Method for transforming tea tree somatic embryo based on gene gun mediation and obtaining transgenic plant

Abstract

The invention discloses a method for transforming and obtaining a transgenic plant based on a gene gun mediated tea tree somatic embryo, and belongs to the technical field of tea tree breeding, and the method comprises the following steps: bombarding a carrier containing a target gene into the tea tree somatic embryo by adopting a gene gun method to obtain the transgenic somatic embryo; inoculating the transgenic somatic embryo to a somatic cell induction culture medium for culturing to obtain a secondary embryo; inoculating the secondary embryo to a maturation induction culture medium for culturing to obtain a cotyledon embryo; inoculating the cotyledon embryo to a germination induction culture medium for culturing to obtain a seedling with germinated bud leaves; and inoculating the seedlings with germinated bud leaves to a basic growth culture medium for culturing to obtain the transgenic tea tree plant. The method has the beneficial effects that the method is short in transformation period, can better adapt to the biological characteristics of the tea trees, and has high application value. The invention provides an effective technical approach for construction of a tea tree genetic transformation system, and has wide popularization and application potential.

Description

Technical Field

[0001] The present invention relates to the technical field of tea tree breeding, and particularly relates to a genetic transformation system for tea tree somatic embryos mediated by a gene gun. Background Art

[0002] The tea tree (Camellia sinensis), as an important cash crop, is widely planted in China and is rich in various secondary metabolites with economic value and health care functions, such as catechins and tea polyphenols. These substances endow the tea tree with unique flavors and health benefits. However, the biological characteristics of the tea tree, such as a long growth cycle and self-incompatibility, limit the application and effectiveness of traditional breeding methods in tea tree variety improvement. Especially in the case of a long breeding cycle, traditional cross-breeding methods have problems such as low efficiency and long cycle. It is worth noting that compared with model crops such as rice and corn, the lack of an efficient genetic transformation system in the tea tree has led to a lag in functional genomics research and slow progress in the molecular mechanism analysis of key traits, further hindering the development of its biological research level and research efficiency. For example, soybeans have achieved precise improvement of traits such as insect resistance and herbicide tolerance through transgenic technology, while the tea tree still relies on traditional methods in the fields of secondary metabolite synthesis regulation and stress resistance, with a long research cycle. Therefore, the development of efficient genetic transformation technology has become a key requirement for improving the speed and quality of tea tree variety improvement.

[0003] Currently, the bottleneck of the tea tree genetic transformation system is the difficulty of organ regeneration and low transformation efficiency. Although a system for direct regeneration of tea tree somatic embryos has been established, there are still many challenges in efficient and stable genetic transformation. Plant genetic transformation refers to the process of transferring foreign genes from one organism to another through biological means, followed by stable expression and integration in the genome and the ability to be stably inherited. Currently, plant genetic transformation technologies mainly include methods such as Agrobacterium-mediated transformation, virus-mediated transformation, and gene gun methods. Among them, Agrobacterium-mediated genetic transformation is widely used in gene improvement of various plants due to its simple operation and relatively high transformation efficiency. However, the tea tree is rich in secondary metabolites, especially chemical components such as catechins, which have a significant inhibitory effect and can interfere with the growth and infection process of Agrobacterium, thus significantly reducing the transformation efficiency. Therefore, Agrobacterium-mediated transformation of the tea tree is mainly limited to the application of gene transient expression systems, and stable transformation and gene integration have not been achieved.

[0004] In contrast, since the biolistic method was first proposed by Sanford et al. in 1987, it has become an effective plant gene transformation technology. The biolistic method uses the acceleration of heavy metal particles to directly introduce the foreign genes carried on their surfaces into plant recipient cells, thereby achieving gene transfer and integration. This method avoids the limitations of Agrobacterium and is particularly suitable for plants rich in secondary metabolites such as tea trees. Although the biolistic method can penetrate cell walls and cell membranes, due to the randomness of particle impact, only a few cells can successfully receive and express foreign genes, resulting in relatively low transformation efficiency. In addition, the impact of high-speed particles may cause a certain degree of damage to cells, affecting cell growth and activity. Although the operation process of the biolistic method is relatively simple, precise control of experimental conditions is required to ensure transfection effects and reduce cell damage. There are few reports on successfully obtaining transgenic plants by transforming tea tree somatic embryos mediated by gene gun. Summary of the Invention

[0005] The technical problem to be solved by the present invention is how to provide a rapid, efficient and stable genetic transformation method for tea trees.

[0006] The present invention solves the above technical problem by the following technical means:

[0007] The present invention provides a method for tea tree genetic transformation mediated by gene gun, comprising the following steps:

[0008] S1: Bombard a vector containing a target gene into tea tree somatic embryos by the biolistic method to obtain transgenic somatic embryos; the specific parameters of the gene gun are as follows: the diameter of gold powder is 0.6 μm to 1.6 μm, the bombardment pressure is 1100 to 1550 psi, the bombardment distance is 3 to 9 cm, and the vacuum condition is 26 to 32 Hg;

[0009] S2: Inoculate the transgenic somatic embryos onto a somatic embryo induction medium for culture to obtain secondary embryos;

[0010] S3: Inoculate the secondary embryos onto a maturation induction medium for culture to obtain cotyledon embryos;

[0011] S4: Inoculate the cotyledon embryos onto a germination induction medium for culture to obtain seedlings with bud and leaf germination;

[0012] S5: Inoculate the seedlings with bud and leaf germination onto a basic growth medium for culture to obtain transgenic tea tree plants.

[0013] Preferably, step S1 is specifically as follows: construct a fusion expression vector containing a target gene, then embed the vector into gold powder, and use a gene gun to introduce the target gene into tea tree somatic cells by means of high-speed bombardment with helium gas.

[0014] Preferably, the specific parameters of the gene gun are further preferably bombardment pressure: 1100, 1300, 1550 psi; bombardment distance: 3, 6, 9 cm; vacuum condition: 26, 29, 32 Hg.

[0015] Preferably, the diameter of the gold powder is 0.6, 1.0, 1.6 μm, and further preferably 0.6 μm.

[0016] Preferably, in step S1, the variety of the tea tree is any one or more of Shucha Early and Longjing 43.

[0017] Preferably, the somatic embryos of the tea tree are also pretreated before gene gun bombardment. Specifically, the somatic embryos of the tea tree are inoculated on a pretreatment medium and cultured for 4 - 6 hours; the somatic embryos of the tea tree are healthy somatic embryos with a diameter of 0.3 - 0.5 cm; the composition of the pretreatment medium is: MS basal medium, 30 g·L -1 sucrose, 100 mM mannitol, phytagel, pH adjusted to 5.8; the culture conditions are 23 °C, in the dark.

[0018] Preferably, the target gene is the CsAlaDC gene, and the CDS sequence of the CsAlaDC gene is as shown in SEQ ID No.1.

[0019] Preferably, in step S2, the composition of the somatic induction medium is: WPM medium, 0.2 mg·L -1 2,4 - D, 0.1 mg·L -1 6 - BA, 30 g·L -1 sucrose, phytagel, antibiotics, pH adjusted to 5.8; cultured for 2 - 3 weeks at 23 °C in the dark, and subcultured every 14 - 21 days during the culture process.

[0020] Preferably, in step S3, the composition of the maturation induction medium is: MS medium, 0.3 mg·L - 1 ABA, 1 g·L -1 graphite powder, 30 g·L -1 sucrose, phytagel, antibiotics, pH adjusted to 5.8; cultured for 30 - 40 days at 26 °C under the condition of 8 h darkness and 16 h light, and subcultured every 14 - 21 days during the culture process.

[0021] Preferably, in step S4, the composition of the germination induction medium is: MS medium, 1 mg·L -1 GA, 0.05 mg·L -1 2,4 - D, 0.5 mg·L -1 6 - BA, 1 g·L -1Graphite powder, 30 g·L -1 Sucrose, plant gel, antibiotics, adjust the pH to 5.8; Cultivate under the conditions of 26°C, 8 h of darkness and 16 h of light for 30 - 40 days, and subculture every 14 - 21 days during the cultivation process.

[0022] Preferably, in step S5, the composition of the basic growth medium is: MS medium, 1 g·L -1 Graphite powder, 30 g·L -1 Sucrose, plant gel, antibiotics, adjust the pH to 5.8; Cultivate under the conditions of 26°C, 8 h of darkness and 16 h of light, and subculture every 14 - 21 days during the cultivation process.

[0023] The beneficial effects of the present invention are as follows:

[0024] 1. Based on the gene gun genetic transformation system, by optimizing the pretreatment conditions and gene gun parameters, the present invention can efficiently and stably introduce foreign DNA into the somatic embryo tissue cells of tea plants.

[0025] 2. This method can efficiently and rapidly obtain complete transgenic tea plant seedlings, significantly improving the transformation efficiency. The gene gun method has the advantages of wide applicability, short transformation cycle, high efficiency, and the ability to perform multi-gene transformation, etc., making it show great application potential in the genetic transformation of tea plants.

[0026] 3. The transformation cycle of this method is relatively short, which can better adapt to the biological characteristics of tea plants and has high application value. This invention provides an effective technical approach for the construction of a tea plant genetic transformation system and has broad potential for popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a GUS tissue staining diagram of transgenic somatic embryos of tea plants in Example 1 of the present invention.

[0028] Figure 2 It is a qRT-PCR verification diagram of transgenic somatic embryos of tea plants in Example 1 of the present invention.

[0029] Figure 3 It is a dd-PCR verification diagram of transgenic somatic embryos of tea plants in Example 1 of the present invention.

[0030] Figure 4 It is a diagram of the growth process (A - I) of transgenic plants in Example 1 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0031] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will describe the technical solutions in the embodiments of the present invention clearly and completely in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0032] The test materials, reagents, etc. used in the following embodiments can be obtained from commercial sources without special instructions.

[0033] For those not specifying specific technologies or conditions in the embodiments, they can all be carried out according to the technologies or conditions described in the literature in this field or according to the product instructions. Without special instructions, the quantitative tests in the following embodiments are all set with more than three repeated experiments, and the results are averaged.

[0034] The main instruments and reagents used in the embodiments of the present invention:

[0035] (1) Gene gun

[0036] (2) Gold powder, 50% glycerol, 70% ethanol, 100% ethanol, CaCl 2 , spermidine, sterile water

[0037] (3) GUS staining solution: 50 mg X-gluc, 10 mL 100 mM EDTA-Na 2 , 1 mL Triton X-100, 2.05 g Na 2 HPO 4 ·12H 2 O, 0.65 g NaH 2 PO 4 ·2H 2 O, 2 mL 100 mM K 3 Fe(CN) 6 , 2 mL 100 mM K 4 Fe(CN) 6 , add pure water to make up to 100 mL

[0038] The composition of the pretreatment medium is: MS basal medium, 30 g·L -1 sucrose, 100 mM mannitol, plant gel, pH adjusted to 5.8.

[0039] The composition of the somatic cell induction medium is: WPM medium, 0.2 mg·L -1 2,4-D, 0.1 mg·L -1 6-BA, 30 g·L -1Sucrose, plant gel, antibiotics, pH adjusted to 5.8.

[0040] The composition of the maturation induction medium is: MS medium, 0.3 mg·L -1 ABA, 1 g·L -1 Graphite powder, 30 g·L -1 Sucrose, plant gel, antibiotics, pH adjusted to 5.8;

[0041] The composition of the germination induction medium is: MS medium, 1 mg·L -1 GA, 0.05 mg·L -1 2,4-D, 0.5 mg·L -1 6-BA, 1 g·L -1 Graphite powder, 30 g·L -1 Sucrose, plant gel, antibiotics, pH adjusted to 5.8;

[0042] The composition of the basic growth medium is: MS medium, 1 g·L -1 Graphite powder, 30 g·L -1 Sucrose, plant gel, antibiotics, pH adjusted to 5.8.

[0043] Example 1:

[0044] 1. Construction of the fusion expression vector of CsAlaDC

[0045] The CsAlaDC gene fragment was amplified from the tea tree genome, and the ORF frame without the stop codon was fused into the expression vector pBI121 through the restriction enzyme sites Xba I and BamH I. High-concentration (1 μL·μL -1 ) plasmid was obtained through Escherichia coli transformation and large-scale plasmid extraction; the CsAlaDC gene is the theanine synthase gene, and the CDS nucleotide sequence is as shown in SEQ ID No.1; the nucleotide sequence of the pBI121 plasmid is as shown in SEQ ID No.2.

[0046] 2. Somatic embryo pretreatment

[0047] Somatic embryos with a size of 0.3 - 0.7 cm were selected, the somatic embryos were cut in half, and the wound surface was inoculated on the pretreatment medium and cultured in the dark at 23°C for 4 h.

[0048] 3. Gold powder washing and embedding

[0049] Weigh 30 mg of gold powder (diameter 0.6 μm) and put it into a 1.5 mL centrifuge tube, add 1 mL of 70% ethanol, vortex for 5 minutes, let it stand for 15 minutes, and then centrifuge at 5000 rpm·min -1Centrifuge for 5 seconds and remove the supernatant; add 1 mL of sterile water, vortex for 1 minute, let stand for 1 minute, and then centrifuge at 5000 rpm·min -1 Centrifuge for 5 seconds and remove the supernatant. Repeat this step 3 times. Finally, add 500 μL of 50% sterile glycerol to make the concentration of the gold powder suspension 60 mg·mL -1 . Take the above gold powder suspension, add 500 μL of 50% sterile glycerol and resuspend. After resuspension, pipette 100 μL of the gold powder resuspension into a 1.5 mL centrifuge tube, and sequentially add 10 μg of pBI121-CsAlaDC plasmid, 100 μL of 2.5 M CaCl 2 and 40 μL of 100 mM spermidine, vortex for 3 minutes, let stand for 5 minutes, and then centrifuge at 5000 rpm·min -1 Centrifuge for 2 seconds and remove the supernatant. Add 280 μL of 70% ethanol, centrifuge and remove the supernatant. Add 280 μL of 100% ethanol, centrifuge and remove the supernatant. Finally, add 96 μL of 100% ethanol and flick gently to mix well.

[0050] 3. Gene gun-mediated gene transformation of tea tree somatic embryos

[0051] (1) Soak the carrier membrane, carrier disk, 1100 psi rupture disk, and stopping screen in 75% alcohol for 10 minutes, and then place them on sterile filter paper in a laminar flow hood to dry.

[0052] (2) Mount the carrier membrane on the carrier disk, pipette 8 μL of the resuspension from step 3 above, smear it on the carrier membrane, and place it on sterile filter paper to dry the alcohol.

[0053] (3) Place the dried 1100 psi rupture disk, stopping screen, and the carrier disk prepared in (2) above on the gene gun.

[0054] (4) Place the Shuchazao tea tree somatic embryos in the center of a sterile petri dish, place it in the middle of the sample disk, and the sample disk is 6 cm away from the rupture disk. (The tea tree somatic embryos are also pretreated before gene gun bombardment. Specifically: The tea tree somatic embryos are inoculated on the pretreatment medium and cultured for 5 hours; the tea tree somatic embryos are healthy somatic embryos with a diameter of 0.4 cm)

[0055] (5) Start the gene gun vacuum device. When the vacuum reaches 29 Hg, conduct bombardment, and bombard each group of samples 2 times.

[0056] (6) Inoculate the transgenic somatic embryos into the somatic embryo induction medium containing 50 mg·L -1 Kan antibiotic, and culture them in the dark at 23 °C for 2 weeks.

[0057] 4. GUS tissue staining to identify transgenic somatic embryos

[0058] (1) Take the samples cultured for two weeks in the above step (6) and rinse the surface of somatic embryos with PBS buffer solution.

[0059] (2) Put the rinsed somatic embryos into the GUS staining solution and stain them at 37 °C in the dark until the somatic embryos turn blue.

[0060] (3) Rinse the chromosomal somatic embryos in step (2) three times with PBS buffer solution and take pictures of the somatic embryos for preservation.

[0061] As Figure 1 shown, the transgenic positive embryos show blue, while the embryos of the non-transgenic control group remain white.

[0062] 5. Verification of the expression pattern of CsAlaDC in transgenic somatic embryos by qRT-PCR

[0063] (1) Design quantitative primers according to the CsAlaDC gene sequence

[0064] Forward primer: 5’-CAACAAGCCTCACCATCACG-3’ (SEQ ID No.3)

[0065] Reverse primer: 5’-ATAGACGGGCAAACCAATCC-3’ (SEQ ID No.4)

[0066] (2) qRT-PCR amplification reaction: Reaction system: 5 μl of SYBR Mix enzyme, 0.6 μl of primer mixture, 1.5 μl of template DNA, make up to 10 μl with sterilized deionized water. Put the above reaction tubes into the qRT-PCR instrument for reaction. Reaction procedure:

[0067] 1) Pre-denaturation at 95 °C for 30 s;

[0068] 2) Denaturation at 95 °C for 10 s;

[0069] 3) Annealing temperature: 58 °C, annealing time: 30 s:

[0070] 4) Repeat steps ② and ③ for 40 cycles:

[0071] As Figure 2 shown, the expression level of CsAlaDC in transgenic somatic embryos is significantly up-regulated compared with that of non-transgenic somatic embryos.

[0072] 6. Culture of positive seedlings

[0073] (1) Inoculate the transgenic somatic embryos onto the somatic embryo induction medium containing 50 mg·L -1 Kan antibiotic. As Figure 4 shown in E, secondary embryos occur after 2 weeks of culture.

[0074] (2) Inoculate the somatic embryos undergoing secondary embryogenesis onto a maturation induction medium containing 50 mg·L -1 Kan antibiotic, as shown in Figure 4 Figure F, and cotyledon embryos will occur after 28 days of culture.

[0075] (3) Inoculate the cotyledon embryos onto a germination induction medium containing 50 mg·L -1 Kan antibiotic, as shown in Figure 4 Figure G, and seedlings with germinated buds and leaves will be obtained after 34 days of culture.

[0076] (4) Inoculate the transgenic seedlings with germinated buds and leaves onto a basic growth medium for culture to obtain CsAlaDC transgenic seedlings.

[0077] (5) Verify the integration of the exogenous plasmid in the transgenic somatic embryos by PCR

[0078] 1) Design amplification primers according to the pBI121-CsAlaDC plasmid sequence

[0079] Forward primer: 5’-AGCCACTTACTTTGCCATCT-3’ (SEQ ID No.5)

[0080] Reverse primer: 5’-TTTGCTCGGAAGAGTATGAA-3’ (SEQ ID No.6)

[0081] 2) PCR amplification reaction: Reaction system: 12.5 μl of Taq Mix enzyme, 2 μl of primer mixture, 1 μl of template DNA, and make up to 25 μl with sterilized deionized water. Put the above reaction tubes into a PCR instrument for reaction. Reaction program:

[0082] ① Pre-denature at 94 °C for 3 min;

[0083] ② Denature at 94 °C for 15 s;

[0084] ③ Annealing temperature: 58 °C, annealing time: 30 s;

[0085] ④ Extend at 72 °C for 25 s,

[0086] ⑤ Cycle steps ②, ③, and ④ for 30 cycles;

[0087] ⑥ Extend at 72 °C for 5 min;

[0088] ⑦ Store at 4 °C.

[0089] As shown in Figure 3As shown, there is a band at 327 bp, indicating that the exogenous plasmid has been successfully introduced into the tea tree seedlings. If there is no band at 327 bp, it means that the exogenous plasmid has not been successfully transformed in the tea seedlings.

[0090] Example 2:

[0091] The difference between this example and Example 1 is that the specific parameters of the gene gun are as follows: gold powder size 1.0 μm, bombardment pressure 1300 psi, bombardment distance 9 cm, and vacuum condition 26 Hg.

[0092] Example 3:

[0093] The difference between this example and Example 1 is that the specific parameters of the gene gun are as follows: gold powder size 1.6 μm, bombardment pressure 1550 psi, bombardment distance 3 cm, and vacuum condition 32 Hg.

[0094] The CDS sequence of the theanine synthase CsAlaDC gene (SEQ ID NO.1)

[0095]

[0096] The CDS sequence of the pBI121 plasmid (shown as SEQ ID NO.2)

[0097] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for genetic transformation of tea plants mediated by gene gun, characterized in that: The following steps are involved: S1: using a gene gun method to bombard a vector containing a target gene into tea somatic embryos to obtain transgenic somatic embryos; the specific parameters of the gene gun are: the diameter of the gold powder is 0.6-1.6 μm, the bombardment pressure is 1100-1550 psi, the bombardment distance is 3-9 cm, and the vacuum condition is 26-32 Hg; S2: The transgenic somatic embryos are inoculated into somatic cell induction medium to obtain secondary embryos; S3: inoculating the secondary embryos onto a maturation induction medium and culturing them to obtain cotyledon embryos; S4: inoculating the cotyledon embryos onto a germination induction medium and culturing them to obtain seedlings with bud and leaf germination; S5: The seedlings with sprouted buds and leaves are inoculated onto a basic growth medium for cultivation to obtain transgenic tea plants.

2. The method according to claim 1, characterized in that: Step S1 specifically comprises: constructing a fusion expression vector containing the target gene, embedding the vector into gold powder, and using a gene gun to introduce the target gene into tea plant somatic cells by high-speed bombardment of helium.

3. The method according to claim 1, characterized in that The specific parameters of the gene gun are: bombardment pressure 1100psi, bombardment distance 6cm, vacuum condition 29Hg.

4. The method according to claim 1, characterized in that The diameter of the gold powder is 0.6, 1.0 or 1.6 μm.

5. The method according to claim 1, characterized in that In step S1, the variety of tea tree is any one or more of Shucha Zao and Longjing 43; the target gene is CsAlaDC gene, and the CDS sequence of CsAlaDC gene is shown in SEQ ID No.

1.

6. The method according to claim 1, characterized in that The tea tree somatic embryos are also pretreated before the gene gun bombardment, specifically: the tea tree somatic embryos are inoculated on the pretreatment medium and cultured for 4-6 hours; the tea tree somatic embryos are robust somatic embryos with a diameter of 0.3-0.5 cm; the pretreatment medium is composed of: MS basic medium, 30 g·L -1 Sucrose, 100 mM mannitol, plant gel, pH adjusted to 5.8; culture conditions are 23°C and protected from light.

7. The method according to claim 1, characterized in that In step S2, the somatic cell induction medium is composed of: WPM medium, 0.2 mg·L -1 2,4-D, 0.1mg·L -1 6-BA, 30g·L -1 Sucrose, plant gel, antibiotics, pH adjusted to 5.8; culture at 23°C in the dark for 2 to 3 weeks, with subculture every 14 to 21 days during the culture process.

8. The method according to claim 1, characterized in that In step S3, the composition of the maturation induction medium is: MS medium, 0.3 mg·L -1 ABA, 1 g L -1 Graphite powder, 30g·L -1 Sucrose, plant gel, antibiotics, pH adjusted to 5.8; cultured at 26°C, 8h dark 16h light conditions for 30-40 days, with subculture every 14-21 days during the culture process.

9. The method according to claim 1, characterized in that: In step S4, the germination induction medium is composed of: MS medium, 1 mg·L -1 GA, 0.05mg·L -1 2,4-D, 0.5mg·L -1 6-BA, 1g·L -1 Graphite powder, 30g·L -1 Sucrose, plant gel, antibiotics, pH adjusted to 5.8; cultured at 26°C, 8h dark 16h light conditions for 30-40 days, with subculture every 14-21 days during the culture process.

10. The method according to claim 1, characterized in that In step S5, the basic growth medium is composed of: MS medium, 1 g·L -1 Graphite powder, 30g·L -1 Sucrose, plant gel, antibiotics, pH adjusted to 5.8; cultured at 26°C, 8h dark 16h light, with subculture every 14 to 21 days during the culture process.

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