits of blueberries and conducting precision breeding of blueberries. Agrobacterium-mediated genetic transformation is one of the most widely used plant genetic transformation methods. The transformation principle is that Agrobacterium tumefaciens carrying Ti plasmid or Agrobacterium rhizogenes carrying Ri plasmid can integrate the T-DNA fragment in the plasmid into the plant genome after infecting the plant, thereby inducing transgenic plants. In the Agrobacterium-mediated genetic transformation system, the concentration of the bacterial solution is one of the main factors affecting the transformation efficiency. Different types of plants tolerate different bacterial solution concentrations. If the bacterial solution concentration is too low, the transformation efficiency is low, and if the concentration is too high, it is easy to damage plant cells. The injection transformation method (RAPID) using stem segments and other organs as explants proposed by Guoguo Mei et al. is to achieve rapid transformation of plants such as potatoes and sweet potatoes by directly injecting the bacterial solution into the corresponding parts of the plant, and at the same time, it shows that surfactant is an important variable for injection transformation. Compared with the Agrobacterium tumefaciens transformation method, the Agrobacterium rhizogenes transformation method has the advantages of simple operation and low cost. It can induce the transformed single cells to produce independent hairy roots and avoid the problem of chimeras. Hairy root transformation systems have been established for plants such as citrus, apple, and peach, but Agrobacterium rhizogenes is rarely used in the genetic transformation of blueberries.

[0004] The southern high-bush blueberry variety 'Lanmei No. 1' is a national forest tree variety in my country (number: Guo R-ETS-VC-006-2018), with a complex genetic background, strong heat resistance, high nitrogen utilization efficiency, strong adaptability, good yield, and high fruit anthocyanin content. The development and utilization of its genetic resources is of great significance to the innovation of blueberry germplasm in my country. However, the existing blueberry genetic transformation method is mainly the Agrobacterium tumefaciens transformation method with leaves as explants, which is relatively cumbersome and time-consuming, requires a corresponding tissue culture system, and the adventitious bud conversion rate does not exceed 10%. Therefore, it is urgent to provide a blueberry genetic transformation system and method with a high conversion rate. Summary of the invention

[0005] The purpose of the present invention is to provide a method for constructing a blueberry genetic transformation system mediated by Agrobacterium rhizogenes to solve the problems existing in the above-mentioned prior art. The construction method provided by the present invention can simply, quickly and efficiently construct a blueberry genetic transformation system, with a maximum conversion rate of 29.75%.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for constructing a blueberry genetic transformation system, comprising the following steps:

[0008] preparing blueberry explants;

[0009] Mixing the bacterial solution of Agrobacterium rhizogenes, acetosyringone and SL-77 to obtain an infection solution;

[0010] Infecting the blueberry explants with the infection solution;

[0011] The infected explants are cultured and mildew-proofed to obtain transgenic strains;

[0012] The transgenic strain is verified to obtain the blueberry genetic transformation system.

[0013] Preferably, the preparation of the blueberry explant comprises: selecting semi-lignified green branches of blueberry, removing the young and tender stem segments at the top, trimming them to a length of 6-8 cm, retaining the number of axillary buds not less than 2, and removing leaves, to obtain the blueberry explant.

[0014] Preferably, the OD of the Agrobacterium rhizogenes culture is 600 is 1.0; the Agrobacterium rhizogenes is Agrobacterium rhizogenes expressing green fluorescent protein.

[0015] Preferably, the Agrobacterium rhizogenes is Agrobacterium rhizogenes K599.

[0016] Preferably, in the infection solution, the concentration of acetosyringone is 200 μmol / L.

[0017] Preferably, in the infection solution, the mass fraction of SL-77 is 0.05%.

[0018] Preferably, the infection comprises: injecting the infection solution into both ends of the explant and the bud point of each axillary bud.

[0019] Preferably, the amount of infection solution injected each time is 0.2-0.3 mL.

[0020] Preferably, the variety of blueberry is Blue Beauty No. 1.

[0021] The present invention also provides an application of a blueberry genetic transformation system obtained according to the construction method in blueberry breeding.

[0022] The present invention discloses the following technical effects:

[0023] The construction method provided by the present invention comprises taking a semi-lignified green branch of blueberry, removing the top and cutting it into 6-8 cm long cuttings with more than two buds and removing leaves, and using OD 600 The infection solution composed of 1.0% Agrobacterium rhizogenes K599 bacterial solution, 200 μmol / L acetosyringone and 0.05% SL-77 is injected into both ends of the cutting and the axillary buds for infection, and then the steps of culturing, mildew prevention and transgenic strain verification are carried out. The construction method provided by the present invention can efficiently construct a blueberry genetic transformation system, and the highest conversion rate is 29.75%. The present invention provides technical support for achieving the purpose of simple, rapid and efficient blueberry genetic transformation and breeding, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 This is a brief flow chart of the injection transformation method; a is cutting preparation; b is infection; c is cutting; d is screening for GFP-positive plants; e is material identification; the bright green part in de represents GFP-positive new tissue;

[0026] Figure 2 The results of screening and identification of transgenic plants; wherein, AD in (1) are fluorescence photographs taken under a stereoscopic fluorescence microscope (excitation wavelength 488 nm), ad are bright field photographs corresponding to AD; A is a negative lateral bud leaf; B is a lateral bud leaf expressing green fluorescence; C is a negative hairy root; D is a hairy root expressing green fluorescence; scale bar = 1000 μm; M in (2) is DNAMarker DL2000; 1 is a blank control; 2 is a negative control; 3 is a positive control; 4-10 are PCR products of transformed plant DNA. DETAILED DESCRIPTION

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0030] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0031] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0032] Example 1

[0033] 1. Materials and Methods

[0034] 1.1 Materials

[0035] 1.1.1 Plant materials

[0036] The experimental materials were from the ground-planted plants of 'Lanmei No. 1' in the blueberry experimental nursery of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province. On the day of the experiment, the green branches of the plant with a diameter of 3-5 mm and semi-lignified growth were selected, the top tender stem segments were removed and the cuttings with a length of 6-8 cm, more than 2 axillary buds and leaves removed were used as explants, and moisturized for later use.

[0037] 1.1.2 Obtaining Agrobacterium

[0038] The Agrobacterium rhizogenes strain was K599, carrying the pMDC83 plasmid expressing kanamycin resistance gene, hygromycin resistance gene and reporter gene GFP (Green Fluorescent Protein). The glycerol strain was donated by the College of Agriculture, Nanjing Agricultural University.

[0039] 1.2 Agrobacterium activation and infection fluid preparation

[0040] Take out the glycerol culture of Agrobacterium rhizogenes K599 from the -20℃ refrigerator, streak it on LB solid medium containing 50mg / L kanamycin for activation, and culture it in the dark at 28℃ for 1-2d. Pick a single colony and add it to LB liquid medium containing 50mg / L kanamycin, and culture it overnight at 28℃ and 120r / min. Shake the culture solution again at a ratio of 1mL of culture solution per 100mL of culture medium, and culture it overnight at 28℃ and 120r / min until the color of the culture solution turns orange. Centrifuge the culture solution at 6500r / min for 10min, pour off the supernatant, add the precipitated bacteria to the resuspension solution (WPM+100μMAS, pH 5.3), pipette and mix, and adjust the OD 600 The value was adjusted to 1.0, and the injection was performed after activation for 2-3 hours at 28°C and 120 r / min in dark conditions.

[0041] 1.3 Genetic transformation of blueberry stem segments

[0042] 1.3.1 Effect of different bacterial concentrations on transformation efficiency

[0043] Set the concentration of K599 infection solution (OD 600 The value) gradient was 0.5, 1.0, and 1.5. A 1 mL sterile syringe was used to inject the infection solution into both ends of the cuttings and the axillary bud points. 0.2-0.3 mL of infection solution was injected into each injection point, and 26 explants were inoculated for each treatment.

[0044] 1.3.2 Effects of different additive combinations on conversion efficiency

[0045] K599 infection solution OD 600 The value is 1.0. An orthogonal experiment of the additive combination AS (Acetosyringone) and SL-77 (Surfactant, Silwet L-77) was set up, and the following four treatments were added to the resuspension: ① 100 μM AS without SL-77; ② 200 μM AS without SL-77; ③ 100 μM AS with 0.05% SL-77; ④ 200 μM AS with 0.05% SL-77; a total of 4 treatments.

[0046] Use a 1 mL sterile syringe to inject the infection solution into both ends of the cuttings and the axillary buds. Inject 0.2-0.3 mL of infection solution into each injection point, and inoculate 40 explants for each treatment.

[0047] 1.3.3 Cutting culture of blueberry stem segments

[0048] After infection, the cuttings were incubated in the substrate soil for 4 days in the dark, and then switched to light culture. The seedling tray was covered with a moisturizing transparent plastic cover. In the first week after dark culture, 1000 times carbendazim solution was sprayed daily to prevent mildew. Before rooting, the stem segments were sprayed with water regularly to keep them moist. The cuttings were cultured at 25°C, 16h light / 8h dark. After about 45 days of culture, the cuttings took root and germinated, and fluorescence screening was performed.

[0049] 1.3.4 Identification of transgenic plants

[0050] To identify the effectiveness of the genetically transformed blueberry strains, a stereo fluorescence microscope (Zeiss SteREODiscovery.V8) was used to observe the fluorescence expression of infected plants under 488nm blue light. The autofluorescence of wild-type plants was used as a negative control, and new lateral buds and roots expressing GFP signals were screened and photographed.

[0051] To identify whether the target gene is integrated into the blueberry genome, the lateral stems and leaves of the pseudo-positive cuttings were further extracted, ground into powder under liquid nitrogen freezing conditions, and DNA was extracted using a plant genomic DNA extraction kit (FastCleanPlantGenomicDNAKit, Kangwei Century CWBIO, CW05715). The specific steps refer to the kit instructions. Primers Kgfp-F and Kgfp-R were designed, and the amplified band length was 302bp. The primers were synthesized by Anhui General Biological Co., Ltd.

[0052] Kgfp-F: 5'-GAAGTTCGAGGGCGACA-3' (SEQ ID NO. 1);

[0053] Kgfp-R: 5'-CGTTGGGGTCTTTGCTTA-3' (SEQ ID NO. 2).

[0054] The GFP gene fragment was cloned by PCR using a high-fidelity enzyme (CWBIO, CW2969). The reaction system (25 μL) was 2×SuperPfxMasterMix (Dye) 10 μL, Kgfp-F 0.8 μL, Kgfp-R 0.8 μL, ddH2O 7.4 μL, and DNA 1 μL. The reaction program was set as follows: 98°C for 3 min, (98°C for 10 s, 66°C for 30 s, 35 cycles), and 72°C for 1 min. After the reaction, the PCR product was detected by 1% (w / v) agarose gel electrophoresis. The target band was positive, and the photo was taken using a UV gel imager.

[0055] 1.4 Data Statistics and Analysis

[0056] Excel was used to count and calculate the survival rate of explants and the GFP bud induction rate, and Adobe Illustrator 2023 was used to draw the graph. The calculation formulas for survival rate and induction rate are as follows:

[0057] Survival rate = number of surviving explants / total number of explants × 100%;

[0058] Induction rate = number of explants whose new shoots expressed green fluorescence / total number of explants × 100%.

[0059] 2. Experimental results

[0060] 2.1 Effect of different bacterial concentrations on blueberry transformation efficiency

[0061] The green stem segments of 'Lanmei No. 1' were transformed by injection, and the optimal K599 bacterial solution concentration (OD 600 The results showed (Table 1): when the bacterial solution OD 600 When the value ranges from 0.5 to 1.0, the survival rate of explants increases; when the OD 600 When the value ranged from 1.0 to 1.5, the survival rate of explants was the same, which was 96.15%. The GFP bud induction rate of explant new shoots first increased and then decreased with the increase of bacterial solution concentration. 600 When the value was 1.0, the infection effect was the best, the survival rate was 96.15%, and the GFP shoot induction rate was 42.31%.

[0062] Table 1 Effect of bacterial solution concentration on transformation efficiency of stem segments of 'Lanmei No. 1'

[0063]

[0064] 2.2 Effects of different additive combinations on blueberry conversion efficiency

[0065] The green stem segments of 'Lanmei No. 1' were transformed by injection, and the orthogonal treatments of adding AS and SL-77 were performed in the resuspension of K599 Agrobacterium. The results showed (Table 2): the increase of AS concentration and the addition of SL-77 in the resuspension reduced the survival rate of explants, while increasing the GFP bud induction rate. The infection effect was best when 200μM AS and 0.05% SL-77 were added to the resuspension at the same time, with an explant survival rate of 80.00% and a GFP bud induction rate of 42.50%.

[0066] Table 2 Effects of additive combinations on transformation efficiency of stem segments of 'Lanmei No. 1'

[0067]

[0068] 2.3 Screening and identification of transgenic plants

[0069] Fluorescence detection (excitation wavelength 488nm) was performed on the genetically transformed strains of 'Lanmei No. 1', and strains expressing green fluorescence signals in lateral buds and hairy roots were screened out. Figure 2 In order to identify whether the target gene was integrated into the blueberry genome, 20 pseudo-positive plants were randomly selected to extract DNA from GFP buds, and the GFP gene fragment was cloned by PCR for verification. Figure 2 In (2), 14 transformed plants were detected, with a false positive rate of 30%. The highest GFP bud induction rate of the combined genetic transformation lines was 42.50%, and the highest transformation rate of the green stem segments of 'Lanmei No. 1' infected by injection was 29.75%.

[0070] Comparative Example 1

[0071] The only difference from Example 1 is that the stem segments of the tissue culture seedlings of 'Lanmei No. 1' were selected as explants for transformation experiments. Compared with Example 1, the survival rate of the stem segments of the tissue culture seedlings of 'Lanmei No. 1' after injection infection was extremely low, and the trauma caused by the 1mL syringe needle had a greater impact on the stem segments of the tissue culture seedlings, and the explants were prone to wilt and die, resulting in the failure of the transformation experiment.

[0072] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing a blueberry genetic transformation system, characterized in that: The following steps are involved: preparing blueberry explants; Mixing the bacterial solution of Agrobacterium rhizogenes, acetosyringone and SL-77 to obtain an infection solution; Infecting the blueberry explants with the infection solution; The infected explants are cultured and mildew-proofed to obtain transgenic strains; The transgenic strain is verified to obtain the blueberry genetic transformation system.

2. The construction method according to claim 1, characterized in that: The preparation of the blueberry explant comprises: selecting semi-lignified green branches of blueberries, removing the young and tender stem segments at the top, trimming to a length of 6-8 cm, retaining no less than 2 axillary buds, and removing leaves to obtain the blueberry explant.

3. The construction method according to claim 1, characterized in that: The OD of the Agrobacterium rhizogenes culture solution 600 is 1.0; the Agrobacterium rhizogenes is Agrobacterium rhizogenes expressing green fluorescent protein.

4. The construction method according to claim 3, characterized in that: The Agrobacterium rhizogenes is Agrobacterium rhizogenes K599.

5. The construction method according to claim 1, characterized in that: In the infection solution, the concentration of acetosyringone is 200 μmol / L.

6. The construction method according to claim 1, characterized in that: In the infection solution, the mass fraction of SL-77 is 0.05%.

7. The construction method according to claim 1, characterized in that: The infection comprises: injecting the infection solution into both ends of the explant and the bud point of each axillary bud.

8. The construction method according to claim 7, characterized in that: The amount of infection solution used for each injection is 0.2-0.3mL.

9. The construction method according to any one of claims 1 to 8, characterized in that: The variety of blueberry is Blue Beauty No.

1.

10. Use of the blueberry genetic transformation system obtained according to the construction method according to any one of claims 1 to 9 in blueberry breeding.

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