Application of tomato slavt6a gene in regulating amino acid transport to influence terpene content and fruit yield of tomato

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to application of a tomato SlAVT6A gene in regulating amino acid transport to affect terpene content and fruit yield of the tomato. The tomato SlAVT6A gene regulates amino acid into a secondary metabolic pathway by affecting amino acid transport in the tomato vacuole, and further affects volatile terpene accumulation and fruit yield. By means of a whole genome association analysis (mGWAS) method, the application locates the SlAVT6A gene which is significantly related to the amino acid content of the tomato, and further experimental verification finds that the gene is located on the vacuole membrane and has the function of expelling amino acids. Overexpression and knockdown of the gene in the tomato plant find that changing the expression amount of the SlAVT6A gene can change the amino acid content in the tomato, affect the synthesis of secondary volatile terpenes and the fruit yield, and confirm the biological function and application approach of the gene.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology. Specifically, it relates to the application of the tomato SlAVT6A gene in regulating the entry of amino acids into secondary metabolic pathways by affecting amino acid transport within tomato vacuoles, thereby further influencing the accumulation of volatile terpenoids and fruit yield. Background Technology

[0002] Tomato (Solanum lycopersicum), originating in South America, is one of the world's most widely cultivated horticultural crops, favored for its unique sweet and sour flavor. In actual tomato production, its metabolites play a crucial role in growth, development, quality formation, and resistance to adverse environments. Tomato metabolites mainly include primary metabolites such as proteins, amino acids, fats, carbohydrates, and nucleic acids, as well as secondary metabolites such as phenols, terpenes, and alkaloids, which are further generated from the reactions of primary metabolites. When the external environment changes, amino acid metabolism undergoes plasticity changes to meet specific cellular needs and regulate specific growth and development pathways. Amino acids form the protein skeleton, participate in various physiological and biochemical reactions as enzymes, and act as transporters, carrying different substances. Furthermore, amino acids play a role in maintaining normal cell morphology, oxidative energy production, and mitigating heavy metal ion toxicity, thereby enhancing the plant's resistance to stress.

[0003] Amino acid metabolism and redistribution in plants is a complex process involving the coordinated action of multiple organelles and tissues, requiring transmembrane transport to distribute amino acids to different organelles. The transport of amino acids within or between cells is facilitated by specialized membrane proteins—amino acid transporters. These transporters have different affinities and specificities for different amino acids and play important roles in plant growth and development. Vacuoles are the main reservoirs of excess amino acids within cells and are crucial for maintaining cytoplasmic amino acid homeostasis and responding to metabolic demands. Amino acid vacuole transporters (AVTs), belonging to the amino acid transporter (ATF) family, are responsible for the exchange of amino acids between the cytoplasm and vacuoles, thereby ensuring the proper distribution of amino acids among organelles to maintain cytoplasmic homeostasis and other metabolic functions. AVTs have been cloned in Arabidopsis thaliana and rice, belonging to the amino acid transporter-like (ATL) family and aromatic and neutral amino acid transporter (ANT) subtypes, respectively. AtANT1 and AtAvt3A-C facilitate the transport of aromatic and neutral amino acids, as well as auxins, although their physiological significance is not yet fully elucidated. Studies in rice have shown that OsATL6 is involved in the temporary storage of glutamine in root vacuoles under NH4+ enrichment conditions. Furthermore, OsATL14 is associated with changes in seed coat color during rice domestication, while OsATL15 is related to THX uptake and distribution, thus conferring resistance to brown planthoppers. However, these studies have primarily focused on the identification of translocation activities and their effects on plant growth, while the molecular mechanisms underlying the dynamic changes of amino acids within vacuoles and their impact on plant secondary metabolism and growth and development remain unclear. Summary of the Invention

[0004] The purpose of this invention is to isolate a tomato SlAVT6A gene, verify its broad-spectrum amino acid transport function in yeast mutants, and discover its localization in the vacuolar membrane. Overexpression and knockdown of this gene in tomato plants revealed that altering the expression level of the SlAVT6A gene can change the amino acid content in tomatoes, affect the synthesis of secondary volatile terpenes, and fruit yield, thus confirming the biological function and application pathways of this gene.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] The first aspect of this invention is to locate the tomato SlAVT6A gene using genome-wide association analysis (mGWAS), the nucleotide sequence of which is shown in SEQ ID No. 1, and to discover the application of this gene in regulating tomato fruit yield; the application is to increase tomato fruit yield by overexpressing the SlAVT6A gene.

[0007] Furthermore, the overexpression of the SlAVT6A gene increases tomato fruit yield, including the following steps:

[0008] (1) Extract total RNA from tomato leaves, reverse transcribe to obtain cDNA, and use cDNA as a template to perform PCR amplification with primers SlAVT6A-F and SlAVT6A-R;

[0009] SlAVT6A-F: 5'-aaaaagcaggcttaATGGCTCTAGCCAAAAATGG-3';

[0010] SlAVT6A-R: 5'-agaaagctgggtaCTATAGCCTATTTCCACTCATATTG-3';

[0011] (2) The PCR product obtained in step (1) was ligated into the pDonr207 entry vector by the BP reaction of GATEWAY cloning technology, positive clones were screened and sequenced to confirm, and the full-length cDNA of the SlAVT6A gene was obtained. Then, it was ligated into the overexpression vector PBI121 by the LR reaction of GATEWAY cloning technology to construct the PBI121-SlAVT6A vector.

[0012] (3) The PBI121-SlAVT6A vector constructed in step (2) was introduced into tomato plants through the Agrobacterium-mediated tomato genetic transformation system.

[0013] The second aspect of this invention provides the application of the above-mentioned tomato SlAVT6A gene in regulating the content of terpenoids in tomato secondary metabolites; the application method is to increase the total terpenoid content of tomato lines by overexpressing the SlAVT6A gene.

[0014] Furthermore, the application involves the tomato SlAVT6A gene regulating amino acid transport, affecting the metabolic flux of the MVA and MEP pathways, and thus influencing downstream secondary metabolic pathways, thereby affecting the content of terpenoids in tomato secondary metabolites.

[0015] Furthermore, the overexpression of the SlAVT6A gene increases the total terpene content of tomato lines, including the following steps:

[0016] (1) Extract total RNA from tomato leaves, reverse transcribe to obtain cDNA, and use cDNA as a template to perform PCR amplification with primers SlAVT6A-F and SlAVT6A-R;

[0017] SlAVT6A-F: 5'-aaaaagcaggcttaATGGCTCTAGCCAAAAATGG-3';

[0018] SlAVT6A-R: 5'-agaaagctgggtaCTATAGCCTATTTCCACTCATATTG-3';

[0019] (2) The PCR product obtained in step (1) was ligated into the pDonr207 entry vector by the BP reaction of GATEWAY cloning technology, positive clones were screened and sequenced to confirm, and the full-length cDNA of the SlAVT6A gene was obtained. Then, it was ligated into the overexpression vector PBI121 by the LR reaction of GATEWAY cloning technology to construct the PBI121-SlAVT6A vector.

[0020] (3) The PBI121-SlAVT6A vector constructed in step (2) was introduced into tomato plants through the Agrobacterium-mediated tomato genetic transformation system.

[0021] The third aspect of this invention provides a tomato SlAVT6A gene overexpression vector containing the above-mentioned gene and its application in regulating tomato fruit yield.

[0022] Furthermore, the application involves transferring the overexpression vector into tomato plants to increase tomato fruit yield.

[0023] The fourth aspect of this invention provides a tomato SlAVT6A gene overexpression vector containing the above-mentioned gene and its application in regulating the content of terpenoids in tomato secondary metabolites.

[0024] Furthermore, the application method involves transferring the overexpression vector into tomato plants to increase the total terpene content of the tomato lines.

[0025] Compared with the prior art, the present invention has the following effects:

[0026] The transgenic material testing further verified the function of SlAVT6A in regulating amino acid transport in vacuoles and its role in influencing the synthesis of secondary terpenoid volatiles and fruit yield. This provides a way to influence secondary metabolic pathways and enhance the stress resistance of tomatoes by changing the flux of primary metabolite amino acids. It provides a strategy for improving crop environmental adaptability by utilizing metabolites and will have important guiding significance for breeding high-quality new tomato varieties.

[0027] The present invention will be further described below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0028] Figure 1 mGWAS localization and phylogenetic analysis of the SlAVT6A gene Figure 1Figure A in the figure shows the results of the genome-wide association analysis of phenylalanine content, with the dashed line representing the significance threshold (p = 2.14E-07). Figure 1 Figure B in the diagram shows the phylogenetic analysis of the located genes.

[0029] Figure 2 Subcellular localization and expression profile analysis of SlAVT6A Figure 2 Figure A in the figure shows the subcellular localization of SlAVT6A, which coincides with the AtTIP3 fluorescence localization of Arabidopsis thaliana vacuoles; Figure 2 Figure B in the figure shows the expression level of SlAVT6A in different tissues and at different developmental stages. NL represents new leaves, ML represents mature leaves, R represents roots, S represents stems, IF represents immature flowers, MF represents mature flowers, F1 represents fruits with a diameter of 1 cm, F2 represents fruits with a diameter of 2 cm, IGF represents immature green fruits, MG represents mature green fruits, BR represents the color-breaking stage; BR3, 7, and 10 represent the number of days after color breaking. RF represents red fruits.

[0030] Figure 3 Functional validation of SlAVT6A as a vacuole amino acid efflux protein. Figure 3 Figure A shows the validation of the efflux function of SlAVT6A in the yeast mutant strain 22Δ10α; Figure 3 In Figure B, the efflux activity of SlAVT6A in the yeast mutant strain 22Δ10α was validated. Results were based on three replicates. Error bars represent standard deviation (SD). ** indicates a P-value less than 0.01, *** indicates a P-value less than 0.001, and **** indicates a P-value less than 0.0001. All three indicate highly significant differences.

[0031] Figure 4 The construction, identification, phenotypic and yield observation of SlAVT6A transgenic materials were performed. Figure A shows the expression level detection of SlAVT6A overexpressing and knockout plants; Figure B shows the phenotypic observation of SlAVT6A overexpressing and knockout plants; Figure C shows the fruit yield of SlAVT6A overexpressing and knockout plants; Figure D shows the quantitative data of yield. The results were based on three replicates. The error bars represent the standard deviation (SD). ** indicates a P value less than 0.01, *** indicates a P value less than 0.001, and **** indicates a P value less than 0.0001. All three indicate extremely significant differences.

[0032] Figure 5The content of 20 amino acids in SlAVT6A transgenic plants; the results are based on three replicates, and the error bars represent the standard deviation (SD). ** indicates a P value of less than 0.01, *** indicates a P value of less than 0.001, and **** indicates a P value of less than 0.0001. All three indicate extremely significant differences.

[0033] Figure 6 The content of terpenoids in SlAVT6A transgenic plants. Results were based on three replicates. Error bars represent standard deviation (SD). ** indicates a P-value less than 0.01, *** indicates a P-value less than 0.001, and **** indicates a P-value less than 0.0001. All three indicate highly significant differences.

[0034] Figure 7 SlAVT6A regulates terpene metabolism by influencing the MVA and MEP pathways. Figure A shows the expression levels of MVA and MEP pathway genes in SlAVT6A overexpressing and knockout plants; Figure B shows the contents of IPP, FPP, and GGPP in SlAVT6A overexpressing and knockout plants. Results are based on three replicates, and the error bars represent standard deviation (SD). ** indicates a P-value less than 0.01, *** indicates a P-value less than 0.001, and **** indicates a P-value less than 0.0001, all three indicating highly significant differences.

[0035] Figure 8 : Image of the introductory vector pDonr207-SlAVT6A plasmid.

[0036] Figure 9 : Overexpression vector PBI121-SlAVT6A.

[0037] Figure 10 Image of the knockdown vector pk7WGIGW2R(II)-SlAVT6A plasmid. Detailed Implementation

[0038] The following examples define the present invention and describe the methods for isolating and cloning DNA fragments containing the complete coding region of the SlAVT6A gene, and for verifying the function of the SlAVT6A gene. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0039] Preparation of reagents and culture media:

[0040] (1) Abbreviations of reagents and solutions: All amino acid solutions used in this invention are prepared in aqueous solution with a concentration of 100 μm; the abbreviations of plant hormones used in the culture medium are as follows: KN (Kanamycin); KT (Kinetin); IAA (Indole-3-acetic acid); 2,4-D (2,4-Dichlorophenoxyacetic acid); AS (Acetosringone); DMSO (Dimethyl Sulfoxide); Tim (Timentin); ZR (trans-Zeatin-riboside); IBA (3-Indolebutyric acid); KH2PO4 (Potassium dihydrogen phosphate); MS (Murashige & Skoog); GA3 (gibberellins).

[0041] (2) Main solution formulation:

[0042] 1) Nitrogen-deficient yeast culture medium

[0043] YNB (for mutant strains) 6.7 g

[0044] 20 g glucose

[0045] Dissolve at room temperature and bring the volume to 1000 mL with distilled water.

[0046] 2) Preparation of MS liquid culture medium

[0047] MS Base salt 4.33 g;

[0048] MS Vitamin 0.1031 g;

[0049] 30 g of sucrose;

[0050] Dissolve at room temperature and bring the volume to 1000 mL with distilled water.

[0051] 3) ZR stock solution, IBA stock solution 2 g / L; IAA stock solution, KH2PO4, 2,4-D stock solution, KT stock solution 1 g / L; Tim stock solution 320 g / L; KN stock solution 50 g / L; AS stock solution 200 mM / L. All require filtration and sterilization.

[0052] 3) Culture medium formulation for tomato genetic transformation

[0053] Pre- / co-culture medium

[0054] 1 L of MS liquid culture medium;

[0055] Agar powder 6 g;

[0056] ZR 0.5 mL;

[0057] IAA 0.1 mL;

[0058] Adjust the pH to 5.8, bring the volume to 1000 mL, and seal for sterilization.

[0059] Screening culture medium

[0060] 1 L of MS liquid culture medium;

[0061] Agar powder 6 g;

[0062] ZR 0.5 mL;

[0063] IAA 0.1 mL;

[0064] Tim 0.6 mL;

[0065] KN 0.5 mL;

[0066] Adjust the pH to 5.8, bring the volume to 1000 mL, and seal for sterilization.

[0067] Rooting medium

[0068] 1 L of MS liquid culture medium;

[0069] Plant-based gel (gum powder) 2.5 g;

[0070] IBA 0.5 mL;

[0071] Tim 0.5 mL;

[0072] KN 0.5 mL;

[0073] Adjust the pH to 5.8, bring the volume to 1000 mL, and seal for sterilization.

[0074] 4) YEB medium

[0075] Yeast extract 1 g;

[0076] Tryptone 5 g;

[0077] Beef extract 5 g;

[0078] 5 g of sucrose;

[0079] MgSO4•7H2O 0.5 g;

[0080] 15 g of agar powder;

[0081] Adjust the pH to 7.4, bring the volume to 1000 mL, and seal for sterilization.

[0082] 5) 1.5×CTAB extract

[0083] CTAB 15 g

[0084] 1 M Tris-HCl (pH 8.0) 75 mL

[0085] 0.5 M EDTA (pH 8.0) 30 mL

[0086] NaCl 61.4 g

[0087] Add ddH2O to bring the volume to 1000 mL.

[0088] Example 1: Discovery and localization of the SlAVT6A gene

[0089] The applicant conducted metabolite analysis on tomato materials from around the world, performed statistical analysis on the measurement data, and selected varieties with significant differences in amino acid synthesis for genome-wide association analysis. The results showed a significant association between phenylalanine and a loci on chromosome 5. Among these genes, we discovered an unknown gene, Solyc05g052300, annotated as an amino acid transport haplotype. Through phylogenetic analysis, we named it SlAVT6A and speculated that the SlAVT6A gene may affect the amino acid content of tomatoes by transporting amino acids.

[0090] Example 2: Isolation and cloning of the SlAVT6A gene, identification of its expression pattern and subcellular localization

[0091] To obtain the coding sequence of the SlAVT6A gene, the applicant planted the sequenced tomato variety MicroTom for one month and extracted total RNA from tomato leaves using TRIZOL reagent (purchased from Beijing TransGen Biotech Co., Ltd.) (extraction method according to the TRIZOL reagent instructions). The RNA was then reverse transcribed into cDNA using the Script All-in-one RT EasyMix for qPCR kit from Tolo Harbour. The reaction conditions were: 42℃ for 30 min, 80℃ for 5 s. Using this cDNA as a template, PCR amplification was performed using primers SlAVT6A-F: 5'-aaaaagcaggcttaATGGCTCTAGCCAAAAATGG-3' and SlAVT6A-R: 5'-agaaagctgggtaCTATAGCCTATTTCCACTCATATTG-3', yielding the full-length coding sequence of the SlAVT6A gene (1317 bp, as shown in SEQ ID No. 1). PCR reaction conditions: 94 ℃ pre-denaturation for 2 min; 98 ℃ for 10 sec, 58 ℃ for 30 sec, 68 ℃ for 2 min, 35 cycles; 68 ℃ extension for 5 min. The amplified PCR products were ligated into the pDonr207 entry vector using a BP reaction with GATEWAY cloning technology. Positive clones were screened and confirmed by sequencing to obtain the entry vector pDonr207-SlAVT6A containing the full-length cDNA of the SlAVT6A gene.

[0092] SlAVT6A was ligated into a pH7WGF vector containing a GFP tag via the LR reaction of Gateway cloning technology, followed by transient injection into tobacco. The injected tobacco was first cultured in the dark for 24 h, then under light for 24 h, and then observed. Figure 2 Experimental results showed that, compared with the empty vector, SlAVT6A and Arabidopsis thaliana vacuolar aquaporin AtTIP3 exhibited specific fluorescence on the vacuolar membrane, indicating that SlAVT6A was localized on the vacuolar membrane.

[0093] RNA samples were collected from various tissues at different developmental stages of tomatoes. RNA was extracted according to the method described in Example 2, and cDNA was obtained from different samples through reverse transcription. The cDNA was detected using a SYBR quantitative PCR kit (purchased from Vazyme) (the usage method is as described in the kit instructions). The real-time quantitative PCR software Quantstudio™ was used, with the tomato actin gene as an internal control. The detection primer sequences were Slactin-qRT-F: 5'-GCCAAAGAAGATCAAGCACA-3' and Slactin-qRT-R: 5'-TCAGCATT AGGGCACTCCTT-3'. The relative expression level of SlERF.H6 in different samples was calculated using the ΔΔCt method. Data processing and plotting were performed using GraphPad Prism 8 software. (See attached image). Figure 2 . Figure 2 Figure B shows the expression profile of SlERF.H6, indicating that SlAVT6A is highly expressed in trophic tissues.

[0094] Example 3: SlAVT6A is an amino acid vacuole efflux protein

[0095] SlAVT6A was ligated into the yeast expression vector ADH1 using the LR reaction of GATEWAY cloning technology. The applicant named the correctly sequenced clone ADH1-SlAVT6A. The mutant strain 22Δ10α could not grow on media containing arginine and γ-aminobutyric acid as the sole nitrogen source. To determine the amino acid transport activity of SlAVT6A, we heterologously expressed SlAVT6A in the yeast amino acid transporter mutant 22Δ10α, and cultured the transformed yeast on YNB solid medium containing a mixture of (NH4)2SO4 and 2 mM amino acids as the sole nitrogen source. The results showed that the SlAVT6A-22Δ10α yeast transformant could grow normally on medium containing (NH4)2SO4, while 2 mM amino acids did not restore the growth of SlAVT6A-22Δ10α, indicating that SlAVT6A cannot transport amino acids into yeast cells.

[0096] Therefore, we conducted an amino acid efflux experiment using yeast mutants. Yeast mutants containing ADH1-AtAAP3 and ADH1-SlAVT6A were cultured in SD / -Ura liquid medium until an OD600 of 1 was reached. The collected samples were then washed three times with sterile water. The yeast samples were then transferred to a new liquid medium with 3 mM NH4+ as the sole nitrogen source and cultured for 24 h. The bacterial culture was then collected and centrifuged to separate yeast samples and yeast culture medium samples. The amino acid content of each sample was determined. The results are shown below. Figure 3Experimental results showed that after 12 h of liquid culture in 3 mM NH4+, the amino acid concentration in SlAVT6A-22Δ10α decreased by 32% compared with AtAAP3-22Δ10α, while the amino acid content in the yeast culture medium of SlAVT6A-22Δ10α increased by 9 times.

[0097] In addition, ADH1-SlAVT6A cultured in liquid medium with 3 mM NH4+ as the sole nitrogen source for 12 h was washed three times with sterile water and then transferred to nitrogen-free medium. Two mL of bacterial culture was collected at 0, 2, 4, 6, and 8 hours. The collected bacterial culture was centrifuged to separate yeast samples and yeast culture medium samples, and the amino acid content of each was determined. Amino acid efflux activity was calculated as the ratio of amino acid content in yeast culture medium to (amino acid content in yeast sample + amino acid content in yeast culture medium). The results showed that after 12 h of nitrogen-free culture, the efflux activity of SlAVT6A was higher than that of the control.

[0098] Example 4: Identification and Observation of Growth Phenotype of SlAVT6A Transgenic Material

[0099] The overexpression vector was constructed as follows: the overexpression vector pBI121 was ligated into the LR reaction of the GATEWAY cloning technology, and the applicant named the correctly sequenced clone PBI121-SlAVT6A.

[0100] The knockdown expression vector was constructed as follows: the knockdown expression vector pk7WGIGW2R(II) was ligated into the LR reaction of the GATEWAY cloning technology. The applicant named the correctly sequenced clone pk7WGIGW2R(II)-SlAVT6A.

[0101] PBI-SlAVT6A and pk7WGIGW2R(II)-SlAVT6A were used for transformation. Specifically, they were introduced into the tomato variety MicroTom through an Agrobacterium-mediated tomato genetic transformation system. After pre-culture, infection, co-culture, screening for kanamycin-resistant calluses, differentiation, rooting, transplanting, and identification, transgenic plants were obtained.

[0102] Specific steps: (1) Soaking: Soak several seeds of the MicroTom tomato variety in water for 3-4 hours in advance, then pour out the water and add 75% alcohol for 45 seconds for disinfection, rinse five times with sterile water, add 20% NaClO for 18 minutes (in the dark), after disinfection, wash the seeds five times with sterile water, soak for about two minutes each time, pour out the sterile water, absorb the water slightly, and then transfer to 1 / 2 MS germination medium for dark culture; (2) Cutting seedlings for pre-culture: When the seeds germinate and grow to have two mature cotyledons, cut off the leaf tips and leaf bases with scissors, place the cotyledons upside down with the back of the leaves facing up on the pre-culture medium, and pre-culture in the dark for about 1-2 days; (3) Agrobacterium transformation and culture: Add the constructed PBI121-SlAVT6A and pk7WGIGW2R(II)-SlAVT6A vectors to 20 μl of EHA105 competent cells (from Shanghai Weidi Biotechnology, commercial strain), place on ice and let stand for 5 minutes. After 5 min, transfer to liquid nitrogen for 5 min, then incubate in a 37 ℃ water bath for 5 min, and finally stand on ice for 5 min. Add 100 μL of YEB liquid medium and incubate in a shaker at 28 ℃ for 1 h. Spread the bacterial culture onto YEB solid plates with resistance and incubate at 28 ℃ for 1-2 days. After the single colony appears, transfer the colony to YEB liquid medium and culture it on a shaker at 28 ℃ for 12 hours; (4) Agrobacterium infection and co-culture: use liquid MS medium to adjust the suspension of Agrobacterium to OD600 = 0.2-0.3, gently shake the cotyledons in the Agrobacterium suspension for 12-15 min, then transfer them to sterilized filter paper to dry, and place them on the co-culture medium in the dark for 2 days; (5) Screening culture: transfer the cotyledons on the co-culture medium to the screening medium, change the medium every two weeks until adventitious buds grow from the callus tissue; (6) Rooting: cut off the adventitious buds, remove all the surrounding callus tissue, keep them as intact as possible, and transfer them to the rooting medium (insertion medium) for rooting culture; (7) Transplanting: select healthy plants that have grown roots and transplant them into nutrient soil. Avoid a large loss of water for 5 days after transplanting. After the plants grow normally, identify them. The specific steps for identification are as follows: (1) Vector transformation identification: In order to verify whether the pBI121 overexpression vector has been transformed into the plant, PCR amplification identification was performed using vector-specific primers OJX487: 5'-CGCAAGACCCTTCCTCTATATAAG-3' and OJX488: 5'-TAAAACGACGGCCAGTGAATTCCC-3'. PCR reaction conditions: 94 ℃ pre-denaturation for 5 min; 94 ℃ for 30 sec, 60 ℃ for 30 sec, 72 ℃ for 1 min, 35 cycles; 72 ℃ extension for 5 min.Then, agarose gel electrophoresis was performed, and the presence of a 1317bp band indicated vector transfection; (2) RNA extraction and reverse transcription: The RNA extraction, reverse transcription method in Example 2 and real-time fluorescence quantitative PCR were used to detect the relative expression level of SlAVT6A in transgenic plants. The results are as follows. Figure 3 As shown in Figure A, SlAVT6A overexpression and knockdown expression plants were successfully constructed.

[0103] Phenotypic observation and photography were conducted on six-week-old transgenic tomato plants, such as... Figure 3 As shown in Figure B, plants overexpressing SlAVT6A exhibited superior agronomic traits compared to wild-type plants, while the opposite was true for SlAVT6A knockout materials. Furthermore, fruit yield was assessed 120 days after transplanting for both wild-type and transgenic materials at the same growth stage. Figure 4 C and Figure 4 As shown in Figure D, plants overexpressing SlAVT6A had higher fruit yields than wild-type plants, while the opposite was true for SlAVT6A knockdown materials.

[0104] Example 5: Detection of amino acid and terpene content in SlAVT6A transgenic material

[0105] Metabolic samples were collected from 6-week-old T2 generation transgenic material. Fresh tomato leaf tissue was mixed with 70% methanol (containing 0.1 mg / L lidocaine) at a ratio of 0.1 g / 1 mL and vortexed. The mixture was then incubated overnight at 4 °C for extraction. The samples were then centrifuged at 4 °C (1000 g, 10 min), and the supernatant was transferred to a syringe and filtered through a filter (SCAA-104, 0.22 μm pore size, ANPEL). The amino acid content of the filtered samples was determined using a liquid chromatography-tandem mass spectrometry (LC-MS / MS) system. (See figure...) Figure 5 The results showed that the content of most amino acids in the leaves of SlAVT6A overexpressing plants decreased, with significant decreases in glycine, leucine, isoleucine, tyrosine, serine, threonine, cysteine, valine, proline, phenylalanine, tryptophan, methionine, lysine, arginine, and histidine, but an increase in aspartic acid content. Conversely, the amino acid content in the leaves of SlAVT6A knockout materials showed the opposite trend.

[0106] The extraction and detection of terpenoids were performed according to the method of Kang et al. Leaves from the same leaf position of 6-week-old transgenic materials and wild-type plants were selected and immersed in 2 mL of methyl tert-butyl ether (MTBE) containing 10 ng / μL naphthalene as an internal standard. After gently shaking for 5 minutes at room temperature, the leaves were removed and their dry weight was measured. The liquid was transferred to a sample vial, which was the terpenoid extract. The extract was analyzed using an Agilent 7890B gas chromatography-mass spectrometry system. The GC program used an injector temperature of 280℃. The initial column temperature was held at 40℃ for 1 min, then increased to 90℃ at a rate of 40℃ / min, to 110℃ at a rate of 15℃ / min, to 250℃ at a rate of 25℃ / min, and finally increased to 320℃ at a rate of 40℃ / min and held for 2 min. The helium carrier gas flow rate was set to 0.4 mL / min. The collected data were compared with the database, and substances were identified based on the elution time of the nuclei. (See...) Figure 6 The results showed that the total terpene content of the SlAVT6A overexpression lines was significantly increased, reaching 4.32 times that of the wild-type. Specifically, the contents of α-thujene and β-pinene in the leaves of the SlAVT6A overexpression lines were 12.11 times and 15.98 times that of the wild-type, respectively. These results indicate that SlAVT6A affects terpene biosynthesis by altering amino acid availability.

[0107] Example 6: SlAVT6A affects terpene metabolism by influencing the MVA and MEP pathways.

[0108] Considering that terpenoids originate from the cytoplasmic mevalonate (MVA) and plastosolic methyl erythritol phosphate (MEP) pathways, we used 6-week-old T2 generation transgenic materials as samples, collecting RNA and metabolic samples. RNA extraction and reverse transcription were performed as in Example 2. The expression of published MVA and MEP pathway-related genes in the SlAVT6A transgenic plants was detected, with specific procedures identical to Example 2. Figure 7 In the SlAVT6A-OE material, MVA and MED biosynthetic pathways are represented. SlHMGS, SlHMGG1, SlHMGR2, and SlFPPS belong to the MVA pathway, while DXS1, DXS2, DXR, HDR, GPPS, and GGPPS2 are genes related to the MEP pathway, responsible for monoterpene and diterpene synthesis. Experimental results showed that the expression levels of genes such as SlHMGS, SlHMGG1, SlHMGR2, DXS1, DXS2, and DXR were significantly increased in the SlAVT6A-OE material.

[0109] After lyophilizing the metabolic sample, 50 mg was weighed and added to 500 μL of extraction buffer (methanol:ammonia = 70:30, v / v). The mixture was vortexed for 30 s, sonicated in ice water for 30 min, and then centrifuged at 8000 rpm for 10 min at 4℃. The supernatant was collected into a sample vial after centrifugation and stored for later use. Detection method: Samples were detected using an AB Sciex 7500 Qtrap high-sensitivity, low-resolution liquid chromatography-mass spectrometry (LC-MS / MS). We used scheduled MRM mode with a fixed retention time window in negative ion mode for relative quantification of IPP, FPP, and GGPP substances. LC conditions were as follows: sample loading volume was 5 μL; the column was a Shimadzu (Japan) shim-pack VP-ODS C18 column (1.9 μm, 2.1 x 100 mm); mobile phase A was 5 mM ammonium bicarbonate aqueous solution; and mobile phase B was pure acetonitrile. The time gradient was as follows: mobile phase B increased linearly from 5% to 20% within 0–2 min; from 20% to 100% within 2–6 min; 100% pure acetonitrile was maintained for 1 min; mobile phase B decreased from 100% to 5% within 0.1 min; and 5% mobile phase B was maintained for 12 min. The flow rate was 0.35 mL × min⁻¹, and the column temperature was 25 ℃. Data were analyzed using OS software (Sciex). Experimental results are shown below. Figure 7 In SlAVT6A-OE plants, B, IPP, FPP, and GGPP are highly accumulated. This indicates that SlAVT6A affects the metabolic flux of the MVA and MEP pathways by regulating amino acid allocation, thereby influencing downstream secondary metabolic pathways.

[0110] The present invention has been described in detail above. Unless otherwise defined, 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 invention pertains.

[0111] The term "protein" is used interchangeably herein to mean a polymer of amino acid residues. The term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids. As used herein, the term encompasses amino acid chains of any length, including full-length proteins (i.e., antigens), wherein the amino acid residues are linked via covalent peptide bonds.

[0112] The term "conversion" refers to the method of introducing a heterologous DNA sequence into a host cell or organism.

[0113] The term "expression" refers to the transcription and / or translation of endogenous genes or transgenes in plant cells.

[0114] For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments are given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Tomato SlAVT6A The application of genes in regulating tomato fruit yield is characterized by, The tomato SlAVT6A The nucleotide sequence of the gene is shown in SEQ ID No. 1; the application method is through overexpression. SlAVT6A Genes that increase tomato fruit yield.

2. The application according to claim 1, characterized in that, The hyperexpression SlAVT6A Genes that increase tomato fruit yield include the following steps: (1) Extract total RNA from tomato leaves, reverse transcribe to obtain cDNA, and use cDNA as a template to perform PCR amplification with primers SlAVT6A-F and SlAVT6A-R; SlAVT6A-F: 5'-aaaaagcaggcttaATGGCTCTAGCCAAAAATGG-3'; SlAVT6A-R: 5'-agaaagctgggtaCTATAGCCTATTTCCACTCATATTG-3'; (2) The PCR product obtained in step (1) was ligated into the pDonr207 entry vector using the BP reaction of GATEWAY cloning technology. Positive clones were screened and confirmed by sequencing to obtain the desired product. SlAVT6A The full-length cDNA of the gene was then ligated into the overexpression vector PBI121 via the LR reaction of GATEWAY cloning technology to construct the PBI121-SlAVT6A vector. (3) The PBI121-SlAVT6A vector constructed in step (2) was introduced into tomato plants through the Agrobacterium-mediated tomato genetic transformation system.

3. Tomato SlAVT6A The application of genes in regulating the content of terpenoids, a secondary metabolite in tomatoes, is characterized by: The tomato SlAVT6A The nucleotide sequence of the gene is shown in SEQ ID No. 1; the application method is through overexpression. SlAVT6A Genes that increase the total terpene content of tomato strains.

4. The application according to claim 3, characterized in that, The application method is through tomatoes. SlAVT6A Genes influence the metabolic flux of the MVA and MEP pathways by regulating amino acid transport, thereby affecting downstream secondary metabolic pathways and thus the content of terpenoids in tomato secondary metabolites.

5. The application according to claim 3, characterized in that, The hyperexpression SlAVT6A Genes that increase the total terpene content in tomato strains include the following steps: (1) Extract total RNA from tomato leaves, reverse transcribe to obtain cDNA, and use cDNA as a template to perform PCR amplification with primers SlAVT6A-F and SlAVT6A-R; SlAVT6A-F: 5'-aaaaagcaggcttaATGGCTCTAGCCAAAAATGG-3'; SlAVT6A-R: 5'-agaaagctgggtaCTATAGCCTATTTCCACTCATATTG-3'; (2) The PCR product obtained in step (1) was ligated into the pDonr207 entry vector using the BP reaction of GATEWAY cloning technology. Positive clones were screened and confirmed by sequencing to obtain the desired product. SlAVT6A The full-length cDNA of the gene was then ligated into the overexpression vector PBI121 via the LR reaction of GATEWAY cloning technology to construct the PBI121-SlAVT6A vector. (3) The PBI121-SlAVT6A vector constructed in step (2) was introduced into tomato plants through the Agrobacterium-mediated tomato genetic transformation system.

6. The application of an overexpression vector in regulating tomato fruit yield, characterized in that, The overexpression vector contains the tomato as described in claim 1. SlAVT6A Gene.

7. The application according to claim 6, characterized in that, The application involves transferring the overexpression vector into tomato plants to increase tomato fruit yield.

8. The application of an overexpression vector in regulating the content of terpenoids in tomato secondary metabolites, characterized in that, The overexpression vector contains the tomato as described in claim 1. SlAVT6A Gene.

9. The application according to claim 8, characterized in that, The application method involves transferring the overexpression vector into tomato plants, thereby increasing the total terpene content of the tomato lines.

Images