Application of MdHT13.2 gene in regulation and control of plant growth

Abstract

The invention belongs to the field of molecular biology, and particularly relates to application of an MdHT13.2 gene in regulation and control of plant growth. According to RNA-seq (Ribonucleic Acid-Seq) data analysis, MdHT13.2 with high expression quantity in apple leaves is screened out. Mutant yeast sugar absorption complementation experiments show that MdHT13.2 not only has transport activity on hexose such as glucose, fructose and the like, but also has stronger transport capacity on cane sugar. The results of in-situ hybridization of apple leaves and GUS staining experiments of arabidopsis show that MdHT13.2 has a high expression level in the phloem of the vein. The MdHT13.2 as a member in a hexose transporter gene family can regulate and control sugar accumulation in plants and promote growth of the plants.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, and specifically relates to a... MdHT13.2 The application of genes in regulating plant growth. Background Technology

[0002] apple( Penalty × domestic As one of the world's four major fruits, the apple is highly popular among consumers due to its unique flavor, rich nutrition, and certain medicinal value. Carbohydrates in apples are not only the source of energy but also the core determinants of their sweetness, crispness, flavor profile, and storability. The dynamic transformation of sugar, starch, and pectin occurs throughout the entire process of apple development and ripening, while varietal characteristics and cultivation techniques further shape the composition of carbohydrates, ultimately resulting in the diverse and delicious qualities of apples.

[0003] The yield and quality of apples are largely influenced by the distribution and accumulation of photosynthetic assimilates. The distribution pathway of carbon assimilates from the leaf (the "source") to the storage organs (the "sink") plays an indispensable role in the overall growth and development of the plant. When mature apple leaves perform photosynthesis, they produce large amounts of sugars. These sugars are transported to various sink organs via the phloem for utilization or storage for later use. The transported assimilates are non-reducing sucrose or sorbitol. Sorbitol, as a characteristic transport sugar in Rosaceae plants, shares the physiological function of long-distance transport of photosynthetic assimilates with sucrose. When sugar is transported to sink organs, it causes an increase in the sugar concentration inside the fruit cells. This phenomenon is detrimental to maintaining efficient transport between source and sink organs, as an excessively high sugar concentration gradient can create an obstacle.

[0004] Therefore, identifying genes in apples related to carbohydrate metabolism has profound scientific significance and industrial application value for regulating apple sugar transport and controlling apple quality and yield. Summary of the Invention

[0005] In view of the above technical problems, the present invention provides a MdHT13.2 The application of genes in regulating plant growth MdHT13.2 Genes can mediate sugar transport, increase sugar accumulation, and promote plant growth.

[0006] The specific technical solution provided by this invention is as follows: In a first aspect, the present invention provides a MdHT13.2 The application of genes in regulating plant growth, the aforementioned MdHT13.2 The accession number of the gene in the Rosaceae Genome Database is MD13G1189100.

[0007] In a preferred embodiment of the present invention, the plant is a tomato or an apple.

[0008] As a preferred embodiment of the present invention MdHT13.2 Genes are used to promote photosynthesis in plants.

[0009] As a preferred embodiment of the present invention MdHT13.2 Genes are used to promote sugar accumulation in plants.

[0010] More preferably, overexpression MdHT13.2 Genes are used to promote the accumulation of fructose, glucose, galactose, and sucrose in tomato fruits; silencing these genes... MdHT13.2 The gene is used to promote the accumulation of glucose, galactose, and fructose in apple plants.

[0011] In a second aspect, the present invention provides a method for increasing the sugar content of apples or regulating apple growth, comprising silencing the apple plant as described above. MdHT13.2 Gene 。

[0012] In a third aspect, the present invention provides a method for increasing the sugar content of tomato fruit or regulating tomato growth, comprising overexpressing the sugar content of tomato plants as described above. MdHT13.2 Gene 。

[0013] In a preferred embodiment of the present invention, the sugar content is the content of fructose, glucose, and galactose.

[0014] In a fourth aspect, the present invention provides a method for cultivating dwarf apple lines, comprising silencing apple plants as described above. MdHT13.2 Gene 。

[0015] In a fifth aspect, the present invention provides a method for cultivating dwarf tomato lines, comprising overexpressing the [specific expression] in tomato plants. MdHT13.2 Gene 。

[0016] The beneficial effects of this invention are as follows: 1. Based on RNA-seq data analysis, this invention screens for high expression levels in apple leaves. MdHT13.2 .right MdHT13.2Amino acid sequence and phylogenetic analysis with homologous genes from different species revealed that MdHT13.2 possesses an MFS domain containing 12 transmembrane domains, making it a typical hexose transporter. Subcellular localization experiments confirmed its location on the cytoplasmic membrane. Mutant yeast sugar uptake complementation experiments showed that MdHT13.2 not only exhibits transport activity for hexoses such as glucose and fructose but also demonstrates a strong transport capacity for sucrose. In situ hybridization of apple leaves and GUS staining experiments in Arabidopsis thaliana showed high expression levels of MdHT13.2 in the phloem of leaf veins. These results suggest that MdHT13.2, as a member of the hexose transporter gene family, may play a role in phloem sugar transport, thereby regulating sugar accumulation in plants and promoting plant growth.

[0017] 2. Obtaining overexpression through Agrobacterium-mediated genetic transformation MdHT13.2 The results of sugar content determination in tomato plants showed that overexpression MdHT13.2 It significantly enhanced the glucose, fructose, sucrose, and galactose content of tomato fruits. To further explore the function of apple MdHT13.2, [further research was conducted]. MdHT13.2 Silent apple plants were observed to have an increased number of fine veins in their leaves and more prominent cell protrusions on the leaf surface. Compared to the wild type, the silent plantlets showed nearly twice the glucose and galactose content in their leaves, along with a slight increase in fructose content, while sucrose and sorbitol content were significantly reduced. Attached Figure Description

[0018] Figure 1 It's an apple. MdHT13.2 Expression characteristic analysis; A, MdHT13.2 Specific expression in different tissues of 'Fuji'; B, MdHT13.2 Specific expression in different tissues of 'Greensleeves'; DAB indicates the number of days after flowering, and 12pm indicates midnight.

[0019] Figure 2 It's an apple. MdHT13.2 Tissue-specific expression analysis; MdHT13.2 Relative expression levels in different tissues of 'GL-3' apple (root, stem, leaf, and shoot tip) using RT-qPCR; * indicates significant differences between leaves and other tissues. p <0.05).

[0020] Figure 3 This is an amino acid sequence alignment analysis of MdHT13.2; the purple part indicates that all sequences at this site are completely identical, the red or yellow part indicates that some sequences are identical, and the white part indicates... MdHT13.2 The sequence does not match the amino acid sequences of other species; the red horizontal line represents the MFS domain.

[0021] Figure 4 yes MdHT13.2 Phylogenetic analysis of homologous genes; amino acid sequence analysis of different species such as apple: MdHT13.2, tomato: SlSTP2, Arabidopsis: AtSTP13, and rice: OsMST4.

[0022] Figure 5 yes MdHT13.2 Transmembrane domain prediction; the horizontal axis (0-500) represents the position of amino acids, and the vertical axis represents the confidence level of the prediction result. Twelve purple rectangles represent predicted transmembrane domains; blue and orange indicate whether the amino acid sequence is located inside or outside the membrane, respectively.

[0023] Figure 6 It is the subcellular localization of MdHT13.2; A, MdHT13.2-GFP Subcellular localization in tobacco; B. MdHT13.2-GFP Subcellular localization of protoplasts. GFP (green fluorescence); Bright field; Merge map.

[0024] Figure 7 yes MdHT13.2-pro Arabidopsis thaliana GUS staining; A, MdHT13.2-pro GUS staining image of Arabidopsis thaliana leaf; magnified view of leaf veins in images B and A: the blue area indicated by the arrow is the location of GUS staining.

[0025] Figure 8 yes MdHT13.2 In situ hybridization experiment; A, MdHT13.2 A) Bright-field observation of leaf vein sections using antisense probes; B) Autofluorescence observation of leaf vein xylem under ultraviolet light; C) Fusion-field observation of sections; using MdHT13.2 Cross-section of apple leaf veins hybridized with positive probes as negative control (D / E / F); xylem (Xy) xylem; phloem (Ph) phloem; parenchyma cell (PC) parenchyma cell; palisade tissue (PT) palisade tissue. Scale bar, 200 μm.

[0026] Figure 9 This is an experiment on sugar uptake by a hexose-deficient yeast strain; A. Expression MdHT13.2 mutant yeast EYB.VW4000 The strains were cultured on media containing 1 mM, 10 mM, 100 mM Glc (glucose), Fru (fructose), and 100 mM Maltose, respectively; B, MdHT13.2 mutant yeast EYB.VW4000 The strains were cultured in liquid media containing 1 mM, 10 mM, 100 mM Glc (glucose), and Fru (fructose); the empty vector (pDR196) and mutant yeast served as controls.

[0027] Figure 10 This is an experiment on sugar uptake by sucrose-deficient yeast strains; A. Sucrose-deficient yeast strains SUSY7 heterologous expression MdHT13.2 . SUSY7 Mutant yeast was cultured on media containing 1 mM, 10 mM, 100 mM sucrose, and 100 mM glucose; B, mutant yeast culture was cultured overnight on liquid media containing 1 mM, 10 mM, 100 mM sucrose, and 100 mM glucose; empty vector (pDR196) and mutant yeast served as controls.

[0028] Figure 11 yes MdHT13.2 Identification of transgenic tomato lines; A. Heterologous overexpression in tomato MdHT13.2 Phenotypic characteristics of the three strains (L1, L4, L5); B. Quantitative verification at the RNA level. MdHT13.2 Expression levels of overexpressed tomato lines; ** indicates highly significant differences between wild-type and transgenic lines. p <0.01); C. Design specific primers at the DNA level for identification. MdHT13.2 Overexpression was performed on three tomato lines (L1, L4, and L5).

[0029] Figure 12 yes MdHT13.2 Determination of tomato sugar-related indicators by overexpression; A. Tomato heterologous overexpression MdHT13.2 The contents of Fructose, Glc, Suc, and Gal in fruits and wild-type fruits were determined at the expansion stage (17 DAB), color-changing stage (30 DAB), and maturity stage (45 DAB). DAB represents the number of days after flowering; B, MdHT13.2 Determination of soluble solids in tomato fruit; C, wild type and three MdHT13.2 Determination of Fru (fructose), Glc (glucose), and Suc (sucrose) content in the roots of overexpressing tomato lines (L1, L4, L5); different letters indicate significant differences. p <0.05).

[0030] Figure 13 yes MdHT13.2 Identification of transgenic apple plants; A, wild-type WT apple and three MdHT13.2 A. Detecting silent apple strains at the DNA level using specific primers. B. Detecting silent apple strains at the RNA level. MdHT13.2 The expression level in WT was set to 1.0; different letters indicate significant differences ( p <0.05).

[0031] Figure 14 yes MdHT13.2 Phenotypic observation of transgenic apple leaves; wild-type and three [types] at the same time. MdHT13.2 Observation of silent genetically modified apple leaves.

[0032] Figure 15 yes MdHT13.2 Determination of leaf sugar content in silent apple lines and wild types; A~F represent the determination of Fru (fructose), Glc (glucose), Suc (sucrose), Sor (sorbitol), Gal (galactose), and starch content, respectively; different letters indicate significant differences. p <0.05). Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] 1. Materials and Methods 1.1 Experimental Materials 1.1.1 Plant materials, strains, and vectors Test plant: 'GL-3' Malus domestica Apple tissue culture seedlings, Tobacco Benzovia ( ) Nicotiana benthamiana Apple callus tissue, 'Micro-Tom' tomato ( Solanum lycopersicum Arabidopsis thaliana ( Arabidopsis thaliana (e.g., Columbia type).

[0035] Tested strains: Escherichia coli: DH5α (Tulugang Biotechnology Co., Ltd.); Agrobacterium tumefaciens: GV3101, EHA105 (Weidi Biotechnology); Agrobacterium rhizogenes: K599 (Weidi Biotechnology); Yeast hexose-deficient strains: EYB.VW4000 Yeast sucrose-deficient strains: SUSY7All of these were preserved in the laboratory of the invention team and recorded in the following literature: Fakher B, Ashraf MA, Wang L, Wang X, Zheng P, Aslam M, Qin Y. Pineapple SWEET10 is a glucose transporter. Hortic Res. 2023Apr 12;10(10):uhad175. ;Tian X, Zou H, Xiao Q, Xin H, Zhu L, Li Y, Ma B, CuiN, Ruan YL, Ma F, Li M. Uptake of glucose from the rhizosphere, mediated by apple MdHT1.2, regulates carbohydrate allocation. Plant Physiol. 2023 Aug 31;193(1):410-425.) The test vectors: pGWB406 (gateway vector construction), pDR196 (Kanglang Biotechnology), and pk7wwG2D (II) were constructed according to the literature record "Tian X, Zou H, Xiao Q, Xin H, Zhu L, Li Y, Ma B, Cui N, RuanYL, Ma F, Li M. Uptake of glucose from the rhizosphere, mediated by appleMdHT1.2, regulates carbohydrate allocation. Plant Physiol. 2023 Aug 31;193(1):410-425."

[0036] 1.2 Experimental Methods 1.2.1 RNA extraction, reverse transcription and qRT-PCR RNA was extracted using the CTAB method.

[0037] Reverse transcription: Perform the procedure on ice, taking care to avoid RNA contamination. Use the kit (ToloScript RTEasyMix for qPCR) to reverse transcribe and obtain cDNA.

[0038] The total reaction volume (20 µL) consisted of the following components: template RNA 1 pg ~ 1 µg, 5× All-in-One RTBuffer 4 µL, All-in-One Enzyme Mix 1 µL, and H2O to bring the volume to 20 µL.

[0039] Quantification was performed using cDNA as a template. The reaction mixture consisted of the following components: 2×FastSYBR Mixture 25 µL, forward and reverse primers 1 µL each, Template DNA 2 µL, and H2O 22 µL.

[0040] Place the reaction mixture on ice and mix well. The PCR reaction program is as follows: 95℃ pre-denaturation for 20s, 95℃ denaturation for 3s, and 60℃ annealing / extension for 30s.

[0041] Follow the instructions and use 2 -△△t The method processes the data.

[0042] 1.2.2 DNA Extraction The specific steps for extracting DNA from plants are as follows: (1) Add approximately 0.1 g of plant tissue flash-frozen in liquid nitrogen to a 2 mL centrifuge tube, and add 2 µL of liquid nitrogen. β Add mercaptoethanol and 1 mL of DNA extraction buffer Buffer 1, vortex to mix, and let stand for 10 min, inverting the container 2-3 times during this period; centrifuge at 4°C for 15 min at 12000 rpm, and remove the supernatant. (2) Add 0.5 mL of preheated extraction buffer Buffer2 (65℃) and 2 µL of preheated extraction buffer to the centrifuge tube. β Resuspend the precipitate in mercaptoethanol, mix well, and incubate at 65°C for 10 min, inverting 3-5 times during incubation. Add chloroform:isoamyl alcohol = 24:1 to the centrifuge tube, mix well, and centrifuge at 12000 rpm for 15 min. Transfer 450 µL of the supernatant to a new centrifuge tube. Add 300 µL of isopropanol pre-chilled at -20°C, mix gently, and incubate at 4°C overnight. Centrifuge at 12000 rpm at 4°C for 15 min, and discard the supernatant. (3) Add 70% ethanol to rinse, centrifuge at 4℃ for 10 min, 12000 rpm, repeat this step three times; pour off the supernatant solution, let it air dry in a fume hood for 10 min, blow dry and add 20 µL H2O; measure the extraction concentration and store in a -20℃ refrigerator.

[0043] 1.2.3. Gene Cloning Search and download from the Rosaceae genome database. MdHT13.2 The full-length CDS sequence and promoter sequence, MdHT13.2The gene's accession number in the Rosaceae Genome Database is MD13G1189100. Design MdHT13.2 Full-length primers for the gene sequence (attb-HT13.2-F full-length: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGGCCGGCGGAGGGTTC-3', SEQ ID NO.8; attp-HT13.2-R full-length: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTACAACTGAGAAACACC-3', SEQ ID NO.9) were used to amplify the full-length gene (MdHT1) using cDNA as a template. 3.2-CDS Primers were designed using apple genomic DNA extracted by the CTAB method as the basic template for subsequent experiments.

[0044] The reaction system for amplifying the target gene is as follows: 1 μL template, 1 μL MdHT13.2-CDS, 1 μL MdHT13.2-pro R, 1 μL MdHT13.2-pro F, 25 μL mixed enzyme, and 22 μL H2O.

[0045] MdHT13.2-pro F: 5'-GTGCGCTATTGTTGCTTAAGTTC-3', SEQ ID NO.4; MdHT13.2-pro R: 5'-CAAGAAGAAGGACGCTTGGTG-3', SEQ ID NO. 5.

[0046] PCR reaction program: 94℃ pre-denaturation for 2 min, 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 72℃ final extension for 2 min. The number of denaturation, annealing, and extension cycles was 35.

[0047] The PCR products were recovered by electrophoresis.

[0048] 1.2.4 Target gene linked to T vector Select a PCR tube and add 1.5 µL (concentration of 50 ng / µL) of pMD19-T vector, 5 µL of Solution I enzyme (produced by TaKaRa) and 3.5 µL of target gene in sequence. Mix well, set the reaction temperature to 16℃, and react overnight to obtain the recombinant vector.

[0049] 1.2.5. Escherichia coli transformation Remove competent cells and place them on ice (0-4℃). Once the cell suspension has completely thawed and remains uniformly suspended, add 5 μL of the ligation product of the target gene (concentration ≥50 ng / μL). Mix well, incubate on ice for 30 min, then quickly transfer to a 42℃ water bath for heat shock for 90 s. After heat shock, immediately place the cells on ice and hold for 1-2 min. Add 400 μL of LB medium and incubate at 37℃ in a shaker for 60 min. Centrifuge to collect the cells, and spread the remaining 100 μL of supernatant onto resistant LB plates. Air dry, seal, and incubate upside down in a 37℃ incubator overnight.

[0050] 1.2.6 PCR-positive monoclonal colonies Approximately seven single-clone bacterial spots were selected and cultured in LB medium containing 200 μL of antibiotics for 4 h.

[0051] Transformed monoclonal colonies were validated by PCR using a 2×Es Taq MasterMix (Dye) premixed enzyme system. The PCR reaction mixture consisted of: 1 μL template, 1 μL R, 1 μL F, 10 μL mix enzyme, and 8 μL H2O.

[0052] MdHT13.2-F: 5'-ATGGCCGGCGGAGGGTTCCA-3', SEQ ID NO.2; MdHT13.2-R: 5'-CAACTGAGAAACACCATCAAATCCA-3', SEQ ID NO.3.

[0053] The PCR amplification procedure is the same as the gene cloning process described above.

[0054] 1.2.7 Plasmid Extraction 200 μL of bacterial culture was pipetted and sent to the company for sequencing. The sequence was aligned using SnapGene, and the alignment was successful. 200 μL of *E. coli* was added to 10 mL of LB medium and incubated overnight. The culture was then flash-frozen in liquid nitrogen at a 1:1 ratio of glycerol to bacteria and stored at -80°C. Plasmids were extracted from the *E. coli* culture using a plasmid extraction kit (HLingene).

[0055] 1.2.8 Constructing a Carrier Using the Gateway method, the designed MdHT13.2 specific fragment (257-bp) was inserted into the silencing vector pk7wwG2D(II), resulting in... MdHT13.2 Silencing vector. The constructed recombinant plasmid was transformed into Agrobacterium competent cells.

[0056] 1.2.9 Determination of tissue expression patterns Different tissues (roots, stems, leaves, and shoot tips) of 'GL-3' apples were sampled, flash-frozen in liquid nitrogen, and RNA was extracted using the CTAB method. cDNA was reverse transcribed using a kit, and primers were designed and their specificity determined using NCBI and SnapGene. Quantification was performed using real-time quantitative PCR (RT-qPCR).

[0057] 1.2.10. Agrobacterium-mediated transformation Add 5 μL of recombinant plasmid carrying the target gene to 100 μL of Agrobacterium competent cells, incubate on ice for 5 min, transfer to liquid nitrogen for 5 min, quickly place in a 37℃ water bath for 5 min, and finally place on ice for 5 min to stabilize the cell state.

[0058] In a sterile laminar flow hood, 700 μL of LB medium was added to Agrobacterium and cultured in a shaker at 37°C for 1 h. After centrifugation at 5000 rpm for 60 s, approximately 100 μL of supernatant remained. The supernatant was resuspended and spread onto plates containing the appropriate antibiotic. The plates were incubated upside down in a 28°C incubator for 3 days. Once single colonies had grown, they were picked for PCR detection to determine whether the transformation was successful.

[0059] 1.2.11. Arabidopsis GUS staining Will MdHT13.2 promoter ( MdHT13.2-pro ) is linked to the reporter gene ( β The pGWB433 vector contains glucuronidase.

[0060] Arabidopsis genetic transformation: Agrobacterium (GV3101) transformed with recombinant plasmids was cultured to OD. 600 Centrifuge at 6000 rpm for 10 min at room temperature with a concentration of 1.5~2.0; resuspend the bacterial cells in 5% sucrose and add 0.02% Siluet-77 (100 μL per 500 ml) and mix; immerse unopened Arabidopsis inflorescences in Agrobacterium liquid for 30 s, gently shake, and reinfect again after 30 min interval; after infection, keep Arabidopsis moist in the dark for 24 h, then place in a suitable environment to grow, collect and screen the seeds; sow and culture the screened seeds for about 10~15 days, then stain with GUS and fix with glycerol for subsequent photography and observation.

[0061] 1.2.12 Instantaneous conversion of tobacco leaves (1) MdHT13.2-CDS The recombinant plasmid containing the GFP reporter gene, inserted into the vector pGWB406 with a 35S structure, was transformed into Agrobacterium GV3101 culture overnight. OD 600 Value around 0.6, centrifuge at 5000 rpm for 5 min; (2) Preparation of tobacco resuspension: 0.01 mol / L MgCl2·6H2O, 0.01 mol / L ES·H2O, add water to make up to 400 mL, adjust pH to 5.6 with KOH, and add acetylsuccione solution after completion; (3) The bacterial suspension was placed in the dark at room temperature for 2-3 days to resuspend the bacterial solution; (4) Inject the back of tender tobacco plants, place them in an incubator and incubate in the dark for one day, then expose them to light for two days, and observe the fluorescence signal under a fluorescence microscope.

[0062] 1.2.13. Transient transformation of apple callus tissue 1) Apple callus protoplast extraction: The callus tissue was subcultured for 6 days. 50 ml of the callus tissue was centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and 10 mL of enzymatic hydrolysate was added. The tissue was then vacuum extracted for 20 min, and enzymatic hydrolysis was carried out for approximately 8 h with continuous gentle agitation. 100 mL of the enzymatic hydrolysate contained the following concentrations: 200 mM MES, 800 mM mannitol, 80 mM KCl, 2 g cellulase, 0.4 g dissociative enzyme, and 0.05 g pectinase.

[0063] The enzymatically digested callus protoplasts were filtered through a 70 μm sieve and transferred to a new 50 mL tube. The tubes were centrifuged at 800 rpm for 5 min, the supernatant was removed, and W5 buffer was added. The centrifugation process was repeated three times until the cell count reached 102. 6 Mix according to the protoplast transformation system. The plasmid to be transformed is MdHT13.2-406, and plasmid 2300-GFP is used as a control. The W5 Buffer solution consists of the following components: 3.08 mL 154 mM NaCl, 12.5 mL 125 mM CaCl2, 2.5 mL 5 mM KCl, 1 mL 2 mM MES, and H2O to 100 mL.

[0064] The protoplast transformation system was as follows: 1 μg of plasmid and 125 μL of protoplast cells were added to a 2 mL centrifuge tube. While adding 125 μL of PEG, the tube wall was gently tapped. The time was 10 min. The mixture was then added to W5 solution and placed in the dark at 25°C for 16-22 h. The cells were observed using a confocal microscope.

[0065] 40% PEG / Ca 2+ Buffer preparation: 4 g PEG-4000 + 0.364 g D-Mannitol + 1 mL CaCl2 (1 M), add H2O to 10 mL.

[0066] 1.2.14. Defective yeast sugar absorption experiment Will MdHT13.2 The full-length clone was inserted into the yeast vector pDR196 (MdHT13.2-pDR196-F: 5'-CCAAGCTTGTGAGGTACTAGAATCAAGTATTC-3', SEQ ID NO.6; MdHT13.2-pDR196-R: 5'-CATGCATGCCTTCCCCAGGTTTACAACTGAG-3', SEQ ID NO.7) to obtain the recombinant vector pDR196- MdHT13.2 Transform to EYB.VW4000 Yeast mutants with hexose uptake deficiency and yeast mutants with sucrose uptake deficiency SUSY7 Recombinant yeast strains were obtained. MdHT13.2-EYB.VW4000 and recombinant yeast; The recombinant yeast strain was cultured in a medium containing glucose, sucrose, and maltose. OD 600 The concentration was 1.0, and the yeast culture was diluted to 10 with sterile physiological saline. -1 10 -2 and 10 -3 Concentration gradient.

[0067] Take 5 μL MdHT13.2-EYB.VW4000 Yeast solutions were spotted onto SD(-URA) solid media containing different concentrations of sugar; similarly, 5 μL of recombinant yeast solution was spotted onto SD(-URA) solid media containing 1 mM, 10 mM, and 100 mM sucrose. After 3 days of incubation, observations and photographs were taken and recorded.

[0068] 1.2.15 In situ hybridization (1) Embed, fix and section the tissue material (note that no RNase should be used during the operation). Cut the leaf into small squares and put them into FAA fixative, add to 10 mL, vacuum for about 15 min, wait for all the tissue to sink to the bottom, and let stand overnight. FAA fixative consists of the following components: 1.35 mL formaldehyde, 0.5 mL glacial acetic acid, 5 mL anhydrous ethanol and 3.15 mL DEPC H2O.

[0069] (2) Dehydration of materials at 4℃: 50% ethanol (material dehydration), 60% ethanol, 70% ethanol, 90% ethanol (0.01 g of eosin added per 10 mL), 100% ethanol gradient dehydration, 30 min per step; (3) Material transparency treatment: 25% xylene-75% ethanol (xylene makes the material transparent), 50% xylene-50% ethanol, 75% xylene-25% ethanol, 100% xylene, and 100% xylene were each treated at room temperature for 60 min. (4) Use xylene to pour the material into the wax cup, add wax flakes of the same volume as xylene, place it in a 42℃ oven to melt slowly, add paraffin every 2 hours, add 3 times, and leave overnight; (5) Replace the mixture in the paraffin cup with paraffin every 6 hours, repeat 4 times, and let it stand overnight; (6) Material embedding: The material is rapidly solidified, cut into pieces, placed on a glass slide, observed under a microscope, dried at 42°C for 2 days, and then sealed for preservation; (7) In the probe synthesis and detection stage, the RNA probe kit produced by Roche was used to complete the probe synthesis (primers anti-sense-MdHT13.2-F: 5'-TTGCTCTTACTGCAAGCCGGTG-3', SEQ ID NO.14; anti-sense-MdHT13.2-R: 5'-TAATACGACTCACTATAGGGGTCTCTAGTGGGAACGTTTCAC-3', SEQ ID NO.15; sense-MdHT13.2-F: 5'-TAATACGACTCACTATAGGGTTGCTCTTACTGCAAGCCGGTG-3', SEQ ID NO.16; sense-MdHT13.2-R: 5'-GTCTCTAGTGGGAACGTTTCAC-3', SEQ ID NO.17), and the operation was carried out in accordance with the kit instructions. Specific primers were used to target... MdHT13.2 A specific sequence of the target gene was amplified, and the T7 promoter sequence (5'-TAATACGACTCACTATAGGG-3', SEQ ID NO.1) was added to the reverse sequence of the specific primers to prepare the antisense probe. The positive control probe was synthesized by adding the T7 promoter to the forward sequence, and the synthesized probes were stored at -80℃.

[0070] (8) Dewax and rehydrate the slides. Treat with preheated proteinase K at 37°C for 0.5 h, followed by treatment with PBS solutions at various levels. The probe needs to be pretreated. For each pair of slides, hybridization requires 5 μl of probe + 95 μl of formamide. Adjust the volume of 5 μl of probe to 100 μl with 50% formamide. Denature the probe at 80°C for 2 min, then immediately place it on ice. Cover the slides with twice the volume (200 μl) of hybridization solution and incubate overnight at 45°C.

[0071] (9) The following elution steps do not require enzyme-free washing. Wash away the hybridization solution, add antibody solution, and incubate at room temperature for 2 h. Place the sample in NBT / BCIP chromogenic solution for overnight treatment, and rinse the sample thoroughly with H2O.

[0072] (10) Microscopic observation results, apply mounting medium, place at room temperature for 2 days and then take a picture.

[0073] 1.2.16. Determination of sugar content (1) Weigh about 0.1 g of the sample stored at -80℃, quickly add 1400 μL of 75% methanol solution and 100 μL of 400 ppm ribitol solution, treat in a metal bath at 70℃ for 30 min at 950 rpm; centrifuge at room temperature at 12000 rpm for 10 min; transfer the supernatant to a dried 10 mL centrifuge tube, add 750 μL of chloroform solution and 1.4 mL of H2O, vortex, centrifuge at 2200 g for 15 min; transfer 1 mL of the supernatant and freeze at -50℃. (2) Take 2 μL of the extracted solution into a 1.5 mL centrifuge tube, vacuum dry for 1 h, add 40 μL of methoxygen (5 mg / mL), and perform a metal bath at 37℃, 950 rpm for 2 h, taking care to avoid light; add 60 μL of LMSTFA to the centrifuge tube, and set the metal bath program at 37℃ to 300 rpm for 30 min; measure within 1~2 days.

[0074] 1.2.17. Genetic transformation and identification of tomatoes (1) Soak the required seeds in water for 10 min, wash the seeds with 75% alcohol for 30 s, wash the seeds with 84 disinfectant and distilled water in a 1:1 ratio for 5 min, and then rinse the seeds repeatedly with sterile water 3 times. (2) Inoculate the seeds onto the inoculation medium and culture for 6-7 days until the two cotyledons of the seed have fully unfolded; (3) After the cotyledons of the sterile seedlings have fully expanded, select the cotyledon part precisely, cut each cotyledon into explant pieces with an area of ​​about 2 to 3 pieces, and place them on the culture medium (1 / 2 MS medium: MS + 2.0 mg / LZT + 0.2 mg / LIAA). (4) Take activated Agrobacterium and infect the cotyledon explants. The infection time is controlled at 20 min and the culture is carried out for 3 days. Wash the bacteria and culture for about 10 days. Change the selection medium every 15 days and culture in the light. (5) Propagate and root, transplant, and conduct DNA and RNA level identification.

[0075] 1.2.18. Genetic transformation and identification of apples (1) Experimental materials were selected from the tender leaves of 'GL-3' tissue culture seedlings with good growth and a rooting time of about 30 days. The strain was activated in advance, and streptomycin and spec were added in a specific ratio during the shaking process. The ratio of the two was set at 2:1 to optimize the efficiency of infection and transformation.

[0076] (2) Select large and thick leaves and immerse them in Agrobacterium resuspension. Make 3-4 cuts on the back of the leaves and gently press the leaves to ensure that the cuts are in full contact with the bacterial solution. The soaking time should be controlled at 8-10 minutes.

[0077] (3) Quickly absorb the excess bacterial solution adhering to the leaf surface. Lay the leaf flat on the surface of the co-culture medium with the back facing up, and use a black cloth to block the light. Incubate for 1-2 days. Co-culture medium: 4.43 g / L MS + 30 g / L sucrose + 8 g / L agar + 2 mL / L TDZ (1 mg / ml) + 0.5 mL / L NAA (1 mg / ml) + 1 mL / L AS (1 mol / L) + 1 mL / L Bet (0.1 mol / L).

[0078] (4) After the co-culture stage, the leaves were promptly transferred to the extension medium and cultured for another 2 days under the environmental conditions set in the medium. Extension medium: 4.43 g / L MS + 30 g / L sucrose + 8 g / L agar + 2 mL / L TDZ (1 mg / ml) + 0.5 mL / L NAA (1 mg / ml) + 1 mL / L Cef (250 mg / ml). (5) The leaves were transferred to the selection medium again and cultured in the dark for 30 days. The selection medium was: 4.43 g / L MS + 30 g / L sucrose + 8 g / L agar + 2 mL / L TDZ (1 mg / ml) + 0.5 mL / L NAA (1 mg / ml) + 1 mL / L Cef (250 mg / ml) + 0.5 mL / L Kana (50 mg / ml). (6) After the 30-day dark culture period, the screening medium carrying the leaves was transferred to a normal light environment until the resistant shoots successfully sprouted. The resistant shoots were propagated and rooted. After they were transferred to the substrate and grew stably, DNA and RNA detection were used for positive identification (primers HT13.2-detect Pk7-F: 5'-CCGTAAGAAGAGGCAAGAGTATGA-3', SEQ ID NO.10; HT13.2-detect Pk7-R: 5'-ACAGAAGGACACAGGAACAGG-3', SEQ ID NO.11).

[0079] 2. Data Statistical Methods IBM SPSS Statistics 25 (version 25.0) was used for data analysis, and GraphPad Prism 8.0 was used to create data charts. Statistical methods included independent samples t-tests and one-way ANOVA, with a significance threshold set at [value missing]. p <0.05. Experimental data are expressed as mean ± standard deviation (mean ± SD) of three independent biological replicates.

[0080] 3. Results and Analysis 3.1 Analysis of biological information and expression patterns of apple MdHT13.2 3.1.1 MdHT13.2 Expression characteristics analysis in Apple Twenty-nine MdHT family members were identified in the apple genome GDDH13v1.1. Further analysis of the transcriptome using RNA-seq data revealed a gene highly expressed in mature leaves, which was named... MdHT13.2 To further understand the expression characteristics of MdHT13.2, transcriptome analysis was performed on two apple varieties, 'Fuji' and 'Greensleeve'. The results showed that... MdHT13.2 The expression level is relatively high in the leaves of 'Fuji'; while in 'Greensleeve', the gene also shows high expression levels in green tissues such as mature leaves and stem tips. Figure 1 Based on the results, it can be inferred that... MdHT13.2 It may have been involved in the sugar unloading process in mature leaves.

[0081] 3.1.2 MdHT13.2 Homologous gene amino acid sequence alignment Screening for and related species in databases of Arabidopsis thaliana, tomato, pear, peach, and rice. MdHT13.2Genes with high homology were identified, and their amino acid sequences were downloaded. Using DNAMAN 8.0, bioinformatics methods were employed to perform multi-species homology sequence alignment analysis of the MdHT13.2 protein. The results showed a high degree of consistency in amino acid sequences among different species. Figure 3 The presence of a conserved MFS domain in the first half of the compared amino acid sequence indicates that MdHT13.2 exhibits typical characteristics of a hexose transporter.

[0082] 3.1.3 MdHT13.2 Systematic evolutionary analysis among different species Search and download from the Rosaceae Database (GDR) (https: / / www.rosaceae.org / ). MdHT13.2 The amino acid sequence. To further confirm the reliability of the gene, the results were obtained from the apple (https: / / www.ncbi.nlm.nih.gov / ) database. Malus domestica Blast comparison of candidates in the Borkh database MdHT13.2 Amino acid sequences. Apple and Arabidopsis thaliana were screened from the database. Arabidopsis thaliana ),tomato( Solanum lycopersicum ), rice ( Oryza sativa Members of the family, such as ), were selected, and their amino acid sequences were used to construct a phylogenetic tree using the NJ method in MEGA 7.0 software. The results showed that MdHT13.2 is closely related to SlSTP2 in tomato and MdSTP13-like in apple, and also related to AtSTP13 in Arabidopsis thaliana. Figure 4 ).

[0083] 3.2 Apple MdHT13.2 Expression patterns and functional verification 3.2.1, MdHT13.2 Transmembrane Domain Prediction and Subcellular Localization MdHT13.2, as a hexose transporter, was analyzed using the TMHMM website to predict its transmembrane domains. The results confirmed the presence of 12 typical transmembrane domains, with the transmembrane region containing 256±3 amino acid residues. The N-terminus was 94.3% located within the cytoplasm. Figure 5 Therefore, it is concluded that the transmembrane domain of MdHT13.2 is mainly located at the C-terminus, which is consistent with the previously predicted region of conserved amino acid residues, indicating that MdHT13.2 conforms to the basic characteristics of a typical membrane protein.

[0084] To clarify the subcellular localization characteristics of MdHT13.2, Agrobacterium (GV3101) was used to transiently transform 4 weeks of Tobacco Benzoinii leaves. Results observed under a laser confocal microscope revealed… MdHT13.2-The GFP green fluorescent signal is located on the plasma membrane, indicating that MdHT13.2 is a plasma membrane-localized sugar transporter. Figure 6 (A). After extracting protoplasts from apple callus using enzymatic hydrolysis, this method observed the same results as transient tobacco injection. Figure 6 (B) This further confirms that MdHT13.2 is located on the plasma membrane.

[0085] 3.2.2, MdHT13.2 Tissue-Specific Identification In order to study MdHT13.2 Tissue-specific expression patterns, to obtain MdHT13.2 Startup sub-driver GUS ( proMdHT13.2 The recombinant plasmid :GUS was used to transform Arabidopsis thaliana using the flower-dipping method. The selected transgenic plasmids were then... MdHT13.2- pro GUS staining experiments were performed on Arabidopsis thaliana (primers MdHT13.2-pro-F: 5'-GTGCGCTATTGTTGCTTAAGTTC-3', SEQ ID NO.12; MdHT13.2-pro-R: 5'-CAAGAAGAAGGACGCTTGGTG-3', SEQ ID NO.13). The results showed that the leaf veins of 10-15 day old Arabidopsis thaliana leaves were more deeply stained, and the fine veins were also stained. MdHT13.2 The promoter exhibits strong activity in leaf veins, and its surface... MdHT13.2 The tissue is located in the veins of the leaf ( Figure 7 ).

[0086] 3.2.3, MdHT13.2 Cell-Specific Identification To further explore MdHT13.2 To determine the cell localization, an antisense probe was designed for in situ hybridization experiments. 'GL-3' leaves were selected, and the midrib was cut for embedding sections. Hybridization signals were observed under a microscope in cross-sectional sections of the apple leaf veins. Hybridization signals were detected in the phloem of the vascular tissue and the palisade tissue cells. MdHT13.2 The antisense probe showed a strong signal, consistent with the results of GUS staining. Figure 8 (A). Under UV (ultraviolet) fluorescence, autofluorescence of the xylem was observed. Figure 8 (B). In contrast, the phloem signal of the control sample obtained using the positive probe hybridization section was relatively diffuse, and no obvious hybridization signal was detected. Figure 8 MdHT13.2 exhibits a specific expression pattern in the phloem tissue of apple leaves, and may be involved in the regulation of sugar transport.

[0087] 3.2.4 Sugar absorption characteristics of MdHT13.2 In order to detect MdHT13.2 The sugar absorption properties of encoded proteins, construction MdHT13.2- The pDR196 recombinant vector was transformed into a defective yeast strain. EYB.VW4000 The results showed that the mutant yeast grew normally on media with low concentrations of fructose and glucose. The mutant yeast did not grow on media with high concentrations of hexose, but it grew normally on media with high concentrations of disaccharide maltose. Figure 9 (A). The mutant yeast strain was cultured in liquid medium, and the bacterial OD was measured. 600 The value was found to be consistent with the results of the yeast plate sample. Figure 9 (B) MdHT13.2 Heterologous expression in yeast restored the yeast strain. EYB.VW4000 The growth defects indicate that MdHT13.2 has the ability to transport low concentrations of glucose and fructose.

[0088] To investigate the sucrose transport capacity of MdHT13.2, the following was used: MdHT13.2- pDR196 recombinant plasmid was transformed into a sucrose transport defective mutant. SUSY7 In the study, the mutant yeast grew on low-concentration sucrose medium, but did not grow on high-concentration sucrose medium, while it grew normally on high-concentration glucose medium. Figure 10 (A). The mutant yeast strain was cultured in liquid medium, and the OD was measured. 600 The value is consistent with the trend shown by the yeast plate. Figure 10 (B). The results showed that MdHT13.2 has the ability to absorb low concentrations of sucrose and is a sugar transport protein with high affinity for low concentrations of hexose and sucrose.

[0089] 3.3 Effects of MdHT13.2 on plant source, glutamic acid content and growth phenotype 3.3.1 MdHT13.2 Heterologous expression alters the growth status of tomatoes. To further investigate the function of MdHT13.2 in tomato sugar transport, the 'Micro Tom' tomato variety, which has a short growth cycle and early fruiting, was selected as the material. An Agrobacterium-mediated genetic transformation system was used to obtain... MdHT13.2- OE overexpression lines were propagated and rooted in transgenic seedlings and cultured in a 23°C light incubator. RNA analysis showed that the expression levels of transgenic tomato lines L1, L4, and L5 were significantly higher than the control; therefore, DNA analysis was performed on these three lines. Figure 11 Three heterologous overexpression tomato lines (L1, L4, and L5) were obtained from B~C. The results showed that, compared with WT, MdHT13.2 Heterologously overexpressed tomato lines are shorter in height. Figure 11 (A) indicates overexpression MdHT13.2 Genes can influence the growth and development of plants.

[0090] 3.3.2 MdHT13.2 Determination of sugar content in overexpressed tomatoes By comparing and analyzing the sugar metabolism characteristics of transgenic tomato lines and wild-type tomatoes, it was found that... MdHT13.2 Heterologous expression of the gene significantly affected carbohydrate allocation in plants. Experimental data showed that, compared with the control, transgenic tomato fruits contained 1-2 times more fructose, glucose, and galactose, and also had a significantly increased sucrose content. The sugar content of the tomato fruits at maturity was even more significantly different from that of the wild type. Figure 12 (A) The soluble solids content of transgenic tomatoes was higher than that of wild-type tomatoes. The fructose content in the roots of transgenic tomatoes was more than twice that of wild-type tomatoes, and the glucose content was also higher than that of wild-type tomatoes, while the sucrose content did not change significantly. Figure 12 (B~C) indicates overexpression MdHT13.2 It can positively regulate sugar accumulation in tomatoes and roots, and promote the distribution of photosynthetic products to sink organs.

[0091] 3.3.3 MdHT13.2 Identification of transgenic apple strains To further explore the functionality of MdHT13.2 in Apple devices, Gateway cloning technology was used to... MdHT13.2 A 257bp specific fragment was inserted into the silencing vector pk7wwG2D(II) to obtain pk7wG2D(II)- MdHT13.2 Silent plasmid. 'GL-3' apple tissue culture seedlings were transformed using the Agrobacterium rhizogenes-mediated transformation of apple leaf discs. Screening and identification were performed at the DNA and mRNA levels. Figure 13 (B~C) Gain three silences (R1, R2 and R3). MdHT13.2 Among the transgenic apple lines, the number of silent lines was found to be slightly lower than that of wild-type (WT). Figure 13 In section A), the transgenic apple line had more leaf veins than the control group, and the leaf surface cells had more protrusions. Figure 14 This indicates that MdHT13.2 affects sugar transport in leaves and thus influences the growth status of apples.

[0092] 3.3.4 MdHT13.2 Determination of sugar content in silent apple strains A comparative analysis of carbohydrate content in the leaves of transgenic silent apple plants and wild-type apples revealed that glucose levels nearly doubled in the transgenic silent plants, fructose content slightly increased, and galactose concentration increased 2-3 times. Meanwhile, transported forms of sorbitol and sucrose significantly decreased, while starch content slightly increased. Figure 15 These results indicate that MdHT13.2 may specifically regulate hexose phloem transport, silencing [the phloem]. MdHT13.2 Genes cause glucose transport to be blocked, resulting in glucose retention in cells.

[0093] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A kind MdHT13.2 The application of genes in regulating plant growth is characterized by, The MdHT13.2 The accession number of the gene in the Rosaceae Genome Database is MD13G1189100.

2. The application according to claim 1, characterized in that, The plant is either a tomato or an apple.

3. The application according to claim 2, characterized in that, MdHT13.2 Genes are used to promote sugar accumulation in plants.

4. The application according to claim 3, characterized in that, overexpression MdHT13.2 Genes are used to promote the accumulation of fructose, glucose, galactose, and sucrose in tomato fruits; silencing these genes... MdHT13.2 The gene is used to promote the accumulation of glucose, galactose, and fructose in apple plants.

5. The application according to claim 2, characterized in that, MdHT13.2 Genes are used to promote photosynthesis in plants.

6. A method for increasing the sugar content of apples or regulating apple growth, characterized in that, Including the silent apple plant as described in claim 1 MdHT13.2 Gene 。 7. A method for increasing the sugar content of tomato fruit or regulating tomato growth, characterized in that, Including the tomato plants overexpressing the expression described in claim 1 MdHT13.2 Gene 。 8. The method according to claim 6 or 7, characterized in that, The sugar content refers to the content of fructose, glucose, and galactose.

9. A method for cultivating dwarf apple strains, characterized in that, Including the silent apple plant as described in claim 1 MdHT13.2 Gene 。 10. A method for cultivating dwarf tomato lines, characterized in that, Including the tomato plants overexpressing the expression described in claim 1 MdHT13.2 Gene 。

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