Application of CaGATA4 gene in regulating development of plant meristem

Abstract

The application discloses application of a CaGATA4 gene in regulation of development of plant meristems, wherein the nucleotide sequence of the CaGATA4 gene is shown as SEQ ID NO. 1. The application applies the CaGATA4 gene in regulation of development of tomato meristems, overexpresses the CaGATA4 gene in the tomato, and makes the stem end meristem development stagnate; with growth and development of the plant, new growth points are grown on part of the plants, and the plants restore growth; the CaGATA4 gene is applied in the pepper, and CaGATA4 silencing plants develop lateral branches in advance, promote development of the pepper plant meristem, and improve early yield of the pepper.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, and particularly relates to a... CaGATA4 Application of genes in regulating the development of plant meristems. Background Technology

[0002] Plant meristems are cell populations located in specific parts of a plant and possess the ability to continuously or periodically divide. They can proliferate and form other tissues and organs through the activity of stem cells within their tissues. Apical meristems are specialized regions formed at both ends of the longitudinal axis of the embryo during plant embryonic development, called shoot apical meristems and root apical meristems, respectively. Plant meristems play a crucial role in plant development; all plant organs originate from the differentiation and division of cells within meristems. Lateral organs, stems, leaves, and flowers in the aboveground parts of plants all differentiate from shoot apical meristems. Therefore, research on pepper meristems not only contributes to a deeper understanding of the growth and development mechanisms of peppers but also provides theoretical support and technical guidance for improving pepper yield and quality.

[0003] In recent years, several genes involved in regulating plant meristem development have been reported. For example... STIP Gene mutation causes the shoot apical meristem to no longer swell excessively; APETALA2 ( AP2 Genes can positively regulate WUS Gene expression, negative regulation CLV3 Expression, showing a phenotype of premature termination of shoot apical meristem; MERISTEM DEFECTIVE ( MDF The mutation leads to abnormal development of the shoot apical meristem, resulting in a premature termination phenotype. ULT1 The mutation produces enlarged inflorescence meristems and floral meristems; FASCIATED ( FAS1 )and( FAS2 Mutations result in severe defects in stem segments of the shoot apical meristem.

[0004] GATA Genes, as one of the key regulators of plant growth and development, contain highly conserved IV zinc finger domains. Many GATA Transcription factors play an important role in plant resistance to abiotic stresses. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above and to provide an application of the CaGATA4 gene in regulating the development of plant meristems.

[0006] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0007] A sort of CaGATA4 The application of genes in regulating plant meristem development, the aforementioned CaGATA4 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] The above CaGATA4 The application of genes in regulating plant meristem development, preferably, the aforementioned CaGATA4 The protein sequence of the gene is shown in SEQ ID NO.2.

[0009] The above CaGATA4 The application of genes in regulating the development of plant meristems, preferably, the plant meristems include at least one of plant shoot apical meristems or plant lateral meristems.

[0010] The above CaGATA4 The application of genes in regulating the development of plant meristems, preferably, the plant includes tomato or pepper.

[0011] The above CaGATA4 The application of genes in regulating plant meristem development, preferably, overexpression of the aforementioned gene in tomatoes. CaGATA4 Genes cause stunted growth of the shoot apical meristem.

[0012] The above CaGATA4 Genes regulate plant meristem Applications in tissue development, preferably, overexpression in tomatoes. CaGATA4 Genes, including the following steps: (1) Homologous recombination of the sequence shown in SEQ ID NO.1 into the pHellsgate8 vector to construct CaGATA4 Gene overexpression vectors; (2) The overexpression vector from step (1) was introduced into tomato cells and cultured. CaGATA4 Transgenic tomato plants with overexpressed genes.

[0013] The above CaGATA4 The application of genes in regulating plant meristem development, with preferred silencing in peppers. CaGATA4 Genes that differentiate into lateral branches prematurely.

[0014] The above CaGATA4 The application of genes in regulating plant meristem development, with preferred silencing in peppers. CaGATA4 Genes, including the following steps: (1) Use the VIGS online tool (http: / / vigs.solgenomics.net) on the Solanaceae Genome website to select CaGATA4 Specific segments of the gene undergo homologous recombination in the pTRV2 vector; (2) After transformation mediated by Agrobacterium, pTRV2- CaGATA4 After mixing Agrobacterium with Agrobacterium containing pTRV1, the mixture was injected into pepper leaves where the cotyledons were fully expanded and new leaves had not yet grown.

[0015] The above CaGATA4 Application of genes in regulating plant meristem development, with pTRV2 being the preferred choice. CaGATA4 The volume ratio of Agrobacterium to pTRV1 Agrobacterium was 1:1.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention will CaGATA4 Genes are being applied to chili peppers. CaGATA4 Silent plants develop lateral branches earlier, which promotes the development of meristematic tissue in pepper plants and increases the early yield of peppers. Attached Figure Description

[0017] Figure 1 For overexpressing genes CaGATA4 Identification of positive plants, detection of expression levels, and analysis of tomato "A57" and overexpression. CaGATA4 Comparison of tomato plants based on genes; among which " "Indicates P < 0.01" Figure 2 For "Zhangshugang" chili peppers and CaGATA4 Comparison of gene-silenced pepper plants and detection of expression levels; among them, " "Indicates P < 0.01" Detailed Implementation To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0018] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0019] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0020] The invention involves CaGATA4 The application of genes in regulating plant meristem development, among which, CaGATA4 The nucleotide sequence of the gene is as shown in SEQ ID NO.1: Nucleotide sequence: ATGGATGTCTACGGAGTATCTGCACCCGACTTGTTTCGTATTGATGATCTTCTCGATTTCTCTAATGATCAAATTTTCTCCACTAACAACACCAACCCCGACTCCTCCTCTCACCAGTACCACCATCAACCTCACTCCCACAACTCTTCCTCCGCCGCCACTGCCACTTACTACGACAATCTTCTTCCTAATTCCTCCGATGACTTCACCGACAACCTCTACGTCCCCAGTGATGATGTAGCGGAGTTAGAATGGCTATCGAACTTTGTAGAAGATTCATTCAGCAATTTTTCAGCCAATTCCATTACCGGAACGATGAATCTCAGTTCCAATACTACGTCGTTTCACGGAAGGTCAAGAAGCAAACGTTCTCGTTCAACTTCAAGCTGGACAACCTCACTCCAAAACCCTAACACAACTACTACTACAAAGAACAAGGAGAGTTCATCAGTACATACCAGAGAAAGGTCATTTTCGCCAATGGATGAAGATGCACCAAGGAGATGCACACACTGCGCTTCGGAGAAGACACCGCAGTGGAGAACCGGGCCTCTAGGCCCGAAAACACTCTGCAATGCATGTGGAGTTAGGTACAAATCGGGCCGGCTTGTACCGGAGTACCGGCCCGCAGCAAGCCCGACTTTCGTGTTAACACAGCATTCAAATTCTCACCGGAAAGTTATGGAGCTCCGTCGACAGAAGGAAATGCTACATCAACCACAGCAGCTTCCGCCGCCGACGACGGAGGAGGGCATGTACGGACGTCACTTTCGGGTCTGCTGA; The protein sequence thereof is (as shown in SEQ ID NO. 2): Protein sequence: MDVYGVSAPDLFRIDDLLDFSNDQIFSTNNTNPDSSSHQYHHQPHSHNSSSAATATYYDNLLPNSSDDFTDNLYVPSDDVAELEWLSNFVEDSFSNFSANSITGTMNLSSNTTSFHGRSRSKRSRSTSSWTTSLQNPNTTTTTKNKESSSVHTRERSFSPMDEDAPRRCTHCASEKTPQWRTGPLGPKTLCNACGVRYKSGRLVPEYRPAASPTFVLTQHSNSHRKVMELRRQKEMLHQPQQLPPPTTEEGMYGRHFRVC .

[0021] Example 1: CaGATA4 Tomato Transformation with Gene Overexpression Vector In this embodiment, the CaGATA4 gene overexpression vector is transformed into tomato, specifically including the following steps: (1) Acquisition of chili cDNA Total RNA was extracted from different tissues of chili peppers (including roots, stems, leaves, flowers, fruits, and seeds), and then the extracted RNA was reverse transcribed into cDNA using the HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (provided by Nanjing Novizan Biotechnology Co., Ltd.) for gene cloning.

[0022] (2) Cloning of the target fragment Amplification CaGATA4 The full-length sequence of the gene and the primer sequences are as follows (as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively): Positive: AACTTCTCGCCCCTCCC; Reverse: GGTTAGCTCAGCGTTGGAAG; The connector sequences are as follows (as shown in SEQ ID NO.5 and SEQ ID NO.6 respectively): Positive: CATTTGGAGAGGACACGCTCGAG; Reverse: TCTCATTAAAGCAGGACTCTAGA.

[0023] The target fragment was amplified using the Novizan gene cloning kit. The reaction system is shown in Table 1, and the PCR reaction conditions are shown in Table 2. The PCR amplification products were identified by 1.5% agarose gel electrophoresis, and the target fragment was recovered using the Novizan product purification kit.

[0024] Table 1 PCR reaction system

[0025] Table 2 PCR reaction procedure

[0026] (3) Construction of overexpression vector The pHellsgate8 vector was digested with XhoI and XbaI enzymes at 37 °C for 2 h, and the digestion products were identified by 1.5% agarose gel electrophoresis. The recovered target fragment was ligated to the digested vector using the Uniclone One Step Seamless Cloning Kit (SC612) containing the homologous recombinase. The reaction mixture consisted of 5 μL enzyme, 3 μL fragment gel-recovered product, and 2 μL linearized vector gel-recovered product. The mixture was rapidly centrifuged and incubated at 50 °C for 15 min. The recombinant product was transformed into Trans T1 E. coli competent cells, incubated on ice for 30 min, heat-shocked at 42 °C for 30 s, and immediately placed on ice for 2 min. 500 μL of antibiotic-free LB medium was added, and the cells were incubated at 37 °C with shaking at 200 rpm for 1 h. The cells were centrifuged at 5000 rpm for 1 min and spread onto a plate containing 100 mg / mL of the enzyme. -1 Spectinomycin was placed on LB solid medium and incubated upside down at 37 °C for 12 h.

[0027] Pick a single colony to 1 mL containing 100 mg / mL -1 In spectinomycin liquid LB medium, the culture was incubated at 200 rpm and 37°C for 12 h using a shaker. Antibiotic liquid medium without colonies was used as a blank control. After the medium containing single colonies became turbid, it was used as a template for bacterial PCR using a 2x Rapid Taq Master Mix kit. The primers were 35S-F (sequence: CATTTGGAGAGGACACGCTCGAG) and the reverse primer for cloning the target fragment. The specific reaction system is shown in Table 3, and the reaction conditions were the same as those for gene cloning.

[0028] Table 3 Bacterial PCR System

[0029] PCR products were analyzed by 1.5% agarose gel electrophoresis. Monoclonal bacterial cultures exhibiting a single bright band in the target region were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. Sequencing results were compared with a reference gene sequence via an online website (http: / / multalin.toulouse.inra.fr / multalin / ). Monoclonal bacterial cultures with perfectly aligned sequences were preserved and plasmids were extracted. Plasmid extraction was performed using the FastPure Plasmid Mini Kit according to the instructions.

[0030] (4) Agrobacterium transformation When using GV3101 Agrobacterium competent cells, both opening the cap and adding the sample must be done in a clean bench, and all reagents and consumables must be sterile. The specific operation is as follows: Take 5 μL of the plasmid obtained in step (3), add 50 μL of partially thawed Agrobacterium competent cells, mix gently, and incubate on ice for 5 min, in liquid nitrogen for 5 min, at 37 ℃ for 5 min, and in an ice bath for 5 min in sequence; add 700 μL of LB liquid medium and incubate at 28 ℃ with shaking for 2 h; centrifuge at 6000 rpm for 1 min, and take 200 μL to spread on a medium containing 100 mg·mL⁻¹ -1 Spectinomycin (Spec) and 50 mg / mL -1 Rif-resistant solid LB agar was cultured upside down at 28 ℃ for 2 days. Several single colonies from the agar were picked and mixed separately into 1 mL of liquid LB agar containing Spec+Rif, with a blank control included. All colonies were then placed in a shaker at 200 rpm and 28 ℃ until the agrobacterium-containing medium became turbid. The culture was then removed (while the blank control remained clear) and colony PCR was performed under the same conditions and system as for E. coli colony PCR.

[0031] Select the bacterial culture that was correctly verified in the previous step (i.e., showed a single bright band consistent with the length of the target fragment), and store it at a ratio of bacterial culture volume to 50% glycerol volume of 1:1 (after mixing, store at -80 ℃). This completes the construction of the overexpression vector.

[0032] (5) Genetic transformation of tomatoes Wild-type tomato variety "A57" Alisa CraigAfter seed disinfection, seeds are sown on 1 / 2 MS inoculation medium. When two cotyledons emerge, the cotyledons are harvested as explants and placed on pre-prepared culture medium. They are then cultured in the dark for 1 day. Agrobacterium is activated and resuspended in a suspension. An appropriate amount of bacterial solution is poured into a glass dish, completely submerging the cotyledons for about 4 minutes. The bacterial solution is then removed, and the explants (cotyledons) that have been dried are returned to the original pre-culture medium, evenly spread, and co-cultured in the dark for 48 hours. The co-cultured explants are then transferred to a selection medium for selection, with the leaf surface facing upwards, and cultured under light and constant temperature until callus formation. Explants that successfully form callus in the selection medium are transferred to a subculture medium and subcultured for about 15 days. Explants that grow well in the subculture are selected, and all parts except for green callus and adventitious buds are removed. These are then placed in a new subculture medium and cultured for another 15 days until 2-3 [unclear text - possibly referring to growth patterns or growth of callus tissue]. Adventitious buds (cm in diameter) were cut from the base of the stem and inserted into a pre-prepared rooting medium to induce rooting. Once the adventitious buds had rooted and grown well into seedlings, the rooted transgenic seedlings were removed, rinsed underwater to remove any remaining medium, and transplanted into a sterilized substrate of approximately 1:2 ratio of nutrient soil to vermiculite. After hardening off in a growth chamber for 3 weeks, the seedlings were transplanted to the field with soil in suitable weather for routine management.

[0033] (6) Identification of transgenic positive plants and detection of expression levels DNA was extracted from transgenic plants, and overexpression-positive plants were identified using colony PCR primers. Wild-type plants, serving as a blank control, showed no target band; samples exhibiting the target band indicated transgenic-positive plants, thus confirming overexpression-positive plants. Additionally, RNA was extracted from transgenic plants and reverse transcribed into cDNA using an Acrylonitrile reverse transcription kit. The cDNA obtained in the previous step was diluted 10-fold and prepared according to a 20 μL ChamQ Universal SYBR qPCR Master Mix (Novazia, Nanjing, China) reaction mixture. Primers were designed online using the GenScript website. The forward primer sequence was (as shown in SEQ ID NO.7): GCCTCTAGGCCCGAAAACAC, and the reverse primer sequence was (as shown in SEQ ID NO.8): CGGAAGCTGCTGTGGTTGAT. Actin (SGN-U580609) was used as an internal control. The forward primer sequence was (as shown in SEQ ID NO.9): GTCCTCTTCCAGCCATCCAT, and the reverse primer sequence was (as shown in SEQ ID NO.10): ACCACTGAGCACAATGTTACCG. The mixture was added to a 96-well half-skirt PCR plate as shown in Table 4, and the reaction procedure was shown in Table 5. Relative gene expression levels were measured using 2... CT The method is used for calculation.

[0034] Table 4 Real-time quantitative polymerase chain reaction system

[0035] Table 5 Real-time quantitative polymerase chain reaction (PCR) procedure

[0036] (7) Phenotypic identification In this embodiment, a total of 10 transgenic plants were obtained, numbered 1-10. Through positive plant identification, 7 plants showed banding, namely plants 1, 2, 4, 5, 6, 9, and 10, which were identified as positive plants. Figure 1 A). The expression levels of 10 transgenic plants were detected. The results showed that the expression levels of individual plants 1, 2, 4, 5, 6, 9, and 10 were significantly higher than those of the wild type. CaGATA4 Overexpression plants ( Figure 1 B). CaGATA4 Overexpression resulted in the arrest of shoot apical meristem development in the plants. As the plants grew, some of them developed new growth points. Figure 1 CF).

[0037] Example 2: CaGATA4 Virus-induced gene silencing (VIGS) in genetically modified peppers This embodiment silences in chili peppers. CaGATA4 Genes, including the following steps: (1) Cloning of silent fragments Use the VIGS online tool (http: / / vigs.solgenomics.net) on the Solanaceae Genome website to select... CaGATA4 The specific fragment of the gene was identified, and specific PCR amplification primers were designed. The primer sequences are as follows (shown in SEQ ID NO.11 and SEQ ID NO.12, respectively): Positive: CGTCCCCAGTGATGATGTAGC; Reverse: GAAGCGCAGTGTGTGCATCTC.

[0038] The connector sequences are as follows (as shown in SEQ ID NO.13 and SEQ ID NO.14 respectively): Positive: CTGTGAGTAAGGTTACCGAATTC; Reverse: CGCGTGAGCTCGGTACCGGATCC.

[0039] The cDNA and PCR reaction system were the same as in Example 1. The PCR reaction conditions were: 95 °C pre-denaturation for 5 min; 95 °C denaturation for 15 s, 58 °C annealing for 15 s, 72 °C extension for 30 s, 35 cycles; 72 °C for 5 min. The PCR amplification products were identified by 1.5% agarose gel electrophoresis, and the target fragment was recovered using the Novizan product purification kit.

[0040] (2) pTRV2 vector digestion The pTRV2 vector was digested with EcoRI and BamHI at 37 ℃ for 2 h.

[0041] (3) Homologous recombination The target fragment was homologously recombined with the vector using Jinsha homologous recombinase. The specific reaction system and procedure were the same as in Example 1.

[0042] (4) Plasmid extraction and Agrobacterium transformation After selecting the correct monoclonal culture and extracting the plasmid, it was transformed into Agrobacterium GV3101 competent cells. The specific reaction system and procedure were the same as in Example 1.

[0043] (5) Virus-induced gene silencing Containing pTRV1, pTRV2-00, pTRV2- CaGATA4 Agrobacterium plasmids in the presence of Kan 50 μg·mL -1 Rif 50 μg·mL -1 Activate the medium with LB medium. Dilute the culture medium 1:100 to LB induction medium (Kan 50 μg·mL). -1 Rif 50 μg·mL -1The culture was incubated at 28 ℃ with shaking at 220 rpm for 12 h using 10 mM MES and 20 μM AS. The culture was then centrifuged at 5000 rpm / min for 10 min to collect the bacterial culture. The supernatant was discarded, and an appropriate amount of infection buffer [10 mM MES (freshly prepared), 10 mM MgCl2 (freshly prepared), pH adjusted to 5.6, then AS to a final concentration of 200 μM] was added to resuspend the culture. The OD600 was adjusted to 1.0. pTRV1 and the infection buffer containing the pTRV2 vector were mixed at a 1:1 ratio and incubated at 28 ℃ for approximately 4 h before infection. pTRV2-00 plants served as a negative control. The optimal infection stage for pepper seedlings was when the cotyledons were fully expanded but the true leaves had not yet emerged. Using a 1 mL sterile syringe, draw an appropriate amount of infection solution, remove the needle, and place the syringe close to the underside of the leaf. Simultaneously, support the front of the leaf with your index finger and push the syringe to inject the infection solution into the leaf, ensuring the entire leaf is thoroughly soaked in one injection to avoid serious damage to the plant from multiple injections. At this point, the leaf will be visibly wetted with the infection solution. Incubate the infected plant in the dark for 24 hours, then switch to a day / night temperature of 22℃ / 18℃, a 16-hour / 8-hour light / dark cycle, and a relative humidity of 60%.

[0044] (6) Phenotypic observation Observe pTRV2- CaGATA4 Phenotypes of pTRV2 empty-load silent plants, such as Figure 2 As shown, there is one silent plantlet with an empty vector and three different silent plants. CaGATA4 The silent plants were numbered 1, 2, and 3 respectively, and the results showed that... CaGATA4 Silent plants develop lateral branches earlier, promoting the development of plant meristems. Figure 2 As shown, this phenomenon may lead to an increase in early chili pepper yields.

Claims

1. A kind CaGATA4 The application of genes in regulating plant meristem development is characterized by, The CaGATA4 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the plant is a tomato or pepper. Overexpression of the gene in tomatoes... CaGATA4 Genes causing arrest of shoot apical meristem development; The silence in chili peppers CaGATA4 Genes that differentiate into lateral branches prematurely.

2. As described in claim 1 CaGATA4 The application of genes in regulating plant meristem development is characterized by, Overexpression in tomatoes CaGATA4 Genes, including the following steps: (1) Homologous recombination of the sequence shown in SEQ ID NO.1 into the pHellsgate8 vector to construct CaGATA4 Gene overexpression vectors; (2) The overexpression vector from step (1) was introduced into tomato cells and cultured. CaGATA4 Transgenic tomato plants with overexpressed genes.

3. As described in claim 1 CaGATA4 The application of genes in regulating plant meristem development is characterized by, Silence in chili peppers CaGATA4 Genes, including the following steps: (1) Using the VIGS online tool on the Solanaceae Genome website to select CaGATA4 Specific segments of the gene undergo homologous recombination in the pTRV2 vector; (2) After transformation mediated by Agrobacterium, pTRV2- CaGATA4 After mixing Agrobacterium with Agrobacterium containing pTRV1, the mixture was injected into pepper leaves where the cotyledons were fully expanded and new leaves had not yet grown.

4. As described in claim 3 CaGATA4 The application of genes in regulating plant meristem development is characterized by, pTRV2- CaGATA4 The volume ratio of Agrobacterium to pTRV1 Agrobacterium was 1:1.

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