Application of bHLH148 gene in regulation of tomato flowering node

Abstract

The invention provides an application of a bHLH148 gene in regulating and controlling a flowering node of a tomato, an ORF (Open Reading Frame) sequence of the bHLH148 gene is shown as SEQ.NO.1, and an amino acid sequence of a protein coded by the bHLH148 gene is shown as SEQ.NO.2. The bHLH148 gene can be used for regulating and controlling the flowering node of the tomato. Through overexpression of the bHLH148 gene in a plant, a tomato plant with reduced initial flower node and advanced flowering can be obtained; compared with a wild plant, the overexpression bHLH148 gene plant shows obvious reduction at the first flower node position, and shows obvious advancement at the first inflorescence budding time. The result provides a new technical direction for improving the flowering node of the plant at a fixed point, completing the regulation and control of the flowering time of the plant and the marketing time of mature fruits.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant molecular biology and specifically relates to bHLH148 Application of genes in regulating flowering node in tomato. Background Art

[0002] tomato( Solanum lycopersicum Tomatoes are important fruits and vegetables, and are also a key model plant for basic research. In recent years, extreme weather has hindered flower bud differentiation in tomatoes, severely impacting fruit yield and quality. Further research into the key genes that regulate flowering node position and timing in tomato plants, while ensuring visual quality, is crucial for understanding tomato fruit yield, quality, maturity, and crop rotation.

[0003] Currently, targeted genetic modification of flowering nodes in plants can regulate flowering timing and the timing of fruit ripening for market. Tomatoes, a day-neutral plant, have cloned genes regulating flowering timing, such as SFT and SPL13. In-depth research has revealed that flowering timing in tomatoes is controlled by multiple genes. However, many genes regulating flowering timing in tomatoes remain undiscovered, hindering the development of a comprehensive genetic regulatory network for flowering nodes and hindering the progress of targeted genetic modification in plants.

[0004] Therefore, the identification and application of flowering node regulatory genes have important theoretical significance and practical application value for improving the quality control and periodic supply cultivation of tomatoes, and are also one of the important development directions of tomato breeding. Summary of the Invention

[0005] In view of this, the present invention provides bHLH148 The application of genes in regulating tomato flowering nodes bHLH148 When the gene is overexpressed, the flowering node of tomato plants can be lowered and flowering can be advanced.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: One of the purposes of the present invention is to provide bHLH148 Application of genes in regulating tomato flowering nodes, the bHLH148 The ORF sequence of the gene is shown in SEQ.NO.1. bHLH148 The amino acid sequence of the protein encoded by the gene is shown in SEQ.NO.2.

[0007] Among them, the sequence information of SEQ.NO.1 is as follows: ATGAATTCGAAAGAAAGAAAGAGAGAGTTTACTCGTCAGCACCAAAGAAAGTTATGAAGCTATCAACTGATCCACAAAGCATAGCTGCTCGCGAAAGAAGGCACAGAATAAGCGATCGTTTTAAAATTTTACAAAGTTTAGTCCCCGGTGGTTCTAAAATGGACACTGTTACTATGTTAGAAGAAGCAATTCACTATGTCAAATTTCTCAA AACACAAATTTGGCTTCACCAAACGATGGTTAATTTAGTCGATATTAATCATGAAATGGTTGGATATTACCCTCTCGTTGATGATGATCAGAATATACACAAAAATAATATTAGTTCAATGGACTATCAACAATGCACAGGTACAAAGTTATGATAACGATGCCTTTCAACAAGTTGAGTTTCCGTTTGAAGAAACTAATATTTCTGGTGATGTTTTTATGTACTATAAT; The sequence information of SEQ.NO.2 is as follows: MNSKEKKERVYSSAPKKVMKLSTDPQSIAARERRHRISDRFKILQSLVPGGSKMDTVLMEEAIHYVKFLKTQIWLHQTMVNLVDINHEMVGYYPLVDDDQNIHKNNISSMDYQQMQQVQSYDNDAFQQVEFPFEETNISGDVFMYYN.

[0008] The second object of the present invention is to provide a method for regulating the flowering node of tomatoes, wherein the method comprises constructing a bHLH148 The gene is overexpressed in a vector and further infected into tomato explants to regulate the flowering node of tomatoes.

[0009] In some specific embodiments, preferably, the construction contains bHLH148 The primers used in the gene overexpression vector contain homologous recombination arms, the sequences of which are as follows: FW: CATTTGGAGAGGACACGCTCGAGTGTGTTGTCAATGAAGTCAAGATCT (SEQ.NO.3); RV:TCTCATTAAAGCAGGACTCTAGACTTAGGCAAACATAATAATAAACTT (SEQ.NO.4).

[0010] In some specific embodiments, preferably, the construction contains bHLH148 The vector used for gene overexpression is pHellsgate8 vector.

[0011] Furthermore, the method further comprises using the gene with sequence number Solyc11g005330 as an internal reference gene to perform bHLH148 Gene expression detection and screening bHLH148 Plants with gene expression increased by 151 to 288 times.

[0012] In some specific embodiments, preferably, the primers for detecting the expression level of the internal reference gene are as follows: Q_FW:GTCCTCTTCCAGCCATCCA; Q_RV:ACCACTGAGCACAATGTTACCG; described bHLH148 The specific primers for gene expression detection are as follows: Q_FW: AGAGAGAGTTTACTCGTCAGCACC (SEQ.NO.5); Q_RV:ATCATCAACGAGAGGGTAATATCC (SEQ.NO.6).

[0013] The third object of the present invention is to provide a bHLH148 The biological material of the gene overexpression vector comprises: bHLH148 The invention relates to a recombinant microorganism containing an overexpression vector of the bHLH148 gene, a transgenic plant cell line containing the overexpression vector of the bHLH148 gene, a transgenic plant tissue containing the overexpression vector of the bHLH148 gene, and a transgenic plant organ containing the overexpression vector of the bHLH148 gene.

[0014] The fourth object of the present invention is to provide a bHLH148 Application of recombinant vectors, expression cassettes or engineered bacteria of gene sequences in regulating the flowering node of tomatoes.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention is achieved by overexpression in tomato plants bHLH148 genes, and further screened bHLH148 Plants with gene expression increased by 151 to 288 times were cultivated to obtain tomato plants with lower flowering nodes and earlier flowering. bHLH148 The average number of flowering nodes of the gene plants is between 3 and 5, while the average number of wild plants is 11; bHLH148The first inflorescence of the genetically modified plants budded within an average of 17-19 days, compared to an average of 43.25 days for wild plants. This result provides a new technical direction for targeted improvement of flowering nodes, regulating flowering time and the time it takes for mature fruit to reach market. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the bHLH148 gene test results of the transgenic plants and AC wild-type plants in Example 3.

[0017] Figure 2 This is a statistical diagram of the flowering node positions of the transgenic plants and AC wild plants in Example 3.

[0018] Figure 3 This is a graph showing the statistical results of the bud appearance time of the first inflorescence of the transgenic plants and AC wild plants in Example 3.

[0019] Figure 4 This is the phenotype of the flowering node of the transgenic plants and AC wild plants in Example 3. DETAILED DESCRIPTION

[0020] The present invention will be further described in detail below in conjunction with specific examples so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0021] Example 1 This embodiment provides bHLH148 The method for constructing a gene overexpression vector is as follows: Primers FW containing homologous recombination arms: CATTTGGAGAGGACACGCTCGAGTGTGTTGTCAATGAAGTCAAGATCT and RV: TCTCATTAAAGCAGGACTCTAGACTTAGGCAAACATAATAATAAACTT were used to clone cDNAs prepared by reverse transcription of total RNA from various tissues of cultivated tomato Ailsa Craig (abbreviated as AC, purchased from the Tomato Genetics Center TGRC (https: / / tgrc.ucdavis.edu / )). bHLH148 gene and obtain the target fragment.

[0022] The PCR amplification system used in this step is as follows:

[0023] The specific PCR amplification procedures are as follows:

[0024] After PCR amplification according to the above steps, the target fragment was recovered by gel excision.

[0025] The pHellsgate8 vector (a gift from CSIRO Plant Industry) was double-digested with X-hoI and X-baI, and the linearized vector was recovered from the gel. The recovered target fragment was then subjected to homologous recombination with the linearized vector. The concentrations of the insert and linearized vector were adjusted. The optimal amount of linearized vector used was 0.02 times the number of base pairs of the linearized vector, and the optimal amount of insert used was 0.04 times the number of base pairs of the insert. The required volume can be calculated based on the amount used and the measured concentration. To a 10 μL system, add 2 μL of 5X CE II Buffer, 1 μL of Exnase II, the calculated insert, and the linearized vector. Make up the remaining volume with ddH2O. After adding all reaction solutions in order, gently pipette to mix thoroughly. Then, place the reaction in a PCR machine at 37°C and 30 min for homologous recombination.

[0026] The ligation product was transformed into E. coli, and the E. coli single clone with the correct sequencing result was amplified and the plasmid was extracted using a DNA miniprep kit to obtain bHLH148 Gene overexpression vector bHLH148-pHellsgate8.

[0027] Example 2 This embodiment provides bHLH148 Genetic transformation for gene overexpression is as follows: (1) Transform the constructed and correctly sequenced recombinant vector into Agrobacterium competent cells (C58) for later use.

[0028] (2) Use of bHLH148 -pHellsgate8 vector was used to infect cotyledonary explants of tomato cultivar AC.

[0029] (3) Cultivate the infected explants in dark.

[0030] (4) Screen the explants until callus tissue grows.

[0031] (5) The callus is subcultured until a growth point emerges.

[0032] (6) Transfer the grown growth points into the rooting medium.

[0033] (7) The rooted plants were transplanted into nutrient soil for growth, and PCR detection was performed using sequencing primers. The PCR product bands were consistent with the length of the bHLH148 gene, and a positive transgenic line was obtained.

[0034] In the above process, plant, E. coli, and Agrobacterium positive detection were all performed using taq-PCR. Positive plants and clones were detected using a primer pair (SEQ. NO. 7 and SEQ. NO. 4), and positive clones were sequenced using a sequencing primer pair (SEQ. NO. 7 and SEQ. NO. 8). The upstream primer sequence of the sequencing primers was: ACGCACAATCCCACTATCCTTC (shown in SEQ. NO. 7); the downstream primer sequence was: CGGTAAGGATCTGAGCTACACAT (shown in SEQ. NO. 8).

[0035] The taq-PCR detection system is as follows:

[0036] The taq-PCR reaction procedure is as follows:

[0037] The sequence of the sequencing fragment of the recombinant vector is (shown in SEQ.NO.9):.

[0038] Plant tissue DNA extraction method: 1. Take 0.2 g of young leaves from the plant seedlings and place them in 2 mL of EP. Add about 750 µL of CTAB extract (with β-mercaptoethanol) and grind them. Then place them in a 65°C water bath for 50 min. 2. After the water bath, add about 750 µL of 24:1 (volume ratio of chloroform to isoamyl alcohol) to the centrifuge tube, mix by inverting, and centrifuge at 10,000 rpm for 10 minutes. 3. Pipette 500µL of supernatant into a new 1.5mL EP tube, add an equal volume of isopropanol (stored in a -20℃ refrigerator), mix gently, place in a -20℃ refrigerator for 10 minutes, and centrifuge at 12000r / min for 10 minutes; 4. Discard the supernatant and add 800µL 75% alcohol to wash the precipitated DNA. Centrifuge briefly, remove excess alcohol, and dry the DNA in a fume hood until translucent. 5. Add 300µL ddH2O (the amount can be adjusted according to different uses) to dissolve the DNA and store at 4°C until use.

[0039] Example 3 This embodiment provides bHLH148 Gene expression tests are as follows: 3.1 Use the following method to test the expression level of transgenic positive plants The young shoot tip tissues of transgenic plants were taken for RNA extraction. bHLH148 -OE-3, bHLH148 -OE-4 and bHLH148 -OE-6) bHLH148 Gene expression level detection. Expression level detection steps include RNA extraction, reverse transcription and gene expression level detection.

[0040] RNA extraction method: 1. After grinding with liquid nitrogen, take about 0.2 g of sample and transfer it to a 2.0 mL centrifuge tube that has been frozen with liquid nitrogen. Add 1 mL of Trizol, invert, and let stand for 5 minutes to extract. 2. Add 200 µL of chloroform, invert vigorously for 15 seconds, and let stand at room temperature for 3 minutes; 3. Centrifuge at 4°C, 12,000 rpm for 15 min. 4. Transfer 400µL of supernatant to a new 1.5mL centrifuge tube (the amount of supernatant can be reduced to ensure RNA purity), add an equal volume of ice-cold isopropanol (stored in a -20℃ refrigerator), gently invert to mix, and then place at -20℃ for 10 minutes; 5. Centrifuge at 4°C, 12,000 rpm for 10 min. 6. Discard the supernatant and add 1 mL of 75% ethanol (diluted with DEPC water) to suspend the precipitate; 7. Centrifuge at 12000 rpm for 1 minute. 8. Aspirate and discard the supernatant (steps 6 and 7 can be repeated once to improve the purity of the extracted RNA) and place the centrifuge tube in a fume hood to air-dry for 5-10 minutes; 9. Add about 40µL (adjust according to the amount of extracted RNA) of DEPC water to dissolve; 10. Check RNA quality by agarose gel electrophoresis and determine RNA concentration by absorbance, then store at -80°C until use.

[0041] RNA reverse transcription steps: According to the concentration of RNA samples, the RNA samples are uniformly diluted to a specific concentration and reverse transcribed using a reverse transcription kit. The reverse transcription process is as follows: 1) Removal of genomic DNA Prepare the following mixture in an RNase-free centrifuge tube:

[0042] Pipette to mix, centrifuge and place in PCR instrument at 42℃ for 2min.

[0043] 2) First-strand cDNA synthesis Add 2 μL of 10X RT Mix and 2 μL of HiScript II Enzyme Mix to the mixture from the previous step. Pipette to mix thoroughly, centrifuge, and place in a PCR instrument. Set the PCR cycle to: 50°C for 15 minutes; 85°C for 2 minutes. After the reaction is complete, the product can be used directly in qPCR reactions or stored at -20°C.

[0044] bHLH148 Determination of relative expression level The assay was performed using the following procedure and SYBR mix system. bHLH148 The relative expression level of the gene was determined by a conventional method in the art. The internal reference gene Solyc11g005330 was selected.

[0045] The reaction system is as follows:

[0046] The SYBR mix system is as follows:

[0047] In the table, Q_FW and Q_RV are the upstream primer and downstream primer of the expression detection primer, respectively.

[0048] The primers for detecting the expression of the internal reference gene Solyc11g005330 are: Q_FW: GTCCTCTTCCAGCCATCCA Q_RV: ACCACTGAGCACAATGTTACCG bHLH148 The primers for detecting gene expression are: Q_FW: AGAGAGAGTTTACTCGTCAGCACC (SEQ.NO.5) Q_RV: ATCATCAACGAGAGGGTAATATCC (SEQ.NO.6) The test results are as follows Figure 1 As shown, transfer bHLH148 Compared with the wild-type control (AC), the tomato plants carrying the pHellsgate8 vector showed bHLH148 The relative expression of genes increased significantly. bHLH148 The relative expression levels of genes are shown in Table 1. Figure 1 shown.

[0049] Table 1 bHLH148 Relative gene expression test results

[0050] 3.2 bHLH148 Gene overexpression had an effect on the first flowering node of the plant (the first inflorescence node, the results are shown in Table 2, Figure 2 ) and the first inflorescence budding time (results are shown in Table 3, Figure 3 ) were statistically analyzed, and the phenotypes of the flowering node of transgenic plants and AC wild plants were shown in Figure 4 .

[0051] Table 2 Statistics of flowering nodes of tomato plants

[0052] Table 3 Statistical results of the time of budding of the first inflorescence of tomato plants

[0053] According to Table 1-3 and Figure 1-4 The results show: bHLH148 The height of the overexpressing plants was significantly reduced compared to the control AC plants at the first flowering node, and visually showed an early flowering phenotype.

[0054] The above is only a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. bHLH148 The application of the gene in regulating the flowering node of tomatoes is characterized in that: described bHLH148 The ORF sequence of the gene is shown in SEQ.NO.

1. bHLH148 The amino acid sequence of the protein encoded by the gene is shown in SEQ.NO.

2.

2. A method for regulating the flowering node of tomatoes, characterized in that: The method is to construct a bHLH148 The gene is overexpressed in a vector and further infected into tomato explants to regulate the flowering node of tomatoes.

3. The method according to claim 2, characterized in that Build contains bHLH148 The primers used in the gene overexpression vector contain homologous recombination arms, the sequences of which are as follows: FW: CATTTGGAGAGGACACGCTCGAGTGTGTTGTCAATGAAGTCAAGATCT; RV:TCTCATTAAAGCAGGACTCTAGACTTAGGCAAACATAATAATAAACTT.

4. The method according to claim 2, characterized in that Build contains bHLH148 The vector used for gene overexpression is pHellsgate8 vector.

5. The method according to claim 2, characterized in that The method further comprises using the gene with sequence number Solyc11g005330 as an internal reference gene to perform bHLH148 Gene expression detection and screening bHLH148 Plants with gene expression increased by 151 to 288 times.

6. The method according to claim 5, characterized in that The specific primers for detecting the expression of the internal reference gene are as follows: Q_FW:GTCCTCTTCCAGCCATCCA; Q_RV:ACCACTGAGCACAATGTTACCG; described bHLH148 The specific primers for gene expression detection are as follows: Q_FW: AGAGAGAGTTTACTCGTCAGCACC; Q_RV:ATCATCAACGAGAGGGTAATATCC.

7. A method comprising the method according to claim 1 bHLH148 A biological material of a gene overexpression vector, characterized in that The biological material includes: bHLH148 A recombinant microorganism containing a gene overexpression vector bHLH148 A transgenic plant cell line containing a gene overexpression vector bHLH148 Transgenic plant tissue containing a gene overexpression vector bHLH148 Transgenic plant organs containing gene overexpression vectors.

8. Containing the composition according to claim 1 bHLH148 Application of recombinant vectors, expression cassettes or engineered bacteria of gene sequences in regulating the flowering node of tomatoes.

Images