Application of sweet cherry pavag gene related biological agents in creating multi-chamber tomato germplasm

Abstract

This invention belongs to the field of plant molecular biology and genetic breeding technology, specifically relating to sweet cherries. PavAG Application of gene-related bioproducts in the creation of multi-chambered tomato germplasm. PavAG The nucleotide sequence of the gene is shown in SEQ ID NO.1; the related biological product is for promoting... PavAG Substances involved in gene expression. This invention provides the aforementioned... PavAG Genes, containing the aforementioned PavAG Recombinant expression vectors of genes, engineered bacteria containing said recombinant expression vectors, transgenic plant cells, and their application in the genetic improvement of tomatoes. This is achieved by using the sweet cherry source... PavAG Introducing genes into tomatoes and expressing them heterologously can increase the number of locules in tomato fruits, thereby obtaining new multilocular tomato germplasm. This invention provides a new exogenous functional gene resource and molecular breeding approach for improving the multilocular trait in tomatoes, and has good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology and genetic breeding technology, specifically relating to sweet cherries. PavAG Application of gene-related bioproducts in the creation of multi-chamber tomato germplasm. Background Technology

[0002] Tomatoes are an important horticultural crop, and the number of locules in their fruit is a crucial agronomic trait affecting fruit internal structure, yield formation, and varietal type. Multiloculed tomatoes typically exhibit more complex fruit structural characteristics; therefore, identifying functional genes that regulate locule development is of great significance for tomato trait improvement and germplasm innovation.

[0003] AGAMOUS ( AG The gene belongs to the MADS-box transcription factor family, specifically class C floral organ characteristic genes, and plays a crucial role in the determination of floral meristem and the development of stamens and carpels. Previous studies have shown that... AG Its homologs and related genes exhibit strong conservation across different plants, particularly in relation to reproductive organ formation and carpel development. Since the number of fruit locules is closely linked to the number of carpels and their developmental process, screening and utilizing new [genes / genes] is crucial. AG Homologous gene resources hold promise for providing new technical approaches to regulating the number of fruit ventricles. Summary of the Invention

[0004] To develop a novel AG homologous gene resource and discover functional genes that regulate ventricular development, this invention provides a new technical approach for regulating the number of ventriculars in fruits, which can then be used for tomato trait improvement and germplasm innovation. PavAG Application of gene-related biological products in the creation of multi-chambered tomato germplasm. To achieve the above objectives, the present invention adopts the following technical solution.

[0005] This invention provides sweet cherries PavAG The application of gene-related biological products in the creation of multi-chambered tomato germplasm, the aforementioned PavAG The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] The relevant biological products are for promoting PavAG The substance that promotes gene expression, through the transfer of the gene into tomatoes, is... PavAG The gene expression substance was heterologously overexpressed in tomatoes. PavAG Genes were developed to increase the number of chambers in tomato fruits.

[0007] Sweet cherries ( Prunus avium It belongs to the genus *Prunus* of the family Rosaceae. PrunusThe fruit of the subgenus *Cerasus* is characterized by its bright color, tender flesh, and unique flavor. It is rich in nutrients and possesses certain beauty and skin-care benefits, making it a specialty fruit with significant economic value in agricultural production. The inventors of this invention cloned *Cerasus arguta* from sweet cherries during their research. PavAG The gene was identified, and it was found that this gene, under heterologous overexpression conditions, can affect the number of locules in tomato fruits. This invention provides a sweet cherry source... PavAG The study of genes and their application in improving the multi-ventilator trait in tomatoes has expanded the functional gene resources for tomato genetic improvement.

[0008] Preferably, the PavAG The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0009] Preferably, the promoting PavAG Substances involved in gene expression include those containing PavAG Recombinant gene expression vectors.

[0010] Preferably, the containing PavAG Recombinant gene expression vectors are used to express the genes... PavAG It was constructed by cloning the gene into a plant expression vector.

[0011] Preferably, the plant expression vector comprises pHB-GFP.

[0012] The present invention also provides a recombinant expression vector comprising, as shown in SEQ ID NO. 1, operatively linked to a promoter. PavAG Gene.

[0013] Preferably, the promoter is a constitutive promoter or a fruit development-related promoter; more preferably, it is the CaMV35S promoter.

[0014] The present invention also provides an engineered bacterium containing the recombinant expression vector.

[0015] The present invention also provides a transgenic tomato plant cell containing the recombinant expression vector.

[0016] Preferably, the process of creating multi-chamber tomato germplasm is as follows: Construct the containing PavAG Recombinant expression vectors of genes, containing the gene PavAG The recombinant expression vector of the gene was introduced into tomato receptor material, and the desired gene was obtained through screening. PavAG Transgenic plants with increased gene expression levels; the transgenic plants are tomato plants with an increased number of fruit locules.

[0017] Preferably, the tomato receptor material is tomato leaves and / or stem segments.

[0018] Preferably, the method for introducing the recombinant expression vector into the tomato receptor material includes Agrobacterium-mediated transformation.

[0019] Preferably, the Agrobacterium used in the Agrobacterium-mediated transformation method includes Agrobacterium GV3101.

[0020] Preferably, the expression level of the PavAG gene in the transgenic plant is higher than that in the wild-type plant.

[0021] Preferably, the transgenic plant has a higher number of fruit locules than the wild-type plant.

[0022] Compared with the prior art, the present invention has the following beneficial effects: To develop a novel AG homologous gene resource and discover functional genes that regulate ventricular development, this invention provides a new technical approach for regulating the number of ventriculars in fruits, which can then be used for tomato trait improvement and germplasm innovation. PavAG Application of gene-related biological products in the creation of multi-chambered tomato germplasm, wherein the related biological products are used to promote... PavAG The substance that promotes gene expression, through the transfer of the gene into tomatoes, is... PavAG The gene expression substance was heterologously overexpressed in tomatoes. PavAG Genes were used to increase the number of locules in tomato fruits. This application utilizes sweet cherry... PavAG Gene-related biological products (including gene sequences, expression vectors, or proteins) can be genetically transformed or used for gene editing in tomatoes to heterologously express or regulate the AG homologous gene, thereby interfering with the development of the tomato fruit's ventricles. Because... AG This gene family is highly conserved in angiosperms, and its core function is to regulate the development of floral organs and fruits. PavAG As a novel AG homologous gene resource discovered from sweet cherries, its introduction provides a completely new molecular target and pathway for the regulation of ventricular number in tomatoes, breaking through the traditional reliance solely on endogenous tomato genes (such as...). TAG1, TAGL1 The limitations of (etc.) have opened up a new technical path for fruit trait improvement and germplasm innovation.

[0023] This invention achieves the heterologous expression of the sweet cherry in tomatoes. PavAG This invention relates to a gene that can increase the number of locules in tomato fruits, thereby obtaining new multi-loculed tomato germplasm. Furthermore, the gene, recombinant expression vector, engineered bacteria, and method provided by this invention can be used for molecular design breeding and germplasm innovation in tomatoes, demonstrating clear application value. Attached Figure Description

[0024] Figure 1 The wild-type and transgenic tomato plants in this inventionPavAG Gene amplification electrophoresis gel imaging.

[0025] Figure 2 In this invention PavAG Relative expression levels in wild-type and overexpression tomato lines.

[0026] Figure 3 In this invention PavAG A dynamic record of the development of wild-type tomato fruits from color change to maturity, based on overexpression. Detailed Implementation

[0027] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0028] Example 1. Test materials 'Micro Tom' tomato seeds were purchased from Shanghai Sangon Biotech Co., Ltd.

[0029] 2. Culture medium MS1 medium: 0.66g MS medium, 4.5g sucrose, 2.25g agar, pH=5.8, diluted to 300mL with ultrapure water, autoclaved at 121℃ for 21min.

[0030] MS2 medium: 1.32g MS medium, 9g sucrose, 300μL 1mg / mL -1 KT, 2.25g agar, pH=5.8, and ultrapure water were brought to a final volume of 300mL and autoclaved at 121℃ for 21min.

[0031] MS3 medium: 0.44g MS medium, 3g sucrose, pH=5.8, bring the volume to 100mL with ultrapure water, autoclave at 121℃ for 21min.

[0032] MS4 medium: 1.32 g MS medium, 9 g sucrose, 2.25 g agar, pH 5.8, diluted to 300 mL with ultrapure water, autoclaved at 121 °C for 21 min; after cooling to below 65 °C, add 600 μL of 1 mg / mL solution. -1 ZT, 300μL 300mg mL -1 Termetidine, 40 μL 50 mg mL -1 Hygromycin B.

[0033] MS5 medium: 1.32 g MS medium, 9 g sucrose, 2.25 g agar, pH 5.8, diluted to 300 mL with ultrapure water, autoclaved at 121 °C for 21 min; after cooling to below 65 °C, add 60 μL of 1 mg / mL solution. -1 ZT, 300μL 300mg mL -1 Termetidine, 40 μL 50 mg mL -1 Hygromycin B.

[0034] MS6 medium: 1.32 g MS medium, 9 g sucrose, 2.25 g agar, pH 5.8, diluted to 300 mL with ultrapure water, autoclaved at 121 °C for 21 min; after cooling to below 65 °C, add 600 μL of 1 mg / mL solution. -1 IAA, 300 μL 300 mg mL -1 Termetidine, 40 μL 50 mg mL -1 Hygromycin B.

[0035] 3. Test methods Sweet Cherry PavAG Construction of gene overexpression vectors and genetic transformation of tomatoes: The overexpression vector used in this invention is pHB-GFP, and the insertion... PavAG Gene fragments were used to obtain the pHB-PavAG-GFP plant overexpression vector.

[0036] Among them, pHB-GFP is the pHB-GFP plasmid vector, see "pHB-GFP plasmid vector preserved in the Plasmid Vector Bacterial Cell Gene Preservation Center", which comes from Shanghai Sangon Biotech Co., Ltd.

[0037] PavAG The nucleotide sequence of the gene fragment is shown in SEQ ID NO.1: ATGGAGTTCCCAAATCAAGCACCTGAGAGCTCTTCCCAAAGAAAAATTGGAAGAGGCAAGATTGAGATTAAGCGGATTGAAAACACTACAAATCGACAAGTCACCTTCTGCAAGCGCCGCAACGGCTTGCTTAAGAAAGCCTATGAGTTATCTGTTCTATGTGATGCTGAAGTTGCTCTTATTGTCTTCTCTACCCGTGGCCGCCTGTATGAGTATGCTAACAACAGCGTTAGAGCAACGATTGATAGGTACAAAAAAGCATGCACTGATTCTACGAACGGTGGATCTGTTTCTGAAGCTAACACTCAGTTTTATCAGCAGGAATCATCTAAATTACGAAGACAAATCCGCGAAATCCAGAACTCAAACAGGCATATACTGGGTGAAGCTCTTAGCACTTTGAACATCAAGGAACTCAAGAACCTAGAAGGAAGACTGGAGAAAGGGATCAGCAGAATAAGATCCAAAAAGAATGAAATGCTGTTTACTGAAATCGAATTTATGCAAAAGAGGGAGATCGAGCTGCAAAACCATAACAATTATCTGAGAGCAAAGATAGCTGAAAATGAAAGGGCACAACAGCAGCAAACAAATATGATTCAAGGAACTTCTTATGATCAGTCAATGCCTTCACAGTCATACGACAGGAACTTCCTCCCTGTAATGTTGGAGGCCAATAATAATAATAATAATAATAACCATTACTCTCGCCATGACCAGACAGCTCTCCAACTTGTTTGA。

[0038] PavAG The amino acid sequence of the PavAG protein corresponding to the gene fragment is shown in SEQ ID NO.2: MEFPNQAPESSSQRKIGRGKIEIKRIENTTNRQVTFCKRRNGLLKKAYELSVLCDAEVALIVFSTRGRLYEYANNSVRATIDRYKKACTDSTNGGSVSEANTQFYQQESSKLRRQIREIQNSN RHILGEALSTLNIKELKNLEGRLEKGISRIRSKKNEMLFTEIEFMQKREIELQNHNNYLRAKIAENERAQQQQTNMIQGTSYDQSMPSQSYDRNFLPVMLEANNNNNNNNHYSRHDQTALQLV.

[0039] (1) PavAG The steps for constructing a gene cloning vector are as follows: Using sweet cherry leaf cDNA as a template, according to PavAG The nucleotide sequence of the gene was designed using upstream primer PavAG-F and downstream primer PavAG-R. PCR amplification was performed using TOYOBO's KOD Plus high-fidelity enzyme to obtain the target fragment. The PCR amplification program had an annealing temperature of 58℃. Detailed procedures were performed according to the KOD Plus high-fidelity enzyme instruction manual. A blunt-ended cloning vector, pEASY®-Blunt Zero Cloning Kit (TRAN), was selected. 4 µL of the purified PCR amplification fragment was mixed with 1 µL of pEASY®-Blunt Zero Cloning Kit vector and ligated at 30℃ for 10 min. Then, 5 µL of the ligation product was transformed into E. coli competent cells DH5α at 42℃. The sample was sent to the company for sequencing, and the plasmid was returned, yielding sweet cherry. PavAG Sequencing identifies the correct cloned plasmid.

[0040] The nucleotide sequence of the upstream primer PavAG-F is shown in SEQ ID NO.3: 5'-ATGGAGTTCCCAAATCAAGCA-3'.

[0041] The nucleotide sequence of the downstream primer PavAG-R is shown in SEQ ID NO.4: 5'-TCAAACAAGTTGGAGAGCTGT-3',.

[0042] Escherichia coli competent cells DH5α were purchased from Shanghai Sangon Biotech Co., Ltd.

[0043] (2) Construction of pHB-PavAG-GFP plant overexpression vector: The homologous arm sequences of the pHB-GFP vector were analyzed and designed for seamless cloning. Based on the correctly cloned PavAG sequence, upstream homologous arm primer tPavAG-F and downstream homologous arm primer tPavAG-R were designed. These primers retain the pHB-GFP sequence. Hind III restriction site, underlined is homologous arm.

[0044] The nucleotide sequence of the upstream homologous arm primer tPavAG-F is shown in SEQ ID NO.5: 5'-ACCAGTCTCTCTCCAAGCTTATGGAGTTCCCAAATCAAGCA-3'.

[0045] The nucleotide sequence of the downstream homologous arm primer tPavAG-R is shown in SEQ ID NO.6: 5'-GCTCACCATGGATCCAAGCTTTCAAACAAGTTGGAGAGCTGT-3', (3) With sweet cherries PavAG Using the correctly sequenced plasmid as a template, PCR amplification with KOD Plus high-fidelity enzyme yielded the target gene fragment with the added homologous arm of the pHB-GFP vector. Restriction endonucleases from TaKaRa were then used. Hind III. Linearize the pHB-GFP vector to obtain the linearized pHB-GFP vector.

[0046] pHB- was constructed using the homologous arm recombination method. PavAG -GFP plant overexpression vector, recombinase selected from Vazyme. Exnase II. The specific carrier and target fragment usage amount are based on Exnase The formula provided in the product instruction manual (II) shows that after ligation at 37℃ for 30 minutes using a PCR instrument, E. coli competent cells DH5α were transformed. Single clones were then picked for culture PCR and sequencing verification. The upstream verification primer was... PavAG -F, the downstream validation primer was pHB-GFP-R, and the correct plant overexpression vector pHB- was finally obtained. PavAG -GFP. The recombinant plasmid pHB-PavAG-GFP (plant overexpression vector pHB-) was transferred via freeze-transfer. PavAG PavAG-pHB Agrobacterium was obtained by transfecting Agrobacterium GV3101 competent cells with GFP- (-GFP). The plant overexpression vector pHB- PavAG -GFP is equivalent to pHB- PavAG -GFP plant overexpression vector.

[0047] Among them, upstream verification primers PavAGThe nucleotide sequence of -F is shown in SEQ ID NO.7: 5'-ATGGAGTTCCCAAATCAAGCA-3'.

[0048] The nucleotide sequence of the downstream validation primer pHB-GFP-R is shown in SEQ ID NO.8: 5'-ACACGCTGAACTTGTGGCCGTTTA-3'.

[0049] (4) Take a number of plump 'Micro Tom' tomato seeds into a 50mL centrifuge tube, rinse several times with clean water to remove impurities; add distilled water and incubate at 55℃ for 15min. Discard the distilled water in a laminar flow hood, wash three times with sterile water, wash for 30s with 75% (v / v) ethanol, and wash with sterile water until no foam remains; disinfect with NaClO solution for 20min, and then wash five times with sterile water. Transfer the seeds to filter paper to dry, sow them on MS1 ​​medium, and culture at 24℃ in the dark for about 3 days until germination, then culture under normal light. When the cotyledons unfold, cut off the well-grown cotyledons and stem segments with a scalpel in a laminar flow hood, spread them flat on MS2 solid medium, and pre-culture at 24℃ under light for 3 days. Incubate PavAG-pHB Agrobacterium at 28℃ and 200rpm until OD 600 =0.6, centrifuge at 5000 rpm for 15 min, discard the supernatant, resuspend in MS3 liquid medium (containing 0.1 mM acetylsyl syringone), and let stand at room temperature in the dark for 2 h to be used as the infection solution.

[0050] Pre-cultured explants were immersed in the infection solution and kept in the dark for 5 minutes; then transferred to filter paper to blot off any remaining infection solution; finally, they were spread evenly on MS2 solid medium and co-cultured at 24°C in the dark for 1 day. The explants were then transferred to MS4 solid medium for callus induction, with transfers to fresh MS4 solid medium every 20 days until callus formation. After callus formation, they were transferred to MS5 solid medium to induce shoot emergence and seedling development. Once the callus shoots had emerged, the callus mass was surgically removed, and the seedlings were transferred to MS6 solid medium for rooting culture. Well-rooted seedlings were transplanted into nutrient pots, and a small number of transgenic tomato leaf samples were taken for transgenic identification at the DNA and mRNA levels, with upstream verification primers used as a reference. PavAG -F, Downstream validation primers PavAG -R is the primer for PCR amplification. The results showed that the target band of the expected size was amplified in the transgenic lines, while no corresponding band was detected in the wild-type plants, indicating that the sweet cherry origin... PavAG The gene has been successfully integrated into the tomato genome. Figure 1 Positive regenerated seedlings were used for subsequent trials.

[0051] RNA was further extracted from transgenic lines and wild-type plants, reverse transcribed, and then detected by qRT-PCR. The results showed that RNA was present in the transgenic lines. PavAG Gene expression levels were significantly higher in the plant than in the wild type, see Figure 2 .

[0052] Transgenic lines that tested positive for molecular identification were planted simultaneously with wild-type tomatoes under the same conditions. After the fruits matured, the fruits were cut crosswise and the number of ventricles was counted.

[0053] The results showed that the number of locules in transgenic tomato fruits was significantly increased compared to the wild type. In the selected lines, the number of locules increased from 2-4 locules in the wild type to 6-8 locules, exhibiting a stable multiloculed phenotype. Figure 3 .

[0054] The above results indicate that sweet cherry is a source of... PavAG Heterologous expression of genes in tomatoes can increase the number of fruit locules and can be used to create new multi-loculed tomato germplasm.

[0055] In summary, this invention provides a source of sweet cherries. PavAG Genes and their application in creating multi-chambered tomato germplasm. This was achieved by using genes derived from sweet cherries... PavAG Heterologous gene introduction into tomatoes and expression can increase the number of cavities in tomato fruits, thus providing new functional gene resources and molecular breeding methods for improving the multi-cavity trait of tomatoes.

[0056] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0057] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. Sweet cherries PavAG The application of gene-related biological products in the creation of multi-chambered tomato germplasm is characterized by, The PavAG The nucleotide sequence of the gene is shown in SEQ ID NO.1; The relevant biological products are for promoting PavAG The substance that promotes gene expression, through the transfer of the gene into tomatoes, is... PavAG The gene expression substance was heterologously overexpressed in tomatoes. PavAG Genes were developed to increase the number of chambers in tomato fruits.

2. The application according to claim 1, characterized in that, Promote PavAG Substances involved in gene expression include those containing PavAG Recombinant gene expression vectors.

3. The application according to claim 2, characterized in that, The containing PavAG Recombinant gene expression vectors are used to express the genes... PavAG It was constructed by cloning the gene into a plant expression vector.

4. The application according to claim 3, characterized in that, The plant expression vector includes pHB-GFP.

5. The application according to claim 4, characterized in that, The process of creating multi-chamber tomato germplasm is as follows: Construct the containing PavAG Recombinant expression vectors of genes, containing the gene PavAG The recombinant expression vector of the gene was introduced into tomato receptor material, and the desired gene was obtained through screening. PavAG Transgenic plants with increased gene expression levels; the transgenic plants are tomato plants with an increased number of fruit locules.

6. The application according to claim 5, characterized in that, The tomato receptor material is tomato leaves and / or stem segments.

7. The application according to claim 5, characterized in that, Methods for introducing the recombinant expression vector into tomato receptor material include Agrobacterium-mediated transformation.

8. The application according to claim 7, characterized in that, The Agrobacterium-mediated transformation method uses Agrobacterium GV3101.

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