Grape seed development gene VvPHERES1 and application thereof

Abstract

The invention discloses a grape seed development gene VvPHERES1 and an application of the grape seed development gene VvPHERES1. The nucleotide sequence of the developmental gene VvPHERES1 is as shown in SEQ ID NO: 1. The amino acid sequence of the TypeI MADS transcription factor is as shown in SEQ ID NO: 2. According to the invention, the grape seed development related gene VvPHERES1 is cloned for the first time, and the gene function of the grape seed development related gene VvPHERES1 is researched. The method comprises the following steps: constructing a pCAMBIA2300-VvPHERES1 overexpression vector, and carrying out heterologous transformation on a Micro-Tom tomato plant. By comparing the change of seed characters in the overexpressed transgenic tomato and the wild type plant, the result shows that the number of seeds in the overexpressed transgenic tomato is obviously increased. The invention provides a gene related to regulation and control of grape seed development, and a basis is provided for seed selection of new seedless grape varieties.

Description

Technical Field

[0001] The invention relates to the field of plant genetic engineering, and in particular to a grape seed development gene VvPHERES1 and an application thereof. Background Art

[0002] Grapes (Vitis vinifera L.) are widely planted because of their rich nutrition, strong adaptability and high economic benefits. Common grapes can be used for fresh eating, wine making, juice and health care product development. Worldwide, the production and consumption demand of fresh grapes has been increasing, and seedless grapes have become an important development trend in the production and consumption of fresh and dried grapes because of their convenience, suitable taste and increased economic value. In the US market, 80% of fresh grapes and 98% of dried grapes are seedless.

[0003] Common seedless grape breeding methods include conventional hybridization and embryo rescue technology. Conventional hybrid breeding usually uses seeded grapes with seedless tendencies as the female parent and seedless grapes as the male parent for hybridization, but this hybrid breeding method has a long breeding cycle, low breeding efficiency and a low probability of seedless offspring. Embryo rescue breeding technology can use seed-aborted grapes as the female parent. After a certain period of hybrid pollination and fertilization, the ovules of the hybrid young fruits are taken out and cultured in vitro into complete plants under in vitro conditions. This breeding method can increase the probability of seedless hybrid offspring and shorten the breeding cycle, but the seedling rate of its offspring is affected by the efficiency of embryo rescue of the female parent. The currently developed molecular biological breeding technology selects target traits by making targeted changes to genes. Therefore, finding genes with seed development functions has a theoretical basis for the targeted improvement of seedless traits in grapes.

[0004] Currently, there is little research on the genes related to grape seed development, and even fewer genes have been developed that can regulate seedlessness in grape seeds. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a grape seed development gene VvPHERES1 and its application. The present invention cloned the grape VvPHERES1 gene for the first time, and subcellular analysis showed that it was localized in the cell nucleus. In situ hybridization analysis showed that the expression difference in the ovules of seeded grapes and seedless grapes was different. In tomatoes with overexpression of the gene, the number of seeds increased. The technology lays a foundation for grape seed development and seedless grape molecular breeding.

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

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

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

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

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

[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 the expression vector thereof is pCAMBIA2300.

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

[0014] (1) The restriction endonuclease cleavage sites of SacI and XbaI were introduced at both ends of the grape seed development-related gene VvPHERES1.

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

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

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

[0018] 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.

[0019] Any of the following for use in increasing the number of plant seeds:

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

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

[0022] 3) the above-mentioned engineered strains;

[0023] 4) The host bacteria mentioned above.

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

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

[0026] Beneficial effects of the present invention:

[0027] The present invention cloned the grape seed development-related gene VvPHERES1 for the first time and studied its gene function. The pCAMBIA2300-VvPHERES1 overexpression vector and heterologous transformation tomato plants were constructed. By comparing the changes in seed traits between overexpression transgenic tomatoes and wild-type plants, the results showed that the number of seeds in overexpression transgenic tomatoes increased significantly. The present invention provides a gene for regulating grape seed development, providing a basis for the breeding of new seedless grape varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the electrophoresis diagram of VvPHERES1 gene cloning;

[0029] Figure 2 This is a diagram showing the differential expression of VvPHERES1 in seedless grape and seeded grape ovules;

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

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

[0032] Figure 3 This is the electrophoresis diagram of the VvPHERES1 promoter gene cloning of 'seedless white' grape;

[0033] Figure 4This is the PCR detection image of the promoter sequence of VvPHERES1 of 'seedless white' grape in Escherichia coli culture medium;

[0034] In the figure, the target gene fragment size: 2219 bp;

[0035] M: from top to bottom: 5000bp, 3000bp, 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp, 100bp;

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

[0037] Figure 5 This is the PCR detection diagram of Escherichia coli culture liquid of VvPHERES1-2300 gene;

[0038] Figure 6 Detection of subcellular localization of grape VvPHERES1 gene in 'seedless white' grapes;

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

[0040] DAPI field: 358 nm, Bar = 20 μM.

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

[0042] Figure 8 This is a diagram for seed trait identification of VvPHERES1-2300 overexpressing tomato positive plants;

[0043] In the figure, A is a schematic diagram of the number of seeds per fruit of the transgenic strain; B is a schematic diagram of the seed length of the transgenic strain; C is a schematic diagram of the plant and seed development of the transgenic strain. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0045] Example 1 Cloning and expression pattern analysis of grape seed development gene VvPHERES1

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

[0047] The CDS sequences of VvPHERES1 of two different grape varieties, seedless grape 'seedless white' and seeded grape 'Red Globe', were cloned and compared by homologous gene cloning method. 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, 2× Gold PCR Master Mix 10.0 μL, cDNA diluent 1 μL, forward and reverse primers (20 μM) 1 μL each, ddH2O 10 μL.

[0051] The PCR amplification program was: 98°C for 3 min; 98°C for 10 sec, 68°C for 1 min and 30 sec, 35 cycles, and 72°C for 5 min. The PCR amplification product was detected by gel electrophoresis, and a fragment of 708 bp was obtained ( Figure 1 ), after sequencing and comparison, the nucleotide sequence of the gene named VvPHERES1 is shown in SEQ ID NO: 1:

[0052] ATGACTAGAAAGAAGGTGAAGCTTGCATACATCACTAATGATTCTGCAAGAAAAGCAACATTCAAGAAAAGGAAGAAGGGTCTGATGAAGAAGGTGAGTGAGTTGAGCACTCTCTGTGGGATTGATGCCTGTGCCATCCTTTATAGCCCATATGACTCTCAGCCTGAAGTCTGGCCTTCCCCTTTGGGAGTCCAACGTGTGCTTGCCCATTTCAAGAAGATGCCTGAGATGGAGCAAAGCAAGAAGATGGTCAACCAGGAGAGCTTCCTCAGGCAAAGGATAGCAAAAGGCAATGAGCAGCTCAAGAAACAGCGCAAGGACAACCGCGAAAAGGAGATTACTCAAGTCATGTACCAGAGCTTGACTGGTAAGGGCCTGCAGAATCTCAATATAGTTGATTTGAATGATCTTGGGTGGATGATTGACCAGAACTTGAAGGATATTCACAAGAGGATTGAGTCTCTCAACAAAGAGGCTCAGTCCCAGGCAGCAGCAGCGGCCGCAGCTGCAGCAGGACAACTGATAAAGACTGGAGGGAAAGCACAGGAGGAGAAACCAGCCTTTGACAGCATGGATGCCATTCAGAGGCAGCAGTGGTTCGTTGACTTGGTGAACCCCAATGAGCAGATGGGCTTTGCTGGAGATGACATGATGCTGCCATTTGGGGACAACAACCACAACGCTCTCTGGCCTAATGGCTTTTTCCCT;

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

[0054] MTRKKVKLAYITNDSARKATFKKRKKGLMKKVSELSTLCGIDACAILYSPYDSQPEVWPSPLGVQRVLAHFKKMPEMEQSKKMVNQESFLRQRIAKGNEQLKKQRKDNREKEITQVMY QSLTGKGLQNLNIVDLNDLGWMIDQNLKDIHKRIESLNKEAQSQAAAAAAAAAGQLIKTGGKAQEEKPAFDSMDAIQRQQWFVDLVNPNEQMGFAGDDMMLPFGDNNHNALWPNGFFP.

[0055] 2. Analysis of the expression pattern of grape seed development gene VvPHERES1

[0056] The differential expression of VvPHERES1 in 'Seedless White' and 'Red Globe' grapes at eight different stages of ovule development was analyzed by RT-qPCR. In 'Seedless White', the expression of VvPHERES1 first increased from 20 to 25 days after flowering and then gradually decreased, while in 'Red Globe', the expression of VvPHERES1 gradually increased from 20 to 55 days after flowering ( Figure 2 A). Eight weeks after flowering, in situ hybridization analysis of VvPHERES1 mRNA levels showed that it was essentially unexpressed in the endosperm of ‘Flame Seedless’ and ‘Seedless White’, but expressed in the endosperm of ‘Red Globe’ ( Figure 2 B).

[0057] It can be seen from this that the mRNA level of 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 Amplification of the promoter of the gene VvPHERES1 of 'seedless white' grapes

[0059] 1. Primer Design

[0060] The designed primer sequences are:

[0061] VvPHERES1 promoter-F:TTGGGCCCGGCGCGCCAAGCTTTTAGAAGTTTGTTCTCTATTTCACTTTGTTTTTAAAAAC,

[0062] VvPHERES1 promoter-R:GAATTCCCGGGGATCCGTCGACGA TGCAGGCAAAAATAGTTAGAGAGG;

[0063] 2. PCR amplification and electrophoresis were performed using the genomic DNA of 'seedless white' grapes to obtain the amplified fragment ( Figure 3 );

[0064] 3. According to the principle of constructing vectors by homologous recombination method, the vector used is PCAMBIA1391-GUS vector, wherein the vector construction PCAMBIA1391-GUS uses HindIII and SaII double restriction digestion, and the primers are designed with PCAMBIA1391-GUS as the backbone:

[0065] The sequence added to the upstream linker was: TTGGGCCCGGCGCGCCAAGCTT (HindIII);

[0066] The sequence added to the downstream linker was: GAATTCCCGGGGATCCGTCGAC (SaII);

[0067] The above amplified fragment was used as a template and the above adapter sequence was used for amplification to obtain the recombinant vector PCAMBIA1391-GUS-VvPHERES1;

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

[0069] 5. PCR detection and sequencing comparison to obtain the promoter sequence of VvPHERES1 ( Figure 4 ), whose 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] According to the principle of constructing vectors by homologous recombination method, when designing primers, a SacI restriction site was added in front of the 5' end of the upstream primer, and the restriction site corresponded to the front AGAACACGGGGGAC vector fragment in the pCAMBIA2300 vector, and an XbaI restriction site was added in front of the 5' end of the downstream primer, and the restriction site corresponded to the back 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, 2× Gold PCR Master Mix 10.0 μL, cDNA diluent 1 μL, forward and reverse primers (20 μM) 1 μL each, ddH2O 10 μL.

[0076] The PCR amplification program was: 98°C for 3 min; 98°C for 10 sec, 68°C for 1 min and 30 sec, 35 cycles, and 72°C for 5 min. The VvPHERES1 gene fragment containing the restriction site was obtained. The 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 sites was connected to the downstream of the 35S promoter of the PCAMBIA2300-GFP vector by homologous recombination to obtain the PCAMBIA2300-VvPHERES1 recombinant vector, which was transformed into Escherichia coli and positive transformants were screened ( Figure 5 ), transformed it into Agrobacterium GV3101, and further screened positive strains using PCR.

[0081] 3. Subcellular localization of grape VvPHERESE1 gene

[0082] Using the tobacco transient transformation method, the Agrobacterium solution of the PCAMBIA2300-VvPHERES1 recombinant vector was injected into tobacco leaves. The expression level was the highest 2-3 days after injection. The VvPHERES1 gene was located in the cell nucleus under a laser confocal microscope ( Figure 6 ).

[0083] 4. Genetic transformation to obtain VvPHERES1 overexpressing tomato plants

[0084] Micro-Tom tomato sterile seedlings were obtained by tissue culture technology. After 6-7 days, the cotyledons of the sterile seedlings that had just unfolded were taken, the two ends were removed, and the middle part was cut into sections and placed in a pre-culture medium for 24 hours. The cotyledons were infected with Agrobacterium and co-cultured. Then, buds were induced in a bud induction differentiation medium. When the resistant buds grew to 2 cm, the buds were cut and transferred to a rooting medium. When a complete plantlet was formed, some leaves were cut and genomic DNA was extracted using the TransDirect Plant Tissue PCR Kit. The positive plants ( Figure 7 ).

[0085] Example 3

[0086] Identification of seed traits in transgenic tomato plants

[0087] After the fruits of the obtained positive VvPHERES1 overexpressing tomato plants matured, the fruit and seed sizes were measured and the seed numbers were counted.

[0088] After analysis, the number of seeds in the overexpression strain VvPHERES1-2300-2# was significantly higher than that in the wild type ( Figure 8 A), but no significant change in seed length ( Figure 8 B).

[0089] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A grape seed development gene VvPHERES1, characterized in that: The nucleotide sequence of the developmental gene VvPHERES1 is shown in SEQ ID NO:

1.

2. A Type I MADS transcription factor encoded by the developmental gene VvPHERES1 according to claim 1, characterized in that: The amino acid sequence of the Type I MADS transcription factor is shown in SEQ ID NO:

2.

3. A promoter of the gene VvPHERES1 according to claim 1, characterized in that: The nucleotide sequence of the promoter of the gene VvPHERES1 is shown in SEQ ID NO:

3.

4. A primer pair for obtaining the sequence of grape seed development gene VvPHERES1, characterized in that: The primer pairs are: VvPHERES1-F:ATGACTAGAAAGAAGGTGAAGCTTGCAT, VvPHERES1-R:AGGGAAAAAAGCCATTAGGCCAGAG.

5. An overexpression vector containing the VvPHERES1 gene, characterized in that: The overexpression vector is pCAMBIA2300-VvPHERES1, and its expression vector is pCAMBIA2300.

6. An engineered strain containing the overexpression vector pCAMBIA2300-VvPHERES1 according to claim 4, characterized in that: The engineered strain is Escherichia coli.

7. A host bacteria containing the overexpression vector pCAMBIA2300-VvPHERES1 according to claim 4, characterized in that: The host bacteria is Agrobacterium GV3101.

8. Use of any one of the following for increasing the number of plant seeds, characterized in that: 1) The grape seed development gene VvPHERES1 according to claim 1; 2) The overexpression vector containing the VvPHERES1 gene according to claim 5; 3) The engineered strain of claim 6; 4) The host bacteria according to claim 7.

9. The use according to claim 8, characterized in that: The plant is a seeded grape.

10. Use of the deletion or interference of the gene VvPHERES1 according to claim 1 in breeding new grape varieties with seedless traits.

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