Vv pheres1 and use thereof

Abstract

The application discloses a grape seed development gene VvPHERES1 and application thereof. The nucleotide sequence of the development gene VvPHERES1 is shown as SEQ ID NO:1. The amino acid sequence of a Type I MADS transcription factor is shown as SEQ ID NO:2. The application clones a grape seed development related gene VvPHERES1 for the first time and studies the gene function. A pCAMBIA2300-VvPHERES1 overexpression vector and a heterologous Micro-Tom tomato plant are constructed. By comparing the changes of seed traits in the overexpression transgenic tomato and the wild type plant, the result shows that the seed quantity in the overexpression transgenic tomato is obviously increased. The application provides a grape seed development related gene, and provides a basis for breeding of seedless grape new varieties.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, specifically to a grape seed development gene VvPHERES1 and its applications. Background Technology

[0002] Grapes (Vitis vinifera L.) are widely cultivated due to their rich nutritional value, adaptability, and high economic benefits. Common uses include fresh consumption, winemaking, juice production, and health product development. Globally, the production and consumption demand for fresh grapes has been steadily increasing, and seedless grapes, due to their convenience, palatable taste, and increased economic value, have become a significant trend in both fresh and dried grape production and consumption. In the US market, 80% of fresh grapes and 98% of dried grapes are seedless.

[0003] Common methods for breeding seedless grapes include conventional hybridization and embryo rescue technology. Conventional hybridization typically uses seeded grapes with a tendency to be seedless as the female parent and seedless grapes as the male parent for crossbreeding. However, this method has a long breeding cycle, low breeding efficiency, and a low probability of seedless offspring. Embryo rescue breeding technology allows seed-aborting grapes to be used as the female parent. After a certain period of cross-pollination and fertilization, the ovules of the hybrid young fruit are extracted and cultured in vitro to develop into complete plants. This method can increase the probability of seedless offspring and shorten the breeding cycle, but the seedling survival rate of the offspring is affected by the efficiency of embryo rescue from the female parent. Currently developed molecular biological breeding technology selects for target traits by directionally altering genes. Therefore, finding genes with seed development functions provides a theoretical basis for the targeted improvement of the seedless trait in grapes.

[0004] Currently, there is limited research on genes related to grape seed development, and even less research has been conducted on genes that can regulate seedlessness in grape seeds. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a grape seed development gene, VvPHERES1, and its applications. This invention is the first to clone the grape VvPHERES1 gene, subcellular analysis shows its localization in the cell nucleus, and in situ hybridization analysis reveals differential expression in ovules of seeded and seedless grapes. Overexpression in tomatoes resulted in an increased number of seeds. This technology lays the foundation for grape seed development and molecular breeding of seedless grapes.

[0006] To achieve the above objectives, the technical solution designed by the present invention is as follows:

[0007] This invention provides a grape seed development gene VvPHERES1, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0008] The present invention also provides a Type I MADS transcription factor encoded by the above-mentioned developmental gene VvPHERES1, the amino acid sequence of which is shown in SEQ ID NO:2.

[0009] The present invention also provides a promoter for the above-mentioned gene VvPHERES1, the nucleotide sequence of which is shown in SEQ ID NO:3.

[0010] This invention also provides a primer pair for obtaining the sequence of the grape seed development gene VvPHERES1, wherein the primer pair is as follows:

[0011] VvPHERES1-F: ATGACTAGAAAGAAGGTGAAGCTTGCAT, VvPHERES1-R: AGGGAAAAAGCCATTAGGCCAGAG.

[0012] The present invention also provides an overexpression vector containing the VvPHERES1 gene, wherein the overexpression vector is pCAMBIA2300-VvPHERES1 and its expression vector is pCAMBIA2300.

[0013] The present invention also provides a method for constructing the overexpression vector, comprising the following steps:

[0014] (1) Introduce restriction endonucleases SacI and XbaI at both ends of the grape seed development-related gene VvPHERES1.

[0015] (2) The VvPHERES1 gene fragment containing the restriction site was linked downstream of the 35S promoter of the pCAMBIA2300 vector to obtain the pCAMBIA2300-VvPHERES1 overexpression vector.

[0016] The present invention also provides an engineered strain containing the above-mentioned overexpression vector pCAMBIA2300-VvPHERES1, wherein the engineered strain is Escherichia coli.

[0017] The present invention also provides a host bacterium containing the above-mentioned overexpression vector pCAMBIA2300-VvPHERES1, wherein the host bacterium is Agrobacterium GV3101.

[0018] The mRNA level of the above-mentioned grape VvPHERES1 gene is basically not expressed in the endosperm of seedless grapes, but is expressed in the endosperm of seeded grapes.

[0019] The following are applications of increasing the number of plant seeds:

[0020] 1) The grape seed development gene VvPHERES1 mentioned above;

[0021] 2) The above-mentioned overexpression vectors containing the VvPHERES1 gene;

[0022] 3) The engineered strains mentioned above;

[0023] 4) The host bacteria mentioned above.

[0024] Furthermore, the plant in question is a seeded grape.

[0025] This invention also provides an application of the deletion or interference of the above-mentioned gene VvPHERES1 in the breeding of new grape varieties with seedless traits.

[0026] The beneficial effects of this invention are:

[0027] This invention is the first to clone the grape seed development-related gene VvPHERES1 and study its gene function. An overexpression vector pCAMBIA2300-VvPHERES1 and heterologously transformed tomato plants were constructed. By comparing the changes in seed traits in overexpressed transgenic tomatoes and wild-type plants, the results showed that the number of seeds was significantly increased in the overexpressed transgenic tomatoes. This invention provides a gene regulating grape seed development, laying the foundation for the breeding of new seedless grape varieties. Attached Figure Description

[0028] Figure 1 Electrophoresis image of the VvPHERES1 gene clone;

[0029] Figure 2 Figure showing the differential expression analysis of VvPHERES1 in ovules of seedless and seeded grapes;

[0030] In the figure, A is an analysis of the expression of the VvPHERES1 gene at different stages of 'Nucleate White' (TS) and 'Red Earth' (RG) ovule development;

[0031] B is a diagram showing the in situ hybridization analysis of VvPHERES1 in ovules of seedless grapes 'Thompson Seedless' (a) and 'Flame Seedless' (b) and seeded grapes 'Red Globe' 8 weeks after flowering;

[0032] Figure 3 Electrophoresis diagram of the VvPHERES1 promoter gene clone of 'seedless white' grape;

[0033] Figure 4Image of Escherichia coli culture PCR detection of the VvPHERES1 promoter sequence of 'seedless white' grape;

[0034] The target gene fragment size in the image is 2219 bp.

[0035] M: From top to bottom, they are 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, and 100bp.

[0036] Lanes 1-4 are positive transformants of the VvPHERES1 promoter.

[0037] Figure 5 Image of Escherichia coli culture PCR detection of VvPHERES1-2300 gene;

[0038] Figure 6 A diagram showing the subcellular localization of the VvPHERES1 gene in 'seedless white' grapes;

[0039] In the diagram, GFP represents the green fluorescence field, DAPI represents the DAPI field (nuclear staining), DIC represents the bright field, and Merge represents the superposition field; excitation wavelength: GFP field: 488nm.

[0040] DAPI field: 358nm, Bar = 20μM.

[0041] Figure 7 PCR identification diagram of VvPHERES1-2300 gene positive strain.

[0042] Figure 8 Seed trait identification diagram of tomato plants that are positive for VvPHERES1-2300 overexpression;

[0043] In the figure, A is a schematic diagram of the number of seeds per fruit in the transgenic line; B is a schematic diagram of the seed length of the transgenic line; and C is a schematic diagram of the plant and seed development of the transgenic line. Detailed Implementation

[0044] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0045] Example 1: Cloning and Expression Pattern Analysis of Grape Seed Development Gene VvPHERES1

[0046] 1. Cloning and sequence analysis of the grape seed development gene VvPHERES1

[0047] The CDS sequences of VvPHERES1 from two different grape varieties, 'Seedless White' and 'Red Globe', were cloned and compared using homologous gene cloning methods. Forward and reverse primers were designed based on the sequence information.

[0048] VvPHERES1-F: ATGACTAGAAAGAAGGTGAAGCTTGCAT, as shown in SEQ ID NO:4

[0049] VvPHERES1-R: AGGGAAAAAGCCATTAGGCCAGAG, as shown in SEQ ID NO:5.

[0050] Using grape ovule cDNA as a template, PCR technology was used for amplification. The PCR amplification system was 20 μL, with 2× [missing information - likely referring to specific reagents or components]. 10.0 μL Gold PCR Master Mix, 1 μL cDNA dilution buffer, 1 μL each of forward and reverse primers (20 μM), and 10 μL ddH2O.

[0051] The PCR amplification program was as follows: 98℃ for 3 min; 98℃ for 10 sec, 68℃ for 1 min 30 sec, 35 cycles, 72℃ for 5 min. The PCR amplification product was detected by gel electrophoresis, yielding a 708 bp fragment. Figure 1 After sequencing and alignment, the nucleotide sequence of the gene VvPHERES1 was determined as shown in SEQ ID NO:1.

[0052] ATGACTAGAAAGAAGGTGAAGCTTGCATACATCACTAATGATTCTGCAAGAAAAGCAACATTCAAGAAAAGGAAGAAGGGTCTGATGAAGAAGGTGAGTGAGTTGAGCACTCTCTGTGGGATTGATGCCTGTGCCATCCTTTATAGCCCATATGACTCTCAGCCTGAAGTCTGGCCTTCCCCTTTGGGAGTCCAACGTGTGCTTGCCCATTTCAAGAAGATGCCTGAGATGGAGCAAAGCAAGAAGATGGTCAACCAGGAGAGCTTCCTCAGGCAAAGGATAGCAAAAGGCAATGAGCAGCTCAAGAAACAGCGCAAGGACAACCGCGAAAAGGAGATTACTCAAGTCATGTACCAGAGCTTGACTGGTAAGGGCCTGCAGAATCTCAATATAGTTGATTTGAATGATCTTGGGTGGATGATTGACCAGAACTTGAAGGATATTCACAAGAGGATTGAGTCTCTCAACAAAGAGGCTCAGTCCCAGGCAGCAGCAGCGGCCGCAGCTGCAGCAGGACAACTGATAAAGACTGGAGGGAAAGCACAGGAGGAGAAACCAGCCTTTGACAGCATGGATGCCATTCAGAGGCAGCAGTGGTTCGTTGACTTGGTGAACCCCAATGAGCAGATGGGCTTTGCTGGAGATGACATGATGCTGCCATTTGGGGACAACAACCACAACGCTCTCTGGCCTAATGGCTTTTTCCCT;

[0053] The Type I MADS transcription factor encoded by the grape seed development gene VvPHERES1, and its amino acid sequence is shown in SEQ ID NO:2:

[0054] MTRKKVKLAYITNDSARKATFKKRKKGLMKKVSELSTLCGIDACAILYSPYDSQPEVWPSPLGVQRVLAHFKKMPEMEQSKKMVNQESFLRQRIAKGNEQLKKQRKDNREKEITQVMY QSLTGKGLQNLNIVDLNDLGWMIDQNLKDIHKRIESLNKEAQSQAAAAAAAAAGQLIKTGGKAQEEKPAFDSMDAIQRQQWFVDLVNPNEQMGFAGDDMMLPFGDNNHNALWPNGFFP.

[0055] 2. Expression pattern analysis of grape seed development gene VvPHERES1

[0056] Differential expression analysis of VvPHERES1 at eight different ovule development stages in 'Seedless White' and 'Red Globe' grapes was performed using RT-qPCR. In 'Seedless White', VvPHERES1 first increased and then gradually decreased from 20–25 days after flowering, while in 'Red Globe', it gradually increased from 20–55 days after flowering. Figure 2 A). Eight weeks after flowering, in situ hybridization analysis showed that VvPHERES1 mRNA was essentially not expressed in the endosperm of 'Flame Nucleus-free' and 'Nucleus-free White' varieties, but was expressed in the endosperm of 'Red Globe'. Figure 2 B).

[0057] This indicates that the mRNA level of the grape VvPHERES1 gene is basically not expressed in the endosperm of seedless grapes, but is expressed in the endosperm of seeded grapes.

[0058] Example 2: Promoter amplification of the VvPHERES1 gene in 'Seedless White' grapes

[0059] 1. Primer design

[0060] The designed primer sequences are as follows:

[0061] VvPHERES1 promoter -F: TTGGGCCCGGCGCGCCAAGCTTTT AGAAGTTGTTCTCTATTTCACTTTGTTTTTAAAAAC,

[0062] VvPHERES1 promoter -R: GAATTCCCGGGGATCCGTCGACGA TGCAGGCAAAAATAGTTAGAGAGG;

[0063] 2. Genomic DNA from 'seedless white' grapes was amplified by PCR and electrophoresis to obtain the amplified fragment ( Figure 3 );

[0064] 3. Based on the principle of homologous recombination, the vector used was PCAMBIA1391-GUS. The PCAMBIA1391-GUS vector was constructed using double digestion with HindIII and SaII. Primers were designed using PCAMBIA1391-GUS as the backbone.

[0065] The upstream connector addition sequence is: TTGGGCCCGGCGCGCCAAGCTT(HindIII);

[0066] The downstream connector addition sequence is: GAATTCCCGGGGATCCGTCGAC(SaII);

[0067] Using the above-mentioned amplified fragment as a template and the above-mentioned adapter sequence, the recombinant vector PCAMBIA1391-GUS-VvPHERES1 was amplified to obtain the recombinant vector.

[0068] 4. Transfect Escherichia coli to obtain recombinant Escherichia coli;

[0069] 5. PCR detection and sequencing alignment were performed to obtain the promoter sequence of VvPHERES1. Figure 4 Its nucleotide sequence is shown in SEQ ID NO:3:

[0070]

[0071] Example 3

[0072] Construction of VvPHERES1 overexpression vector and transgenic tomato plants

[0073] 1. Introduce restriction sites at both ends of the VvPHERES1 sequence.

[0074] Based on the principle of constructing vectors using homologous recombination, when designing primers, the upstream primer is prepended to the 5' end with a SacI restriction site and the corresponding AGACACGGGGGAC vector fragment in the pCAMBIA2300 vector. The downstream primer is prepended to the 5' end with an XbaI restriction site and the corresponding ACCATGGTGTCGAC vector fragment in the vector.

[0075] Using grape ovule cDNA as a template, PCR technology was used for amplification. The PCR amplification system was 20 μL, with 2× [missing information - likely referring to specific reagents or components]. 10.0 μL Gold PCR Master Mix, 1 μL cDNA dilution buffer, 1 μL each of forward and reverse primers (20 μM), and 10 μL ddH2O.

[0076] The PCR amplification program was as follows: 98℃ for 3 min; 98℃ for 10 sec, 68℃ for 1 min 30 sec, 35 cycles, 72℃ for 5 min. The VvPHERES1 gene fragment containing restriction enzyme sites was obtained. Primer sequences are as follows:

[0077] PHE-2300-F / Sac I:AGAACACGGGGGAC GAGCTC ATGACTA GAAAGAAGGTGAAGCTTGCAT,

[0078] PHE-2300-R / XbaI: ACCATGGTGTCGAC TCTAGA AGGGAAA AAGCCATTAGGCCAGAG;

[0079] 2. Construction of VvPHERES1 gene overexpression vector

[0080] The VvPHERES1 gene containing restriction enzyme sites was ligated downstream of the 35S promoter of the PCAMBIA2300-GFP vector using homologous recombination to obtain the PCAMBIA2300-VvPHERES1 recombinant vector. This vector was then transformed into *E. coli*, and positive transformants were screened. Figure 5 After transforming it into Agrobacterium GV3101, positive strains were further screened using PCR.

[0081] 3. Subcellular localization of the grape VvPHERESE1 gene

[0082] Agrobacterium tumefaciens containing the recombinant vector PCAMBIA2300-VvPHERES1 was injected into tobacco leaves using a transient transformation method. The highest expression level was observed 2-3 days post-injection. Under a laser confocal microscope, the VvPHERES1 gene was observed to be localized in the cell nucleus. Figure 6 ).

[0083] 4. Obtain VvPHERES1 overexpressing tomato plants using genetic transformation technology.

[0084] Micro-Tom aseptic tomato seedlings were obtained using tissue culture technology. After 6-7 days, newly unfolded cotyledons of the aseptic seedlings were harvested, both ends removed, and segments were cut from the middle and cultured in a pre-medium for 24 hours. The cotyledons were then infected with Agrobacterium and co-cultured. Subsequently, shoots were induced on a shoot differentiation medium. When resistant shoots reached 2 cm in length, they were cut and transferred to a rooting medium. Once complete plantlets had formed, some leaves were harvested, and genomic DNA was extracted using the TransDirect Plant Tissue PCR Kit. Detection was performed using gene-specific primers, yielding positive plants. Figure 7 ).

[0085] Example 3

[0086] Seed trait identification in transgenic tomato plants

[0087] After the fruits of the positive VvPHERES1 overexpressing tomato plants matured, the size of the fruits and seeds was measured and the number of seeds was counted.

[0088] Analysis showed that the overexpression line VvPHERES1-2300-2# had a significantly higher seed count than the wild type. Figure 8 A), but the seed length did not change significantly. Figure 8 B).

[0089] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of any of the following in increasing the number of seeds in tomatoes or seeded grapes, characterized in that: 1) Grape seed development genes VvPHERES1 The grape seed development gene VvPHERES1 The nucleotide sequence is shown in SEQ ID NO:1; 2) Contains the grape seed development gene described in 1). VvPHERES1 overexpression vector pCAMBIA2300- VvPHERES1 The expression vector is pCAMBIA2300. 3) Contains the overexpression vector pCAMBIA2300- described in 2). VvPHERES1 The engineered strain, wherein the engineered strain is Escherichia coli; 4) A host bacterium containing the engineered strain described in 3), wherein the host bacterium is Agrobacterium. Agrobacterium GV3101.

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