Tomato SlGATA6 overexpression vector and application thereof

Abstract

The invention relates to the field of plant genetic engineering and molecular breeding, in particular to a tomato SlGATA6 overexpression vector and application thereof, and the nucleotide sequence of the tomato SlGATA6 overexpression vector is as shown in SEQ ID NO.1. The tomato SlGATA6 overexpression vector is used for improving the fruit setting rate of tomatoes under weak light, compared with an existing technology for improving the fruit setting rate of the tomatoes under the weak light, the tomato SlGATA6 overexpression vector is constructed and infects the tomatoes, an SlGATA6 overexpression tomato plant is obtained, the plant shows the phenotype of delayed falling, and the fruit setting rate is remarkably increased under the weak light. The method established by the invention can be used for regulating and controlling fruits of solanaceae crops, and can improve the biological yield of horticultural crops and accelerate the creation of new varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering and genetic breeding, and more specifically, to a tomato SlGATA6 overexpression vector and an application thereof, and in particular to an application of the tomato SlGATA6 overexpression vector in cultivating tomatoes with a high fruit setting rate under weak light. Background Art

[0002] Plant organ abscission is a common programmed process in the growth and development of plants in nature. During this process, the nutritional organs or reproductive organs of the plant body are separated from the mother plant. The site where the break occurs and its adjacent area are usually called the abscission zone (AZ). The abscission phenomenon has both advantages and disadvantages in agricultural production. In mechanized production, products that are easy to abscission after maturity are cultivated, which is conducive to the mechanical harvesting of agricultural products. However, abscission during the non-mature period, such as the falling of flowers and fruits of tomatoes, will seriously affect the yield and restrict agricultural development. The phenomenon of flower and fruit abscission of tomatoes is affected by many factors, among which environmental factors are an important part. In tomato production, weak light stress is often produced due to restrictions on seasons, regions and other factors. In severe cases, tomato yields can be reduced by 90%, which is an important factor restricting the increase in tomato yields.

[0003] In plants, GATA transcription factors are highly conserved in evolution. The GATA genes of tomato, Arabidopsis and rice can be divided into four subfamilies (I-IV). They are involved in the signal transduction of the plant hormone auxin and in regulating the photomorphogenesis of plants in response to light signals. However, whether GATA transcription factors can regulate plant abscission in response to auxin signals and affect tomato yield under low light and the potential regulatory mechanism remain unknown. Summary of the invention

[0004] In order to make up for the deficiencies of the above-mentioned prior art, the purpose of the present invention is to construct a tomato SlGATA6 overexpression vector for regulating tomato yield under weak light, and transfect the vector into wild-type tomatoes through genetic transformation to make them expressed. The obtained transgenic plants can regulate the tomato flower abscission rate and fruit setting rate under weak light, and then be used for the breeding of high-yield tomato varieties.

[0005] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0006] In a first aspect of the present invention, a tomato SlGATA6 overexpression vector is provided, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The second aspect of the present invention is an application of the above-mentioned tomato SlGATA6 overexpression vector.

[0008] In some embodiments, the use of the tomato S1GATA6 overexpression vector includes any one or more of the following:

[0009] 1) Used to inhibit tomato pedicels from falling off under low light conditions;

[0010] 2) Used to increase the yield of Solanaceae crops under low light conditions.

[0011] In some embodiments, the tomato SlGATA6 overexpression body is used, and the Solanaceae crops include eggplant, pepper, and potato.

[0012] In some embodiments, in the application of tomato SlGATA6 overexpression vector, the illumination intensity of the weak light is 10000-15000 lx.

[0013] In some embodiments, the tomato SlGATA6 overexpression vector is used to transfer the tomato SlGATA6 overexpression vector into tomato plants.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) The present invention clearly shows that the introduction of the SlGATA6 overexpression vector into tomatoes can significantly inhibit tomato pedicel shedding. Compared with the common wild-type plants, which reach a shedding rate of nearly 100% 36 hours after defloration, the SlGATA6 overexpression plants only have about 40% shedding at 36 hours. This significant difference means that in the actual planting process, fruit loss can be effectively reduced, providing a solid guarantee for high tomato yields, and confirming its role in the breeding of high-yield tomato varieties.

[0016] (2) The present invention clarifies that the introduction of the SlGATA6 overexpression vector into tomatoes can significantly reduce the rate of tomato pedicel shedding under weak light and increase the fruit setting rate of tomatoes. Compared with ordinary wild-type plants that only maintain a fruit setting rate of about 40% under weak light, SlGATA6 overexpression plants can maintain a fruit setting rate of about 70% under weak light. This improvement not only helps the selection and breeding of high-yield tomato varieties, but also makes tomato planting no longer overly dependent on light conditions, expands the planting area and planting time of tomatoes, and can achieve stable tomato production even in areas or seasons with insufficient light, which not only helps the selection and breeding of high-yield tomato varieties, but also can further improve land utilization and the stability of agricultural production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the map of the SlGATA6 overexpression vector;

[0018] Figure 2 The results of SlGATA6 expression analysis in the abscission zone of tomato pedicel in SlGATA6 overexpressing transgenic plants;

[0019] Figure 3is the flower drop rate of SlGATA6 overexpressing transgenic plants under low light conditions;

[0020] Figure 4 The phenotype of SlGATA6 overexpressing transgenic plants under low light conditions;

[0021] Figure 5 This is a schematic diagram of the fruit setting of wild-type, SlGATA6-OE#2, SlGATA6-OE#6 and SlGATA6-OE#10 tomato plants in Example 2. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should belong to the scope of protection of the present invention. Unless otherwise specified, the technical means used in each of the embodiments listed below are all conventional technical means familiar to those skilled in the art, and the various materials, reagents, etc. used in the embodiments can be obtained from commercial channels unless otherwise specified.

[0023] The present invention provides a tomato SlGATA6 overexpression vector, the nucleotide sequence of which is shown in SEQ ID NO.1, and the vector spectrum is shown in Figure 1 As shown. The present invention uses genetic transformation to express the tomato overexpression SlGATA6 vector in tomatoes. The tomato SlGATA6 overexpression vector can be used to regulate the shedding of pedicels under weak light, can effectively inhibit the shedding of tomato pedicels, and increase tomato yield. Especially under weak light conditions, the fruit setting rate of tomato plants transferred with tomato overexpression SlGATA6 reaches 70%.

[0024] Example 1: Construction of tomato SlGATA6 overexpression vector and plant

[0025] The nucleotide sequence of the tomato SlGATA6 overexpression vector (SEQ ID NO: 1) is as follows:

[0026]

[0027] The tomato SlGATA6 overexpression vector was obtained by the whole gene synthesis method, and the constructed fusion expression vector plasmid was transformed into the competent Agrobacterium GV1301 by chemical freeze-thaw method, and then coated on the plate culture medium containing 50mg / L Kana and 25mg / L Rif antibiotics. Inverted culture was carried out at 28℃ for 48h, and single clones were selected for PCR detection. The Agrobacterium with the correct electrophoresis band was selected for propagation and reserved for subsequent infection. The detection primers used were:

[0028] F:5'-ACGCACAATCCCACTATCCT-3'

[0029] R:5'-TGTAAAACGACGGCCAGT-3'

[0030] Specifically, the construction of the tomato SlGATA6 overexpression vector includes the following steps:

[0031] (1) Through Anshengda The whole gene synthesis method was to synthesize the 361-1463 bp fragment in the sequence described in SEQ ID NO: 1, and the synthesized fragment was ligated to the pCAMBIA1300-35S (P17949, Miaoling Biotechnology) vector after being digested with BamH I (1010S, Takara) and Sal I (1080S, Takara) using In-Fusion (639649, Clontech) ligase to construct the pCAMBIA1300-SlGATA6 fusion expression vector. The map of the fusion expression vector is shown in FIG. Figure 1 The constructed pCAMBIA1300-SlGATA6 fusion expression vector plasmid was transformed into Agrobacterium GV1301 competent cells by chemical freeze-thaw method. The specific operation was to add 100 ng of plasmid to 100 μl of thawed Agrobacterium GV1301 competent cells, ice water bath for 5 min, liquid nitrogen for 5 min, and then transfer to 37°C water bath for 5 min. Then add 1ml of non-resistant YEP medium, culture at 28℃ for 1h, centrifuge the revived bacterial solution at 6000rpm for 1min to collect the bacterial solution, remove the supernatant and spread it on YEP plate medium (tryptone (10g / L, ST800, Biyuntian), yeast extract (10g / L, Y8020-500, Solebao), NaCl (5g / L, S8210-100, Solebao), agar powder (15g / L, S232-658-1, Solebao)) containing 50mg / L Kana (ST102, Biyuntian) and 25mg / L Rif (ST1632, Biyuntian) antibiotics. Culture at 28℃ inverted for 48h, select single clones for PCR detection. The detection primers used are:

[0032] F:5'-ACGCACAATCCCACTATCCT-3'

[0033] R:5'-TGTAAAACGACGGCCAGT-3'

[0034] (2) Select a monoclonal Agrobacterium with an electrophoresis band of 1500 bp and add it to 1 ml of YEP liquid culture medium containing 50 mg / L Kana and 25 mg / L R If antibiotics. Culture it at 28°C and 150 rpm for 16 h for propagation. Use the Agrobacterium-mediated method to transfer it into tomatoes, and obtain single-copy inserted homozygous overexpression transgenic tomatoes through tissue culture technology.

[0035] (3) Identification of SlGATA6 overexpressing transgenic tomatoes. RNA from the pedicel abscission region of transgenic tomatoes was extracted and cDNA was obtained by reverse transcription. Real-time fluorescence quantitative primers were used:

[0036] F:5'-AGAAACCAAAAAAGAATCCATCAG-3'

[0037] R:5'-AGAAGCTAAGCCGGAGTCAATA-3'

[0038] Using the tomato ACTIN gene (NCBI: NM_001330119.1) as the internal reference gene, real-time fluorescence quantitative PCR was used to detect the change in the expression level of SlGATA6 in the abscission zone of the overexpression strain compared with the wild-type strain, and SlGATA6 overexpression transgenic tomatoes were obtained.

[0039] Specifically, the construction of tomato SlGATA6 overexpression plants includes the following steps:

[0040] Select the tomato wild type (Ailsa Craig) seeds of uniform size and fullness harvested that year, rinse with sterile water 3-5 times in a clean bench, soak the seeds in 70% alcohol for 30 seconds, and then place the seeds in a solution containing 10% sodium hypochlorite for 10 minutes. After disinfection, rinse with sterile water 4-5 times, then use sterile filter paper to absorb the surface moisture, and then inoculate into seed germination medium (MS medium, M7150, Sigma), 40-50 seeds per dish. Incubate in the dark at (24±2)℃ for about 1-2 days, wait until most seeds germinate and turn white, and transfer to a light culture environment, with 16 hours of light per day, light intensity maintained at 1600-1800lx, and temperature controlled at (24±2)℃.

[0041] When the seeds in the germination medium (MS medium) grow to the point where the cotyledons are fully expanded but the true leaves have not yet grown, the explants can be transformed. During the operation, first place the seeds on filter paper, then carefully remove the tips and petioles of the cotyledons with a surgical blade, and cut the remaining cotyledons into 3-4 sections. After cutting, place these leaf pieces horizontally on the pre-culture medium (MS medium + 2,4-D (1 mg / L, ST1760, Biyuntian) + 6-BA (0.5 mg / L, ST1065, Biyuntian)), with 15-20 pieces placed in each culture dish. The culture conditions are the same as before, and a 1-day pre-culture is carried out.

[0042] The previously transformed Agrobacterium was shaken to OD 600 =0.8-1.0, prepare the infection solution. Collect the shaken bacteria by centrifugation and suspend them in the infection solution to infect the cut cotyledons. Pour the bacterial solution into a glass dish. Gently shake the culture dish to allow the cotyledons to fully contact the bacterial solution and infect for 2min 30s. After the infection is completed, remove the excess bacterial solution, leaving 2-3mL of bacterial solution. Use the remaining bacterial solution to scoop the cotyledons onto the filter paper of a 9cm plastic culture dish with tweezers, use the filter paper to fully absorb the bacterial solution on the leaves, ensure that the leaves are dry and not sticky, and transfer the cotyledon end to the original pre-culture medium. After sealing with a single layer of sealing film, place it in a dark box to ensure air circulation and co-culture at 25 degrees for 2 days.

[0043] After the co-cultivation of explants is completed, transfer them to the screening medium (MS medium + 6-BA (1 mg / L) + NAA (0.1 mg / L, ST1758, Biyuntian) + hygromycin (20 mg / L, ST1389, Biyuntian)) for selection culture. The light intensity is maintained at 2000-3000lx for 16 hours per day, and the temperature is controlled at (25±2)℃. After 3-5 days of cultivation, the cotyledons gradually become thicker. The successfully transformed explants will slowly form callus tissue and adventitious buds on this culture medium. Subculture is required every two weeks, and screening is continued for 2-3 generations for a total of 4-6 weeks.

[0044] Adventitious bud elongation culture: Cut the callus with bud primordium into small pieces and transfer to stem elongation medium (MS medium + 6-BA (1mg / L) + NAA (0.1mg / L) + gibberellin GA3 (0.2mg / L, ST1782, Biyuntian) + hygromycin (20mg / L)) for subculture, with 16 hours of light per day, light intensity maintained at 2000-3000lx, and temperature controlled at (25±2)℃. Transfer once every two weeks; if necessary, the transfer interval can be shortened appropriately. After 2-4 weeks of culture, observe the elongation of the adventitious buds.

[0045] Adventitious bud rooting culture: When the adventitious buds grow to about 1 cm, select the healthy regenerated buds, completely remove the callus tissue and culture medium at the base, and transfer them to the rooting medium (MS medium + IBA (0.2mg / L, ST1756, Biyuntian) + hygromycin (20mg / L)), with 16 hours of light per day, the light intensity is maintained at 2000-3000lx, and the temperature is controlled at (25±2)℃. Promote the development of complete plants.

[0046] After 4-7 days of rooting culture, sampling and identification are carried out. PCR identification proves that it has been transferred into the target vector. After the positive seedlings that have been successfully identified grow lateral roots, open the bottle cap, pour a small amount of sterile water into the culture bottle, so that it covers the culture medium by 3-5 mm, and place the culture bottle in a cool and ventilated place to train the seedlings. After 3 days, wash the root culture medium and transplant the seedlings into the soil. Cover the seedlings with transparent plastic film for the first 7 days and culture them in a weak light environment (light intensity is maintained at 1000-2000lx) for a period of time to help the regenerated seedlings adapt to the transition from culture medium to nutrient matrix. After the seedlings survive, move them to the natural environment to continue to train the seedlings, and finally transplant them to the field. The primers required for the detection of positive plants are as follows:

[0047] F:5'-ACGCACAATCCCACTATCCT-3'

[0048] R:5'-TGTAAAACGACGGCCAGT-3'

[0049] When the transgenic tomatoes grow in the field and reach flowering and fruiting, the abscission area of ​​the tomato pedicel is taken, ground with liquid nitrogen, and RNA is extracted from the abscission area using a plant RNA extraction kit (DP432, Tiangen) and referring to the RNA extraction method described in the kit manual; reverse transcription is performed according to the Takara two-step reverse transcription operation steps to obtain cDNA from the abscission area. The obtained cDNA is used as a template for real-time fluorescence quantitative PCR, using real-time fluorescence quantitative primers:

[0050] F:5'-AGAAACCAAAAAAGAATCCATCAG-3'

[0051] R:5'-AGAAGCTAAGCCGGAGTCAATA-3'

[0052] Using the tomato ACTIN gene as the internal reference gene, real-time fluorescence quantitative PCR was used to detect the changes in the expression of SlGATA6 in the abscission zone of the overexpression strain compared with the wild-type strain. Transgenic tomatoes overexpressing SlGATA6 were obtained. By detecting the expression level, homozygous overexpression transgenic tomatoes with a single copy insertion were obtained. The results are shown in Figure 2As shown in the figure, compared with the expression level of SlGATA6 in the abscission zone of wild-type plants, the expression level of SlGATA6 in the overexpression plants increased by 10 to 17 times, indicating that we successfully obtained SlGATA6 overexpression transgenic plants. We identified three SlGATA6 overexpression plants, named SlGATA6-OE#2, SlGATA6-OE#6, and SlGATA6-OE#10.

[0053] Example 2: Detection of shedding rate of SlGATA6 overexpressing plants under weak light

[0054] The wild-type and overexpression plants (SlGATA6-OE#2, SlGATA6-OE#6, SlGATA6-OE#10) were sown in the experimental field and cultured under normal light, 16 hours of light per day, and a temperature controlled at (25±2)°C. When the tomatoes grew to the flowering stage, the flowers that opened on the same day were taken, and the flower organs were manually removed with a blade. The proximal end of the pedicel was inserted into a culture medium containing 1% agarose and placed in a transparent glass cover. The pedicel shedding rate was tested in an environment with a temperature of 25°C and a humidity of 70-80%. The results are as follows: Figure 3 As shown, the rate of pedicel shedding in SlGATA6-overexpressing plants was significantly delayed. At 36 hours after flower removal, the wild-type plants showed nearly 100% shedding, while the SlGATA6-overexpressing plants only had about 40% shedding. This indicates that SlGATA6-overexpressing plants can significantly delay pedicel shedding. In order to detect the shedding of wild-type and SlGATA6-overexpressing plants under low light, shading was started when the first inflorescence of tomatoes grew until the inflorescence completely stopped fruiting. The light intensity under normal light is 30,000-40,000 lx, and under weak light conditions it is 10,000-15,000 lx. After the inflorescence has completely fruited, the number of flowers / fruits that have fallen off in each inflorescence was checked. At least 6 biological replicates were performed for each line. The results are shown in Figure 3 Figure 4 The wild-type tomato had a flower drop rate of about 60% and a fruit set rate of about 40% under weak light, while the SlGATA6 overexpression plant had a flower drop rate of about 30% and a fruit set rate of about 70%, indicating that the SlGATA6 overexpression plant has the effect of inhibiting tomato pedicel shedding under weak light, and the overexpression plant showed a higher fruit set rate.

[0055] The present invention constructs a SlGATA6 overexpression vector and infects tomatoes to obtain SlGATA6 overexpression tomato plants, which show a phenotype of delayed shedding and a significantly increased fruit setting rate under weak light. The method established by the present invention can be used for fruit regulation of Solanaceae crops, and can increase the biological yield of horticultural crops and accelerate the creation of new varieties.

[0056] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A tomato SlGATA6 overexpression vector, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

1.

2. An application of the tomato SlGATA6 overexpression vector according to claim 1.

3. The use of the tomato SlGATA6 overexpression vector according to claim 2, characterized in that: Any one or more of the following: 1) Used to inhibit tomato pedicels from falling off under low light conditions; 2) Used to increase the yield of Solanaceae crops under low light conditions.

4. The use of the tomato SlGATA6 overexpression vector according to claim 3, characterized in that: The Solanaceae crops include tomatoes, eggplants, peppers and potatoes.

5. The use of the tomato SlGATA6 overexpression vector according to claim 4, characterized in that: The Solanaceae crop is tomato.

6. The use of the tomato SlGATA6 overexpression vector according to claim 3, characterized in that: The illumination intensity of the weak light is 10000-15000 lx.

7. The use of the tomato SlGATA6 overexpression vector according to claim 2 or 3, characterized in that: The tomato SlGATA6 overexpression vector was introduced into tomato plants.

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