Application of myrica rubra MrDof5.4 gene in improvement of agronomic traits of tomatoes

Abstract

The invention relates to the field of gene engineering, in particular to application of a waxberry MrDof5.4 gene in improvement of agronomic characters of tomatoes. According to the invention, a key gene MrDof5.4 which responds to a light signal and regulates and controls early development and quality formation of fruits is identified from waxberries through a specific low-intensity red laser processing model in combination with multi-omics analysis for the first time. Furthermore, by heterologous expression of the MrDof5.4 gene in a model crop tomato, it is verified that the MrDof5.4 gene can remarkably improve the plant height, stem diameter, single fruit weight and single plant yield of the transgenic tomato, and therefore the cross-species application potential of the gene in the aspects of improving the comprehensive agronomic traits of crops and improving the yield is verified.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to the Chinese bayberry (Myrica rubra). MrDof5.4 Application of genes in improving agronomic traits of tomatoes. Background Technology

[0002] tomato( Solanum lycopersicum Tomatoes are one of the most important vegetable crops globally, possessing extremely high economic and nutritional value. However, in modern agricultural production, tomato cultivation and industrial development face multiple challenges. Tomatoes are highly sensitive to abiotic stresses such as drought, high salinity, and extreme temperatures. Global climate change has exacerbated the frequency and intensity of these stresses, severely restricting stable tomato yields and the expansion of planting areas. Although some high-yielding varieties have been developed through traditional breeding methods, progress has been relatively slow due to the long breeding cycle, complex process, and frequent linkage with undesirable agronomic traits. In recent years, with the development of molecular biology techniques, directly introducing or regulating key functional genes using genetic engineering has become an effective strategy for crop improvement.

[0003] Yangmei ( Myrica rubra The Chinese bayberry (Myrica rubra) is a subtropical high-value fruit tree with unique flavor and vibrant color. Its fruit development and quality formation are particularly sensitive to light conditions, making it an ideal material for studying light signal regulation mechanisms. This suggests that the Chinese bayberry genome must contain conserved gene resources regulating desirable traits. Compared to tomatoes, Chinese bayberry may possess unique evolutionary pathways and regulatory networks in fruit ripening mechanisms, secondary metabolite synthesis, and stress response pathways. Based on this, this application aims to mine functional genes from the genetically distantly related species of Chinese bayberry and apply them to the genetic improvement of tomatoes. This cross-species utilization of gene resources is expected to overcome the limitations of intraspecific genetic resources in tomatoes, introduce novel modules for controlling desirable traits, and thus provide new technical solutions to common problems faced by the tomato industry.

[0004] The Dof (DNA binding with One Finger) family, an important branch of the Cys2 / His2 zinc finger superfamily, is widely distributed in the plant kingdom but not found in other eukaryotes. A key characteristic of this family of proteins is the N-terminal conserved Dof domain, composed of 50-52 amino acid residues, capable of specifically binding to the 5'-AAAG-3' motif. In recent years, with the application of high-throughput sequencing technology, members of the Dof gene family have been systematically identified in Arabidopsis thaliana, rice, soybean, and various economic crops such as sweet potato, potato, and sunflower. Studies have shown that Dof transcription factors play pleiotropic roles in plant development, metabolic regulation, and stress responses. In Arabidopsis thaliana, overexpression... BnCDF1 By inhibiting CO and FT Gene-delayed flowering, moso bamboo PheDof12-1 By direct combination BottomCOL4 The promoter promotes flowering under long-day conditions. Regarding fruit development, tomatoes... SlDof10 Downregulation triggers parthenogenesis by inhibiting genes involved in vascular tissue development, in durian. DzDof2.2 By activating the ACC synthase gene, ethylene synthesis is upregulated, accelerating fruit ripening. Furthermore, DoF members also play a crucial role in the regulation of sugar metabolism: rice OsDof11 By activating sucrose transport genes OsSUT1 , OsSWEET11 and OsSWEET14 Promotes sucrose distribution in potatoes StCDF1 It affects tuber maturation by regulating the expression of glycoefflux proteins.

[0005] These studies indicate that Dof transcription factors play a crucial role in regulating fruit development in plants, but their specific functions in tomatoes and their regulatory mechanisms on tomato plant height, stem diameter, fruit shape, yield per plant, and fruit weight still require further investigation. Analyzing the functions of Dof transcription factors in tomatoes can provide new molecular breeding strategies for improving tomato plant architecture and increasing yield. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a waxberry... MrDof5.4 Application of genes in improving agronomic traits of tomatoes.

[0007] The present invention provides, in a first aspect, bayberry. MrDof5.4 The use of genes or related biological materials in at least one of the following: A1) Regulating tomato agronomic traits or preparing products that regulate tomato agronomic traits; A2) Cultivate tomatoes with improved agronomic traits or prepare products with improved tomato agronomic traits; A3) Preparation of genetically modified tomatoes; The bayberry MrDof5.4 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] Furthermore, the biomaterial includes one or more of the following: The bayberry MrDof5.4 Gene-encoded proteins and / or containing the aforementioned bayberry MrDof5.4 Gene recombination vectors, recombinant microorganisms, or transgenic tomato cell lines.

[0009] Furthermore, the regulation of tomato agronomic traits includes: positively regulating the growth of bayberry... MrDof5.4 Gene expression is used to improve agronomic traits in tomatoes.

[0010] Furthermore, the aforementioned bayberry MrDof5.4The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2.

[0011] Furthermore, the one containing bayberry MrDof5.4 Gene recombination vectors include basic vectors and MrDof5.4 The basic vector includes the pBWA(V)KS-GFP vector.

[0012] Furthermore, the one containing bayberry MrDof5.4 Recombinant microorganisms include Agrobacterium tumefaciens EHA105.

[0013] Furthermore, the regulation of tomato agronomic traits includes at least one of the following: B1) Plant height; B2) Thick stem; B3) Fruit type; B4) Yield per plant; B5) Single fruit weight.

[0014] The second aspect of the present invention provides positive regulation of bayberry MrDof5.4 The application of gene-expressing biological materials in the cultivation of tomatoes with improved agronomic traits, the aforementioned bayberry MrDof5.4 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0015] Furthermore, the biomaterial includes: the bayberry. MrDof5.4 The gene encodes a protein and / or contains positive regulatory proteins in bayberries. MrDof5.4 Gene recombination vectors, recombinant microorganisms, or transgenic tomato cell lines.

[0016] Furthermore, the agronomic trait improvement includes at least one of the following: B1) Increased plant height; B2) Stem diameter increases; B3) Fruit shape optimization; B4) Increased yield per plant; B5) Increased weight per fruit.

[0017] A third aspect of the present invention provides a method for improving agronomic traits of tomatoes, comprising: The positive regulation of bayberry MrDof5.4 Gene-expressing biological material was transferred into tomatoes.

[0018] A fourth aspect of the present invention provides a method for cultivating tomatoes with improved agronomic traits, comprising: The positive regulation of bayberry MrDof5.4 Gene-expressing biological material was transferred into tomatoes.

[0019] In this application, Agrobacterium-mediated transformation was used to positively regulate the growth of bayberry. MrDof5.4Gene-expressing biological material was transferred into tomatoes.

[0020] Furthermore, the aforementioned bayberry MrDof5.4 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0021] Furthermore, the aforementioned bayberry MrDof5.4 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2.

[0022] Furthermore, the one containing bayberry MrDof5.4 Gene recombination vectors include basic vectors and MrDof5.4 The basic vector includes the pBWA(V)KS-GFP vector.

[0023] Furthermore, the one containing bayberry MrDof5.4 Recombinant microorganisms include Agrobacterium tumefaciens EHA105.

[0024] A fifth aspect of the present invention provides a tomato with improved agronomic traits, the tomato comprising positively regulated bayberry. MrDof5.4 Biological materials for gene expression; The bayberry MrDof5.4 The nucleotide sequence of the gene contains the sequence shown in SEQ ID NO.1; The agronomic trait improvement includes at least one of the following: B1) Increased plant height; B2) Stem diameter increases; B3) Fruit shape optimization; B4) Increased yield per plant; B5) Increased weight per fruit.

[0025] Compared with the prior art, the present invention has the following beneficial effects: This invention, through a specific low-intensity red laser treatment model combined with multi-omics analysis, identified a key gene in bayberry that responds to light signals and regulates early fruit development and quality formation. MrDof5.4 Furthermore, a variety of bayberry species involved in regulating plant height, stem diameter, fruit shape, yield per plant, and / or fruit weight was cloned from tomatoes. MrDof5.4 Genes make bayberries MrDof5.4 Gene overexpression, compared to wild-type plants, in waxberries MrDof5.4 Tomato lines with overexpressed genes showed significant increases in plant height, stem diameter, fruit shape, yield per plant, and fruit weight. (Yangmei) MrDof5.4 The cloning of genes and the discovery of new functions have provided new gene targets and resources for genetic breeding to increase tomato yield and improve plant type. Attached Figure Description

[0026] Picture 1The growth status of bayberry during the fruiting stage is shown in the red laser (2PPFD) treatment experiment. No laser treatment is used as the control (CK). M1 refers to the early color change stage, M2 refers to the middle color change stage, M3 refers to the late color change stage, and M4 refers to the maturity stage.

[0027] Picture 2 The fruit color index is determined by red laser (2PPFD) treatment during the fruiting period of bayberry. The color of the fruit was measured using a CR-10 handheld colorimeter (Konica Minolta Holdings, Inc., Japan), and its photometric intensity (L*), red-green axis (a*), and blue-yellow axis (b*) were recorded. The color (a* / b*) indicates the degree of red pigmentation of the fruit.

[0028] Picture 3 For qRT-PCR analysis MrDOF5.4 Gene expression levels changed at different time points; the gray bar represents the control group (CK), and the pink bar represents the red laser-treated group (R).

[0029] Picture 4 For bayberries MrDof5.4 A schematic diagram of the construction of the overexpression vector.

[0030] Picture 5 Wild type (WT) and MrDof5.4 Overexpression lines (OE8, OE11) MrDof5.4 The relative expression level of genes.

[0031] Picture 6 Wild type (WT) and MrDof5.4 Phenotypic identification of plants and fruit yield at the fruit enlargement and maturity stages of overexpression lines (OE8, OE11).

[0032] Picture 7 Wild type (WT) and MrDof5.4 Fruit type phenotype identification of overexpression lines (OE8, OE11).

[0033] Picture 8 Wild type (WT) and MrDof5.4 Results of fruit longitudinal diameter, transverse diameter, and fruit shape index determination in overexpression lines (OE8, OE11); among which, Picture 8 A represents wild-type (WT) and MrDof5.4 Results of longitudinal diameter measurement of fruits from overexpression lines (OE8, OE11); Picture 8 B represents wild-type (WT) and MrDof5.4 Results of fruit diameter measurement of overexpression lines (OE8, OE11); Picture 8 C represents wild-type (WT) and MrDof5.4 Results of fruit shape index determination of overexpression lines (OE8, OE11).

[0034] Picture 9 Wild type (WT) and MrDof5.4 Results of yield, plant height, and stem diameter measurements of overexpression lines (OE8, OE11); among which, Picture 9 A represents wild-type (WT) and MrDof5.4 Yield determination results of individual plants in overexpression lines (OE8, OE11); Picture 9 B represents wild-type (WT) and MrDof5.4 Results of plant height measurement of overexpression lines (OE8, OE11); Picture 9 C represents wild-type (WT) and MrDof5.4 Results of stem diameter measurement of overexpression lines (OE8, OE11). Detailed Implementation

[0035] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0036] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0037] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0038] In this invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used are commercially available or commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0039] In this application, Yangmei MrDof5.4 The CDS sequence of the gene is shown in SEQ ID NO.1, for the waxberry. MrDof5.4 The amino acid sequence of the gene-encoded protein is shown in SEQ ID NO.2.

[0040] SEQ ID NO.1: ATGCAAGACATCCATTCTATCAAAGGTGGCGGAAGATGGGGAGCGGAACGGAGGCTCCAGCCTCATCAGCACCTCCAGGGCCATCAACAGAACCACCACCAGGCGCTCAAGTGTCCGCGTTGTGACTCTATCAACACCAAGTTCTGTTACTACAACAACTACAACCTCTCCCAGCCCCGCCACTTCTGCAAGAGCTGTCGCCGCTACTGGACCAAAGGCGGCGTCCTCCGCAACGTCCCCGTGGGTGGTGGCTGCCGGAAGGCCAAACGATCCAAGCCGAAGCCTGCGTCCTCCGACGCATCAACTCCGCCGCAGCGGCGAGAACGCAAGTCCAATTCTCATTCTAGCAGCGAGAGCTCCAGTCTCACCGCCACAAACACTACCGCCGCCACAGAGGAAGTGTCGGCGGTCACGGCGGCGCCTTCTTTAACCTCAACTTCGAATCTTTTGAACGTACAAGAGACCAAATTGCTTGCTTCAAATCCTAGTTTCGAGAAAAGGTTGCCGGAACAAGTTTCCGATTGTGGGATATTCTCGGAGATTGGGAGCTTCACGAGTCTGATAATGGCGTCGAACGACGCATTGCCATCTGGGTTTAACCTCATCCCGGAAATTACGCGGTTTAGGTTGAACCAACAAGATCACGATCGTCATCAAGTTCAACAAAATCAACAACAGCACTGGCAGCATGATCAGCAGCAGCAGAAGGTGGTGGATGGTGTCGCTGGTGACGAATTGAATCTGCGGCAAGAGCTCATGGGTGGACTGGTTGATCTATCATCCTCCGAGAACAGATCCAGCTGTGGAGGATTCGGATCGTTGGACTGGCAAGTGAGTGGGGATCAAGGTTTGTTTGATCTTTCTAACACCGTTGATCAAGCTTACTGGAGTCAGAGTCAATGGCCTGATCCAGACCACCCCAGTCTCTATCTCCCGTAA; SEQ ID NO.2: MQDIHSIKGGGRWGAERRLQPHQHLQGHQQNHHQALKCPRCDSINTKFCYYNNYNLSQPRHFCKSCRRYWTKGGVLRNVPVGGGCRKAKRSKPKPASSDASTPPQRRERKSNSHSSSESSSLTATNTTAATEEVSAVTAAPSLTSTSNLLNVQETK LLASNPSFEKRLPEQVSDCGIFSEIGSFTSLIMASNDALPSGFNLIPEITRFRLNQQDHDRHQVQQNQQQHWQHDQQQQKVVDGVAGDELNLRQELMGGLVDLSSSENRSSCGGFGSLDWQVSGDQGLFDLSNTVDQAYWSQSQWPDPDHPSLYLP.

[0041] In this application, the germination medium, YEP medium, co-culture medium, recovery solid medium, screening medium, elongation solid medium, and rooting medium are all conventional commercially available media, and their formulations are shown below: Germination medium (per 1 L): MS medium: Ammonium nitrate (NH4NO3) 1650 mg / L, potassium nitrate (KNO3) 1900 mg / L, calcium chloride dihydrate (CaCl2·2H2O) 440 mg / L, magnesium sulfate heptahydrate (MgSO4·7H2O) 370 mg / L, potassium dihydrogen phosphate (KH2PO4) 170 mg / L, potassium iodide (KI) 0.83 mg / L, boric acid (H3BO3) 6.2 mg / L, manganese sulfate tetrahydrate (MnSO4·4H2O) 22.3 mg / L, zinc sulfate heptahydrate (ZnSO4·7H2O) 8.6 mg / L, sodium molybdate dihydrate (Na2MoO4·2H2O) 0.25 mg / L, copper sulfate pentahydrate (CuSO4·5H2O) 0.025 mg / L, cobalt chloride hexahydrate (CoCl2·6H2O) 0.025 mg / L. 37.3 mg / L of disodium EDTA dihydrate (Na2·EDTA·2H2O), 27.8 mg / L of ferrous sulfate heptahydrate (FeSO4·7H2O), 30 g / L of sucrose, 0.6-0.8% of agar, and pH adjusted to 5.8.

[0042] YEP medium (per 1L): 10 g peptone, 10 g yeast extract, 5 g sodium chloride, pH adjusted to 7.0.

[0043] Co-culture medium (per 1L): MS medium + AS (acetylsyleugenol) 100-200 μM.

[0044] Restoration solid medium (per 1L): MS medium + cephalosporin 250-500 mg / L.

[0045] Screening medium (per 1L): MS medium + cephalosporin 250-500 mg / L + hygromycin 10 mg / L.

[0046] Elongated solid medium (per 1L): MS medium + 6-BA 0.5-1.0 mg / L.

[0047] Rooting medium (per 1L): 1 / 2 MS medium + 0.1-0.5 mg / L 6-BA.

[0048] Example 1: Genes under laser regulation MrDof5.4 Filtering Dongkui bayberry grown in greenhouses ( Myrica rubra Using *Dongkui* as the material, two treatment groups were set up from the flowering period to fruit maturity: the experimental group received continuous irradiation with a 660nm red laser at low quantum flux density (PPFD); the control group received conventional white light irradiation under the same environment. Fruit samples from both groups were collected at the fruit formation stage, pit hardening stage, pre-coloring stage (M1), mid-coloring stage (M2), post-coloring stage (M3), and maturity stage (M4).

[0049] The results showed that, compared with the control group, laser treatment significantly increased the single fruit weight of bayberries, promoted the accumulation of sucrose, fructose, and glucose, and advanced the fruit color-changing period by 3-5 days. Picture 1 , Picture 2 Transcriptome sequencing was performed on fruit samples from each of the aforementioned stages. Bioinformatics analysis showed that the number of differentially expressed genes induced by laser treatment was most significant in the early color-changing stage (M1). By correlating the differentially expressed gene profile with the accumulation dynamics of key metabolites such as fruit sugars and pigments, a core candidate gene with the highest correlation to the laser treatment effect was identified. This gene encodes a Dof family transcription factor, named... MrDof5.4 Expression pattern analysis revealed that in the laser-processed group, MrDof5.4 Gene expression exhibited a specific peak during the early fruit coloring stage, with significantly higher expression levels than the control group, followed by a decline as the fruit matured. qRT-PCR was used to detect the expression pattern of this gene, specifically and significantly induced in the early fruit coloring stage after laser treatment, in independent samples. Picture 3 (Gray bars represent the control group, and pink bars represent the laser-treated group). MrDof5.4 The complete coding sequence (CDS) of the gene is shown in SEQ ID NO.1, and the protein sequence it encodes is shown in SEQ ID NO.2.

[0050] Example 2: Construction of recombinant plasmids Using Primer1 (SEQ ID NO.3) and Primer2 (SEQ ID NO.4) as primers and cDNA from the fruit of *Myrica rubra* as a template, PCR amplification was performed to obtain the target gene. MrDof5.4 The coding sequence (CDS) fragment (with stop codon removed). Design the following primers: Primer 1: 5' CACGGGGGACTTTGCAACGGTACCATGCAAGACATCCATTCTATC 3' (SEQ ID NO. 3); Primer 2: 5' TGAAGCGGCCGCTGTACAGGATCCCGGGAGATAGAGACTGGGGT 3' (SEQ ID NO.4).

[0051] PCR amplification reaction in Bio The PCR was performed using a Rad T100 PCR instrument, with a reaction volume of 50 μL: 2×Hieff Canace Plus PCR Master Mix 25 μL, Primer 1 (10μM) 2μL, Primer2 (10μM) 2μL, Template cDNA (200 ng / μL) 1 μL ddH2O 20μL.

[0052] The procedure is as follows: pre-denaturation at 98℃ for 3 min; denaturation at 98℃ for 10 s, annealing at 60℃ for 20 s, extension at 72℃ for 30 s, repeated 35 times; extension at 72℃ for 5 min; storage at 25℃.

[0053] The vector pBWA(V)KS-GFP was digested with restriction endonucleases Kpn I and BamHI. The digestion reaction system (50 μL) consisted of: 5 μg of vector plasmid, 1 μL of Kpn I, 1 μL of BamHI, 5 μL of 10x NEB Buffer, and ddH2O added to a final volume of 50 μL. After mixing, the mixture was digested at 37°C for 1 h.

[0054] After PCR amplification products and pBWA(V)KS-GFP vector digestion products were recovered and purified, the linearized vector and PCR fragment were recombined using recombinase. The reaction system (10 μL) was as follows: 5 μL of 2x MultiF Seamless Assembly Mix, 2 μL of linearized vector, 1 μL of PCR fragment, and ddH2O added to a final volume of 10 μL. The mixture was reacted in a metal bath at 50 °C for 30 min and then placed on ice.

[0055] The recombinant product was transformed into Escherichia coli DH5α competent cells and cultured overnight at 37°C. Colony PCR was performed using Primer1 and Primer3 as primers, and positive single clones were selected for sequencing.

[0056] Primer 3: 5' GCAGGTCTAGACATGGAGTC 3' (SEQ ID NO.5).

[0057] The above primer Primer1 (SEQ ID NO.3) binds MrDof5.4 At the start of the CDS sequence of the gene, Primer3 (SEQ ID NO.5) is located in the pBWA(V)KS-GFP vector sequence.

[0058] Sequencing results showed that the PCR reaction yielded MrDof5.4 The gene fragment has the nucleotide sequence shown in SEQ ID NO.1, encoding a protein consisting of 188 amino acid residues (SEQ ID NO.2). The obtained recombinant vector was named pBWA(V)KS- MrDof5.4 -GFP (e.g.) Picture 4 ).

[0059] Example 3, Waxberry Gene MrDof5.4 Obtaining and identifying transgenic plants with overexpression The *Myrica rubra* strain constructed in Example 1 was transformed using Agrobacterium EHA105-mediated genetic transformation. MrDof5.4 Gene overexpression vector was transferred into the cotyledonary node of tomato to obtain bayberry. MrDof5.4 Gene overexpression in plants specifically includes the following steps: I. Obtaining Recombinant Agrobacterium pBWA(V)KS- MrDof5.4 - The GFP plasmid was mixed with Agrobacterium EHA105 competent cells and incubated on ice for 5 min. The pBWA(V)KS- plasmid was then electroporated (1500V, 5ms). MrDof5.4 GFP was transformed into Agrobacterium EHA105 competent cells to obtain a recombinant strain. The recombinant strain that was correctly identified by colony PCR was named EHA. pBWA(V)KS- MrDof5.4 -GFP.

[0060] II. Obtaining Transgenic Plants (1) Induction Select healthy, mature tomato seeds and sterilize them using chlorine gas (100 mL sodium hypochlorite (2% effective concentration)) for 5-10 minutes in a fume hood. After sterilization, inoculate the sterilized seeds onto germination medium and place them in a 25°C incubator.

[0061] (2) Pre-culture Once the tomato has sprouted to 4 true leaves, cut the leaves into 2 mm × 2 mm pieces and place them in a pre-culture medium.

[0062] (3) Preparation of Agrobacterium tumefaciens bacterial culture Agrobacterium EHA carrying the target gene plasmid pBWA(V)KS- MrDof5.4 -GFP was initially activated by spreading it on YEP medium. After 48 h of incubation, the cells were collected and activated again on fresh medium. After 24 h of incubation, the cells were collected in the infection solution, vortexed, and the OD600 of the bacterial solution was adjusted to 0.5 using a spectrophotometer before use.

[0063] (4) Infection Pour the prepared bacterial solution into the tomato leaves and incubate for 30 minutes. Discard the inoculum, then place the explants onto a co-culture medium lined with filter paper and co-culture at 25°C in the dark for 3-5 days.

[0064] (5) Restore culture Select explants that have grown well and are free from contamination after co-culture, place them on recovery solid culture medium, and recover culture for 7-10 days.

[0065] (6) Screening and cultivation Explants that had grown clustered shoots after recovery culture were inoculated onto selection medium and cultured under 16 h / 8 h light / dark conditions for 21 days.

[0066] (7) Elongation culture After screening, the well-growing shoots were transferred to a new elongation medium. They were cultured under 16 h / 8 h light / dark conditions for 21 days for selection.

[0067] (8) Rooting culture When the shoots grow to about 5 cm, they are transferred to rooting medium for further selection. The selection process involves 16 h / 8 h light / dark cycles for 21 days.

[0068] (9) Seedling hardening Remove the seedlings from the culture medium, wash off the culture medium adhering to the seedling roots, and transplant them into seedling trays filled with nutrient soil. Incubate at 27℃ for 16 h / 8 h light / dark cycles, hardening off for 3-4 weeks to obtain T0 plants.

[0069] III. Identification of Transgenic Plants 1. MrDof5.4 Identification of overexpressing plants (1) PCR molecular identification Genomic DNA was extracted from the T0 generation plants obtained in step two and used as a template for PCR amplification. The PCR primers were Primer1 (SEQ ID NO.3) and Primer3 (SEQ ID NO.5) from Example 1. The PCR products were detected by 1% agarose gel electrophoresis. The target band was detected in positive plants, with a band size of approximately 700 bp, while it was not detected in negative plants.

[0070] (2) MrDof5.4 Detection of gene expression levels The identified and screened in (1) MrDof5.4 Leaves of homozygous transgenic seedlings were thoroughly ground after being frozen in liquid nitrogen, and RNA was extracted using the Trizol method. An appropriate amount of RNA was then used to obtain cDNA using a reverse transcription kit for qRT. Template for PCR detection. Take an appropriate amount of template cDNA, and... MrTubulin The gene was used as an internal reference gene, and the Genious 2×SYBR Green FastqPCR Mix (No ROX Premixed) (RK21205; AB Clonal) kit was used in Bio Detection in Rad CFX96 real-time PCR instrument MrDof5.4 Gene expression levels. Used MrTubulin Primers for quantitative gene detection (Primer4 and Primer5) and MrDof5.4 The sequences of the primers (Primer6 and Primer7) for gene quantification are as follows: Primer 4: 5' TGTCCCTATTTACGAGGGTTATGC 3' (SEQ ID NO.6); Primer5: 5' CAGTTAAATCACGACCAGCAAGAT 3' (SEQ ID NO.7).

[0071] Primer 6: 5' ATGCAAGACATCCATTCTATC 3' (SEQ ID NO.8); Primer 7: 5' TTACGGGAGATAGAGACTGG 3' (SEQ ID NO.9).

[0072] The relative expression level was calculated using the 2-ΔΔCt method, and the results are as follows: Picture 5 As shown, in wild-type leaves MrDof5.4 Based on the relative expression level, in the two overexpression lines (OE8 and OE11) MrDof5.4 The relative expression levels of all genes increased significantly.

[0073] Example 4, Waxberry Gene MrDof5.4 Agronomic traits identification of overexpressing transgenic plants WT (control group) and MrDof5.4 Homozygous overexpression lines (OE8 and OE11) were grown in a greenhouse. Plant height, stem diameter, fruit shape, single fruit weight, and yield per plant were observed at maturity. Phenotypic data were collected from 20 plants in each system, and photographs were taken. The results are as follows: Picture 6 , 7 As shown.

[0074] The results showed that, compared with WT, the two overexpression lines (OE8 and OE11) exhibited significant improvements in plant height, stem diameter, fruit shape, single fruit weight, and yield per plant. Specifically, the average transverse diameter of WT fruits was 18.72 cm, while the transverse diameter of fruits from the overexpression lines (OE8 and OE11) both exceeded 19.44 cm (e.g., ...). Picture 7 , Picture 8 B); The fruit longitudinal diameter of the overexpression lines (OE8 and OE11) was not significantly different from that of the wild type. Picture 8 A); Compared to the wild type, the overexpression lines (OE8 and OE11) had significantly lower fruit aspect ratios (fruit shape index). Picture 8 C). The average yield per plant for WT was 23.4 g, while the average yield per plant for overexpression lines (OE8 and OE11) was 30.0–36.7 g (e.g., ...). Picture 9 A); The average plant height of WT was 10.8 cm, while the plant height of the overexpression lines (OE8 and OE11) both exceeded 12.5 cm (e.g. Picture 9 B); The average stem diameter of WT was 4.51 cm, while the stem diameter of the overexpression lines (OE8 and OE11) both exceeded 4.57 cm (e.g. Picture 9 C); Explanation of bayberry MrDof5.4 Gene overexpression lines can significantly increase tomato plant height, stem diameter, fruit shape, single fruit weight, and yield per plant, thereby helping to improve tomato yield.

[0075] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Waxberry MrDof5.4 The use of genes or related biological materials in at least one of the following: A1) Regulating tomato agronomic traits or preparing products that regulate tomato agronomic traits; A2) Cultivate tomatoes with improved agronomic traits or prepare products with improved tomato agronomic traits; A3) Preparation of genetically modified tomatoes; The bayberry MrDof5.4 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, wherein the biomaterial comprises one or more of the following: The bayberry MrDof5.4 Gene-encoded proteins and / or containing the aforementioned bayberry MrDof5.4 Gene recombination vectors, recombinant microorganisms, or transgenic tomato cell lines.

3. The application as described in claim 1 or 2, characterized in that, The regulation of tomato agronomic traits includes at least one of the following: B1) Plant height; B2) Thick stem; B3) Fruit type; B4) Yield per plant; B5) Single fruit weight.

4. The application as described in claim 3, characterized in that, The regulation of tomato agronomic traits includes: positive regulation of bayberry... MrDof5.4 Gene expression is used to improve agronomic traits in tomatoes.

5. Positive regulation of bayberry MrDof5.4 The application of gene-expressing biological materials in the cultivation of tomatoes with improved agronomic traits, the aforementioned bayberry MrDof5.4 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. The application as described in claim 5, characterized in that, The biomaterial includes: the bayberry. MrDof5.4 The gene encodes a protein and / or contains positive regulatory proteins in bayberries. MrDof5.4 Gene recombination vectors, recombinant microorganisms, or transgenic tomato cell lines.

7. The application as described in claim 5, characterized in that, The agronomic trait improvement includes at least one of the following: B1) Increased plant height; B2) Stem diameter increases; B3) Fruit shape optimization; B4) Increased yield per plant; B5) Increased weight per fruit.

8. A method for improving the agronomic traits of tomatoes, characterized in that, include: Corrective measures will be implemented for the regulation of bayberry. MrDof5.4 Gene-expressing biological material was transferred into tomatoes.

9. A method for cultivating tomatoes with improved agronomic traits, characterized in that, include: Corrective measures will be implemented for the regulation of bayberry. MrDof5.4 Gene-expressing biological material was transferred into tomatoes.

10. A tomato with improved agronomic traits, characterized in that, The tomato contains positively regulated bayberry MrDof5.4 Biological materials for gene expression; The bayberry MrDof5.4 The nucleotide sequence of the gene contains the sequence shown in SEQ ID NO.1; The agronomic trait improvement includes at least one of the following: B1) Increased plant height; B2) Stem diameter increases; B3) Fruit shape optimization; B4) Increased yield per plant; B5) Increased weight per fruit.

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