Transcription factor slnac078 for regulating flavonoid synthesis in tomato and application thereof

Abstract

The application belongs to the technical field of biology, and discloses a transcription factor SlNAC078 for regulating flavonoid synthesis of tomatoes and application. The CDS sequence of the transcription factor SlNAC078 is shown as SEQ ID NO. 1, the transcription factor SlNAC078 can positively regulate flavonoid synthesis of tomatoes, overexpression of the gene can activate the promoters of SlCHS1, SlCHS2 and SlFLS genes, and significantly improve the flavonoid content in fruits; and the transcription factor SlNAC078 can also improve the root dry weight and single fruit weight of tomato plants, and realize the synergistic improvement of nutritional quality and yield. The transcription factor SlNAC078 for regulating flavonoid synthesis of tomatoes and application directly targets the regulation mechanism of key genes for flavonoid synthesis, the cultivation method is simple, the obtained transgenic line is genetically stable, and the transcription factor SlNAC078 provides a key gene target and technical scheme for tomato breeding, and has an important application prospect in the industrialized production of high-nutrition and high-yield tomato products.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to the transcription factor SlNAC078, which regulates the synthesis of flavonoids in tomatoes, and its applications. Background Technology

[0002] tomato( Solanum lycopersicum Tomatoes (L.) are an important horticultural crop widely cultivated globally. Their fruits are rich in bioactive substances such as carotenoids, vitamin C, and polyphenolic compounds, making them a vital source of nutrition in our daily diet. Flavonoids, key secondary polyphenol metabolites in tomato fruits, are synthesized via the phenylpropanoid pathway and are divided into six subclasses, including anthocyanins, flavan-3-ols, and flavonols. They not only play a crucial role in protecting plants from oxidative stress, ultraviolet radiation, and pests and diseases, but also offer numerous health benefits for humans, including antioxidant, anti-inflammatory, cardiovascular protection, and cancer inhibition effects. Therefore, tomatoes are one of the core indicators for evaluating their nutritional quality.

[0003] The NAC (NAM, ATAF1 / 2, CUC2) transcription factor family is one of the largest and most functionally diverse transcription factor families in plants. They are widely involved in plant growth and development, organogenesis, fruit ripening, secondary metabolism regulation, and responses to various biotic and abiotic stresses. From regulating fruit size and ripening to enhancing drought, salt, and cold resistance, and participating in pathogen defense and nutrient accumulation, NAC transcription factors are involved in almost every key stage of the plant life cycle. In the tomato genome, 101 NAC family genes (SlNACs) have been identified. They are named according to their chromosomal location and are widely distributed across multiple evolutionary subgroups. The expression of these genes exhibits tissue specificity and developmental stage specificity; for example, their expression patterns differ at different stages of root, stem, leaf, flower, and fruit development. This suggests that SlNACs may be widely involved in various physiological processes throughout the tomato life cycle. SlNAC12 overexpression can upregulate the expression levels of genes related to flavonoid biosynthesis and promote flavonoid accumulation (mainly through indirect stress response). However, in tomatoes, there is no clear evidence or in-depth research on NAC family members directly targeting key genes in the flavonoid biosynthesis pathway and directly regulating flavonoid biosynthesis. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a transcription factor SlNAC078 that regulates flavonoid synthesis in tomatoes. Overexpression of the transcription factor SlNAC078 improves the nutritional quality of tomatoes (increasing flavonoid content) and yield traits (increasing root dry weight and single fruit weight), providing a key target for tomato molecular breeding. The breeding method is simple and feasible, and the resulting new tomato lines have significant prospects for industrial application and can be used to produce high-nutrition, high-yield tomato products.

[0005] To achieve the above objectives, the present invention provides a transcription factor SlNAC078, the CDS sequence of which is shown in SEQ ID NO.1.

[0006] A vector containing the CDS sequence of the transcription factor SlNAC078 gene is also provided.

[0007] Preferably, the vector is the plant expression vector pCAMBIA1305-Flag.

[0008] A recombinant strain containing the CDS sequence of the transcription factor SlNAC078 gene is also provided.

[0009] Preferably, the recombinant strain is prepared from Agrobacterium GV3101.

[0010] The study also provides an application of transcription factor SlNAC078 in regulating the synthesis of flavonoids in tomatoes. The CDS sequence of the transcription factor SlNAC078 gene is shown in SEQ ID NO.1.

[0011] Preferably, the regulation is positive regulation, which involves overexpressing the SlNAC078 gene, activating the promoters of the SlCHS1, SlCHS2 and SlFLS genes, and increasing the content of flavonoids.

[0012] The study also provides an application of transcription factor SlNAC078 in increasing the root dry weight of tomato plants. The CDS sequence of the transcription factor SlNAC078 gene is shown in SEQ ID NO.1.

[0013] The study also provides an application of transcription factor SlNAC078 in increasing the weight of a single tomato fruit. The CDS sequence of the transcription factor SlNAC078 gene is shown in SEQ ID NO.1.

[0014] A method for cultivating tomatoes with high flavonoid content is also provided, comprising the following steps: amplifying the nucleotide sequence of the SlNAC078 gene, constructing an overexpression vector of the SlNAC078 gene, transforming Agrobacterium, infecting tomato explants, and obtaining a new tomato variety.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects: 1) This invention, through comparative experiments of CRISPR knockout (KO) and overexpression (OE) lines, for the first time confirmed that SlNAC078 is a positive regulator of flavonoid biosynthesis in tomato, filling the research gap of NAC family members directly targeting key genes in the flavonoid biosynthesis pathway and directly regulating flavonoid biosynthesis. 2) Overexpression of the SlNAC078 gene in this invention can simultaneously achieve multiple excellent agronomic traits. Compared with wild-type tomatoes, the flavonoid content of fruits of the overexpression lines (OE1, OE2) is significantly increased. Among them, the accumulation of key flavonols such as naringenin and quercetin is particularly significant. At the same time, the root dry weight increases by 20.07%-25.17%, and the single fruit weight increases by 37.13%-44.06%, realizing the synergistic optimization of tomato nutritional quality and yield. 3) The SlNAC078 protein of this invention is located in the cell nucleus and can directly and specifically bind to the promoters of the core node genes (SlCHS1, SlCHS2, SlFLS) of the flavonoid synthesis pathway and activate their expression, especially driving the downstream flavonol branch metabolism, reducing non-targeted interference with other metabolic pathways, with high regulatory efficiency and no obvious side effects. 4) This invention provides clear molecular breeding targets and standardized breeding methods, which can be directly applied to the breeding of new tomato varieties with high nutrition and high yield. The resulting new tomato lines meet the diverse market demands of food processing, fresh consumption, etc., and have high application value and great industrialization potential.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 This diagram illustrates the relative expression levels of the SlNAC078 gene-editing CRISPR and OE lines obtained through screening. Figure A shows two independent lines, KO1 (deletion of GAC at bases 172-174 after the SlNAC078 gene start codon at the sgRNA1 target site) and KO2 (deletion of CG at bases 171-172 after the SlNAC078 gene start codon at the sgRNA1 target site), where effective editing of the SlNAC078 gene occurred at the target site. Figure B shows the relative expression levels of the SlNAC078 gene in WT, OE1, and OE2. This represents P < 0.001. This represents P < 0.0001; Figure 2This image shows the phenotypic diagram of the fruit at maturity of the SlNAC078 gene transgenic lines, as well as the root dry weight, single fruit weight, and flavonoid content analysis of the fruits at ripening stage. Figure A shows the overall morphological observation of the WT group compared to KO1, KO2, OE1, and OE2 plants; Figure B shows the morphology and color comparison of the two clusters of fruits in the WT group compared to KO1, KO2, OE1, and OE2 plants; Figure C shows the root dry weight analysis of the fruits at ripening stage in the WT group compared to KO1, KO2, OE1, and OE2 plants; Figure D shows the single fruit weight analysis of the fruits at ripening stage in the WT group compared to KO1, KO2, OE1, and OE2 plants; and Figure E shows the flavonoid content analysis of the fruits at ripening stage in the WT group compared to KO1, KO2, OE1, and OE2 plants. This means P < 0.05. This means P < 0.01. This represents P < 0.001. This represents P < 0.0001; Figure 3 PLS-DA scores plot of 15 flavonoid metabolites in the second fruit clusters of tomato from WT and SlNAC078 transgenic lines (KO1, KO2, OE1 and OE2). Figure 4 Hierarchical clustering heatmap of 15 flavonoids from the two spikes of tomato in transgenic lines (KO1, KO2, OE1 and OE2) of WT and SlNAC078 genes. Figure 5 This figure shows the content of flavonoid metabolites in the fruits of the two spikes of transgenic WT and SlNAC078 gene lines (KO1, KO2, OE1, and OE2). Figure A shows the content analysis of naringenin, B shows the content analysis of hesperidin, C shows the content analysis of dihydroquercetin, D shows the content analysis of quercetin, E shows the content analysis of rutin, and F shows the content analysis of kaempferol. In the figure, ns represents P > 0.05. This means P < 0.05. This means P < 0.01. This represents P < 0.001; Figure 6The figures show the relative expression levels of key genes for flavonoid synthesis in the WT group and the SlNAC078 transgenic lines (KO1, KO2, OE1, and OE2). Figure A shows the relative expression level analysis of the key gene SlCHS1, B shows the relative expression level analysis of the key gene SlCHS2, C shows the relative expression level analysis of the key gene SlCHI, D shows the relative expression level analysis of the key gene SlF3H, E shows the relative expression level analysis of the key gene SlF3'H, and F shows the relative expression level analysis of the key gene SlFLS. Here, ns represents P > 0.05. This means P < 0.05. This means P < 0.01. This represents P < 0.001; Figure 7 The images, from left to right, show the subcellular localization analysis of the SlNAC078 protein: GFP green fluorescence signal, DAPI nuclear staining signal (blue), bright field image, and merged image.

[0018] Figure 8 The experimental results show the binding of His-SlNAC078 to cis-acting elements in the promoter regions of the SlCHS1, SlCHS2, and SlFLS genes. Figure A shows the results of the yeast one-hybrid experiment, and B shows the results of the gel retardation migration experiment.

[0019] Figure 9 The results of a dual-luciferase reporter gene assay for activating transcription of the SlCHS1, SlCHS2, and SlFLS genes using 62SK-SlNAC078 are shown in the figure. Figure A shows the transcriptional activation effect analysis of the SlCHS1 promoter, Figure B shows the transcriptional activation effect analysis of the SlCHS2 promoter, and Figure C shows the transcriptional activation effect analysis of the SlFLS promoter. This means P < 0.01. This means P < 0.0001. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0022] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0023] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0024] Materials and reagents: Tomato variety: Wild-type tomato (Ailsa Craig, AC), which was preserved in the laboratory for a long time and self-propagated for more than 6 generations, with consistent genetic background; Vectors and strains: plant overexpression vector pCAMBIA1305-Flag, CRISPR-Cas9 vector backbone, Escherichia coli DH5α (purchased from Tiangen Biotech (Beijing) Co., Ltd.), Agrobacterium GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd.), yeast strain EGY48, and Escherichia coli Rosetta (DE3). Reagents: Polysaccharide and polyphenol plant RNA extraction kit (purchased from Beijing Polymer Biotechnology Co., Ltd.), high-fidelity PCR enzyme, reverse transcription kit, restriction endonuclease, homologous recombination kit, Ni-NTA Resin packing material (purchased from Beijing TransGen Biotech Co., Ltd.), flavonoid detection kit (purchased from Suzhou Keming Biotechnology Co., Ltd.), LightShift Chemiluminescent EMSA Kit (purchased from Thermo Fisher Scientific), etc.

[0025] SlNAC078 gene: The core gene provided in this invention is derived from tomato ( Solanum lycopersicum L.), whose genome sequence can be found in the Phytozome database Solyc01g021730.2, and is named SlNAC078 in this invention. The coding region (CDS) nucleotide sequence of this gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0026] Example 1 Cloning and expression vector construction of the SlNAC078 gene.

[0027] Gene cloning: Total RNA was extracted from AC tomato leaves using a polysaccharide and polyphenol plant RNA extraction kit, and cDNA was obtained by reverse transcription. Primers were designed based on the sequence (SEQ ID NO.1), and the complete coding region fragment of the SlNAC078 gene was amplified by high-fidelity PCR.

[0028] Construction of expression carrier: 1) Construction of gene overexpression vector: The cloned SlNAC078 gene sequence was ligated into the plant expression vector pCAMBIA1305-Flag using homologous recombination technology to construct the recombinant plasmid p1305-Flag-SlNAC078. This recombinant plasmid was transformed into competent E. coli cells, followed by colony PCR and sequencing (Genewiz). The correct recombinant plasmid was transformed into Agrobacterium strain GV3101 using the heat shock method. After verification by colony PCR, positive Agrobacterium clones were stored at -80℃ for later use.

[0029] 2) Construction of gene editing vectors: Using the online design tool CCTop (https: / / cctop.cos.uni-heidelberg.de:8043 / ), sgRNAs were designed targeting the coding region of the SlNAC078 gene. Two target sites, sgRNA1 (SEQ ID NO.3) and sgRNA2 (SEQ ID NO.4), with high prediction scores and low off-target risk, were selected. The final determined sgRNA target sequences and their corresponding PAM sequences are as follows: SEQ ID NO.3: 5'-AGCCTGCTCTAATGACGACG-3' (corresponding PAM sequence: AGG); SEQ ID NO.4: 5'-GTCCAGATATAGTCGTGATG-3' (corresponding PAM sequence: TGG).

[0030] Using the intermediate vector PCBC-DT1T2 containing a dual sgRNA expression framework as a template, two rounds of PCR amplification were performed using specific primers containing the target sequences described above (the first round amplified DT1 and DT2 fragments separately, and the second round ligated them together) to construct an expression cassette that simultaneously expresses two sgRNAs. The PCR products were purified by gel extraction, cloned using Golden Gate cloning, and assembled into the final plant CRISPR expression vector backbone containing the Cas9 gene, constructing the dual-target knockout vector pCas9-SlNAC078-KO. This vector was transformed into *E. coli* DH5α, and after verification by enzyme digestion and sequencing, the plasmid was extracted and transformed into *Agrobacterium* GV3101 using a heat shock method. After colony PCR verification, the culture was stored at -80°C.

[0031] Example 2 Obtaining, screening and establishing stable lines of transgenic tomato plants.

[0032] 1) Agrobacterium-mediated genetic transformation of tomatoes.

[0033] The *Agrobacterium* strains overexpressing p1305-Flag-SlNAC078 and gene-edited *Agrobacterium* strains (containing pCas9-SlNAC078-KO) constructed and validated in Example 1 were used for genetic transformation of AC tomatoes. The transformation method was *Agrobacterium*-mediated leaf disc method, and the specific steps are as follows: S1. Aseptic seedling culture: Take AC tomato seeds, disinfect the seed surface with 5% NaClO solution for 10 minutes, rinse with sterile water 6-8 times, and then spread them evenly on MS solid medium. Incubate in the dark at 25℃ for 3-4 days until the cotyledons show signs of sprouting, then transfer them to a light incubator (24℃, 16h light / 8h dark) for further culture. Use the seedlings when the cotyledons are fully expanded but before true leaves have emerged.

[0034] S2. Explant preparation and pre-culture: Cut the cotyledons into small pieces of about 0.5cm×0.5cm, place them with the back of the leaves facing up on the pre-culture medium covered with filter paper, and incubate in the dark at 25℃ for 2 days.

[0035] S3. Agrobacterium infection and co-culture: Agrobacterium (overexpressing Agrobacterium engineered strains and gene-edited Agrobacterium engineered strains) in logarithmic growth phase (OD) culture... 600 Collect by centrifugation (0.4-0.6%) and resuspend in sterile water to OD. 600 Approximately 0.1 was used as the infection solution. The pre-cultured explants were immersed in infection solutions containing overexpressed Agrobacterium and gene-edited Agrobacterium engineered bacteria for 5 minutes, respectively. After removing and drying off the excess bacterial solution, the explants were placed with the leaf underside up on the pre-culture medium (the specific composition is shown in Table 1) and incubated in the dark at 25°C for 2 days.

[0036] S4, resistant bud induction, elongation and rooting.

[0037] Bud induction: After co-culture, explants treated with overexpressing Agrobacterium-mediated transformation and gene-edited Agrobacterium-mediated transformation were transferred to the corresponding selection media (overexpression lines used media containing hygromycin B, and knockout lines used media containing glufosinate-ammonium; specific compositions are shown in Table 1). The explants were placed with the leaf underside down and cultured in a light incubator. The media were replaced with fresh media every 7-14 days until resistant buds differentiated.

[0038] Stem elongation: When the buds grow to about 2cm, remove the excess callus tissue and transfer them to the bud elongation medium (the specific composition is shown in Table 1) for continued culture until the stem segments elongate to 5-6cm.

[0039] Rooting: Cut off well-elongated stem segments and insert them into a rooting medium containing antibiotics (the specific composition is shown in Table 1) to induce rooting and obtain complete T0 generation transgenic plants.

[0040] S5. Transplanting of transgenic plants: When the plants have taken root well and are about 10cm tall, wash the culture medium from the roots and transplant them into a sterilized cultivation substrate (peat moss: vermiculite: black soil = 4:1:1). After the plants have recovered from transplant shock by keeping them moist, they are cultured normally in a greenhouse to obtain gene-edited lines and overexpression lines.

[0041] Table 1. Formulas and components of various culture media for transgenic tomato tissues (based on a final volume of 1 L).

[0042] 2) Screening of positive T1 generation single plants.

[0043] The T0 generation plants were self-pollinated, and the seeds were harvested and sown to obtain the T1 generation segregating population.

[0044] Screening of gene-edited lines: PCR and sequencing analysis were performed on the T1 generation population targeting the dual targets designed in Example 1. Figure 1 As shown in Figure A, two independent strains that underwent effective editing at the target site were selected and named KO1 (deletion of GAC at bases 172-174 after the start codon of the SlNAC078 gene at the sgRNA1 target site) and KO2 (deletion of CG at bases 171-172 after the start codon of the SlNAC078 gene at the sgRNA1 target site).

[0045] Screening of overexpression lines: qRT-PCR analysis was performed on the T1 generation population. For example... Figure 1 As shown in Figure B, two independent lines, OE1 and OE2, with significantly and stably elevated SlNAC078 expression levels were screened and named. The SlNAC078 expression level in OE1 was approximately 24.2 times that of the wild type, and the SlNAC078 expression level in OE2 was approximately 16.7 times that of the wild type.

[0046] 3) Establishment of stable T2 generation lines.

[0047] The T1 generation individual plants KO1, KO2, OE1, and OE2 identified above were self-pollinated to harvest T2 generation seeds.

[0048] Genetic stability verification: T2 generation seeds of each line were planted, and the progeny population (20 plants) of each line were subjected to the same molecular identification (target sequencing or qRT-PCR) as above.

[0049] The results confirmed that all tested plants in the T2 generation of KO1 and KO2 lines carried the same Indel mutation with homozygous or biallelic genes at the corresponding target sites; the T2 generation plants of OE1 and OE2 lines maintained the high expression level of their parents and did not show phenotypic segregation.

[0050] Thus, homozygous gene knockout stable lines KO1 and KO2, and homozygous overexpression stable lines OE1 and OE2 were established. In subsequent examples, unless otherwise specified, "OE line" and "KO line" refer to experimental materials derived from these T2 generation stable lines.

[0051] Example 3 Effects of SlNAC078 on tomato phenotype, yield and quality.

[0052] 1) Experimental Design: AC tomato wild-type control (WT), two independent homozygous knockout lines (KO1 and KO2) established in Example 2, and two independent homozygous overexpression lines (OE1 and OE2). All materials were grown simultaneously in the same controlled-environment greenhouse at China Agricultural University, using a randomized block design with 5 replicates for each line. Cultivation management conditions (water, fertilizer, light, and temperature) were kept consistent.

[0053] 2) Phenotypic observation: During the fruit ripening stage, overall morphological observation and image acquisition were conducted on representative plants from each line. Results are as follows: Figure 2 As shown in Figure AB, compared with the WT and KO lines, although the OE1 and OE2 lines have lower plant height, they have more fruits in the second spike, mature earlier, and have larger fruits, while the KO1 and KO2 lines have smaller fruits and mature later.

[0054] 3) Indicator measurement.

[0055] Root dry weight: The dug-up, intact root systems were washed, placed in a 70℃ oven, and dried to constant weight (approximately 72 hours), then weighed using a precision electronic balance. Results are as follows: Figure 2 As shown in Figure C, the average root dry weight of the two OE lines was significantly higher than that of WT (OE1 increased by about 20.07%, and OE2 increased by about 25.17%), while the average root dry weight of the two KO lines was significantly lower than that of WT.

[0056] Single fruit weight: Two clusters of fruit were collected from five replicates of the WT, OE, and KO lines, respectively. The weights were measured using a precision electronic balance, and the number of fruits in each cluster was recorded. The single fruit weight was calculated. Results are as follows: Figure 2 As shown in Figure D, the average single fruit weight of the OE1 and OE2 lines was significantly higher than that of the WT lines (with increases of 37.13% and 44.06%, respectively), while the average single fruit weight of the KO1 and KO2 lines was significantly lower.

[0057] Determination of total flavonoid content in ripe red fruit: A flavonoid reagent kit was used. Ripe red fruit from the same batch used for single fruit weight determination was dried to constant weight, pulverized, passed through a 40-mesh sieve, and approximately 0.02 g was weighed (recorded as [weight value]). Add 2 ml of extraction buffer, extract at 60℃ with shaking for 2 h, centrifuge at 10000 g, 25℃ for 10 min, and collect the supernatant for testing. Add 540 μl of distilled water and 540 μl of sample solution to the blank tube and the test tube, respectively. Add 30 μl of reagent one (5% NaNO2), mix well, and let stand at room temperature for 6 min. Then add 30 μl of reagent two (10% AlCl3), mix well, and let stand at room temperature for 5 min. Finally, add 400 μl of reagent three (1M NaOH), mix well, and let stand at room temperature for 15 min to perform the colorimetric reaction. Measure the absorbance at a wavelength of 510 nm. .in, for Measurement tube - Blank tubes: Only one blank tube is prepared. Calculate the flavonoid content using the following formula: ; Its standard curve is y = 5.02x + 0.0007, R0 2 =0.9996.

[0058] The results are as follows Figure 2 As shown in Figure E, the flavonoid content of fruits from strains OE1 and OE2 increased by approximately 155.19% and 133.89% respectively compared to WT, while the flavonoid content of fruits from strains KO1 and KO2 was significantly lower than that of WT.

[0059] This study, through systematic phenotypic observation and quantitative analysis, has for the first time confirmed that the SlNAC078 gene possesses multiple excellent agronomic functions that synergistically and positively regulate tomato vegetative growth (root dry weight), fruit yield (single fruit weight), and nutritional quality (flavonoid content). Overexpression of the SlNAC078 gene can simultaneously achieve the excellent phenotypes of well-developed root system, high fruit yield, and nutrient enrichment, providing direct phenotypic support for the targeted application of this gene in tomato molecular breeding.

[0060] Example 4 Targeted metabolomics analysis.

[0061] Sample submission and data acquisition: Plants from the same batch used for phenotypic analysis in Example 3 were selected, with six biological replicates for each line. Fruit samples were immediately flash-frozen in liquid nitrogen and stored at -80°C. Tomato fruit samples were sent to Qingdao Standard Testing & Inspection Co., Ltd. for targeted flavonoid metabolomics detection using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). The panel covered 45 flavonoid metabolites, including concentration information for all 45 metabolites in each sample. After data validity screening, data for 15 stably detected metabolites were obtained and used for subsequent analysis.

[0062] 1) Global changes in metabolic profile.

[0063] To assess the overall impact of SlNAC078 on the flavonoid metabolome, partial least squares discriminant analysis (PLS-DA) was performed based on data from the aforementioned 15 metabolites. Figure 3 As shown, the samples from the WT, KO, and OE strains are clearly separated on the score map, indicating systematic differences in the flavonoid metabolite profiles of different genotypes. The hierarchical clustering heatmap of these 15 metabolites (e.g.) Figure 4 As shown in the figure, most metabolites exhibit a highly consistent regulatory direction. Specifically, accumulation is generally upregulated in the OE1 and OE2 overexpression lines (orange), while it is generally downregulated in the KO1 and KO2 knockout lines (blue). The accumulation level in wild-type (WT) is in the middle. This overall trend strongly demonstrates that SlNAC078 is a global positive regulator of the flavonoid metabolic pathway.

[0064] 2) Screening of differential metabolites and identification of key metabolites.

[0065] For 15 metabolites, with WT as the control, the differences between OE and KO lines were analyzed. One-way ANOVA combined with Tukey's multiple comparison test was used to screen out 6 metabolites that were significantly regulated by SlNAC078, with p value <0.05 and fold change >1.5 or <0.67 as the criteria.

[0066] Upstream flavanones (such as Figure 5 As shown in AB): As early core products of flavonoid synthesis, the contents of naringenin and hesperidin in the OE strain increased by about 0.9-1.3 times and 1.7-2.6 times respectively compared with WT, while in the KO strain they decreased by about 64%-85% and 58%-78% respectively.

[0067] Midstream dihydroflavonols (such as Figure 5(As shown in C): Dihydroquercetin, as a direct precursor for flavonol synthesis, also accumulated significantly in the OE strain, with increases of 60.53% and 65.79% in OE1 and OE2, respectively, while it decreased significantly in the KO strain.

[0068] Downstream flavonols and their glycosides (such as...) Figure 5 (As shown in DF): the end products quercetin, its main glycoside rutin, and kaempferol showed the most dramatic accumulation in the OE strain, with increases of approximately 75.15%, 53.21%, and 68.09%, respectively, while these increases decreased sharply in the KO strain. This particularly indicates that SlNAC078 strongly drives the metabolic flux to the downstream flavonol branch.

[0069] It is evident that precise quantification through targeted metabolomics confirmed the global activation of the flavonoid metabolome by SlNAC078, and more accurately elucidated its specific enhancing effect on multiple key nodes in the upstream, midstream, and downstream of the synthetic pathway. In particular, it has a strong promoting effect on the accumulation of flavonol end products such as quercetin, further demonstrating the role of SlNAC078 as a core regulatory factor in improving the nutritional quality of tomato fruits.

[0070] Example 5 Analysis of the expression of key genes in the flavonoid synthesis pathway.

[0071] To elucidate the transcriptional molecular basis of changes in flavonoid metabolite accumulation, we used real-time quantitative PCR (qRT-PCR) to analyze the expression patterns of several key genes in the flavonoid synthesis pathway.

[0072] Experimental Materials and Methods: Fruit samples at the same red-ripe stage as in Examples 3 and 4 were selected to ensure homology of phenotype, metabolite, and gene expression data. Total RNA was extracted from each sample using a polysaccharide-polyphenol plant RNA extraction kit, digested with DNase I (RNase-free), and then reverse transcribed into cDNA. Using the tomato Actin gene (Solyc03g078400) as an internal control, specific primers were designed for qRT-PCR analysis targeting six key genes in the flavonoid synthesis pathway. These genes include: SlCHS1 (chalcone synthase 1), SlCHS2 (chalcone synthase 2), SlCHI (chalcone isomerase), SlF3H (flavonol-3-hydroxylase), SlF3'H (flavonoid-3'-hydroxylase), and SlFLS (flavonol alcohol synthase). Each sample was tested in duplicate, and the experiment was independently repeated four times.

[0073] The results are as follows Figure 6 As shown, SlNAC078 exhibits significant selectivity and specificity in regulating the flavonoid synthesis pathway.

[0074] In the overexpression lines (OE1, OE2), the expression of six genes—SlCHS1, SlCHS2, SlCHI, SlF3H, SlF3'H, and SlFLS—was specifically and significantly upregulated. Specifically, SlCHS1 expression was upregulated by approximately 0.7–1.2-fold, SlCHS2 by approximately 0.7–1.1-fold, and SlFLS by approximately 0.5–0.9-fold. In the knockout lines (KO1, KO2), the opposite trend was observed, with the expression of SlCHS1, SlCHS2, and SlFLS being significantly suppressed.

[0075] It is evident that the upregulation of SlCHS1 and SlCHS2, as the upstream rate-limiting enzymes in the flavonoid synthesis pathway, directly drives the increased synthesis of upstream flavanones (such as naringenin and hesperidin); the upregulation of SlFLS, as a key branching enzyme catalyzing the conversion of dihydroflavonols (such as dihydroquercetin) to flavonols, specifically promotes the strong accumulation of downstream flavonols (such as quercetin and kaempferol) and their glycosides (rutin); while the changes in the corresponding metabolites of genes such as SlF3'H, which have a smaller upregulation, are not significant.

[0076] This further demonstrates that SlNAC078 does not globally activate the entire phenylpropanoid pathway, but rather precisely targets and activates two core nodes in the flavonoid synthesis pathway: the upstream entry gene (CHS) and the downstream flavonol branching determination gene (FLS), thereby efficiently guiding metabolic flow and ultimately leading to a specific and large accumulation of flavonols with high antioxidant activity in the fruit.

[0077] Example 6 Subcellular localization and target gene interaction verification of SlNAC078.

[0078] 1) Subcellular localization of SlNAC078 protein.

[0079] To confirm that SlNAC078 possesses the basic characteristics of a transcription factor, subcellular localization analysis was first performed on it.

[0080] S1. Construction of the fusion expression vector: Using AC tomato cDNA as a template, the full-length coding sequence of the SlNAC078 gene (without a stop codon) was amplified by PCR. This fragment was fused with the coding sequence of green fluorescent protein (GFP) using homologous recombination technology and cloned downstream of the CaMV35S promoter of the plant transient expression vector pCAMBIA1300 to construct the 35S::SlNAC078-GFP fusion expression vector. After amplification and sequencing verification using *E. coli* DH5α (purchased from Tiangen Biotech (Beijing) Co., Ltd.), the plasmid was extracted.

[0081] S2. Agrobacterium transformation and preparation: The plasmid that was verified above was transformed into Agrobacterium strain GV3101 (containing helper plasmid pSoup-p19, purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd.) by heat shock method. Positive engineered strains were obtained by screening on LB solid medium with antibiotics (Kanamycin Kan and Rifampicin Rif) and colony PCR identification.

[0082] S3. Transient transformation and observation of *Nicotiana benthamiana* leaves: Agrobacterium tumefaciens in the logarithmic growth phase was centrifuged and resuspended, and the OD was adjusted to 0.5% using infection solution (containing 0.2M MES, 1M MgCl2, 0.2M acetylsylcholine, pH 5.6). 600 =0.6, incubate at room temperature and in the dark for 2-3 hours. Using a sterile syringe, mix SlNAC078-GFP with Agrobacterium tumefaciens labeled RFP-H2B, and inject into well-grown Nicotiana benthamiana (Nicotiana benthamiana). Nicotiana benthamiana The underside of the leaves (purchased from Beijing Weidi Biotechnology Co., Ltd.) were examined. After culturing in the dark for 8 hours, the plants were transferred to a normal photoperiod (16 hours light / 8 hours dark) and cultured for another 48-72 hours. Before sampling, the lower epidermis of the leaves was stained with DAPI (4',6-diamidinyl-2-phenylindole, purchased from Beyotime Biotechnology) solution.

[0083] S4. Laser confocal microscopy observation: The lower epidermis of the leaf from the injection area was cut off, placed on a glass slide, and observed using a Zeiss laser confocal microscope. GFP (green fluorescence) was excited using a 488nm laser, and DAPI (blue fluorescence) was excited using a 405nm laser. Green and blue fluorescence signals were collected respectively.

[0084] The results are as follows Figure 7 As shown, the green fluorescence signal emitted by the SlNAC078-GFP fusion protein completely overlaps with the blue fluorescence signal in the cell nucleus observed by DAPI staining, while no obvious green fluorescence aggregation was observed in the cytoplasm. This demonstrates that SlNAC078 is a protein located in the cell nucleus, providing cellular localization evidence for its transcriptional regulatory function.

[0085] 2) Yeast one-hybrid experiment.

[0086] S1. Construction of promoter reporter vectors: Using AC tomato genomic DNA as a template, specific fragments containing predicted NAC binding elements (such as CATGG) in the promoter regions of the SlCHS1, SlCHS2, and SlFLS genes were amplified by PCR. Each promoter fragment was then cloned into the yeast reporter vector pLacZi2μ (Amp) using homologous recombination technology. +Multiple cloning sites were identified, and reporter vectors pLacZi-ProSlCHS1, pLacZi-ProSlCHS2, and pLacZi-ProSlFLS were constructed.

[0087] S2. Construction of the transcription factor prey vector: Using AC tomato cDNA as a template, the full-length coding sequence of the SlNAC078 gene, containing a stop codon, was amplified by PCR. This sequence was then cloned into the yeast prey vector pB42AD(Amp) using homologous recombination technology. + In this study, the prey vector pB42AD-SlNAC078 was constructed. The empty pB42AD vector served as a negative control.

[0088] S3, Yeast Transformation and Interaction Detection.

[0089] Preparation of competent yeast cells: Competent yeast cells of strain EGY48 were prepared using the lithium chloride-polyethylene glycol (LiAc-PEG) method.

[0090] Co-transformation: The prey vectors (pB42AD-SlNAC078 or empty pB42AD) constructed above were combined in pairs with each reporter vector (pLacZi-ProSlCHS1, pLacZi-ProSlCHS2, and pLacZi-ProSlFLS) and co-transformed into yeast EGY48 competent cells. The transformation system contained denatured salmon sperm DNA as a vector. The specific steps were as follows: after mixing the plasmids with competent cells, TE / LiAc / PEG solution (TE buffer: lithium acetate: 50% PEG3350 = 1:1:3) was added, and the cells were incubated at 30°C for 30 min. Subsequently, the cells were treated with dimethyl sulfoxide (DMSO) and heat-shocked, and then plated on SD / -Trp / -Ura dual-deficient selection solid medium and incubated at 30°C for 72 h to screen yeast clones that were simultaneously transformed with two plasmids.

[0091] Interaction phenotype observation: Select large single clones that grow well on the dual-deficient medium, streak them on X-gal chromogenic medium (containing galactose, raffinose, BU buffer and X-gal), and observe the color change after incubation at 30℃ for 36 hours.

[0092] The results are as follows Figure 8As shown in Figure A, only yeast colonies co-transformed with the pB42AD-SlNAC078 prey vector and any of the target gene promoter reporter vectors (pLacZi-ProSlCHS1, pLacZi-ProSlCHS2, and pLacZi-ProSlFLS) turned blue on X-gal chromogenic medium. Negative control colonies co-transformed with the empty pB42AD vector and each reporter vector remained white. These results indicate that the SlNAC078 protein can specifically bind to the promoter fragments of SlCHS1, SlCHS2, and SlFLS within the yeast cell nucleus and activate the expression of the downstream reporter gene LacZ.

[0093] 3) Gel retardation migration assay (EMSA).

[0094] Expression and purification of S1 and His-SlNAC078 recombinant proteins.

[0095] Vector construction and transformation: The full-length coding sequence of the SlNAC078 gene was cloned into the prokaryotic expression vector pET-28a(+) to construct the N-terminal His-tagged expression vector pET-28a-SlNAC078. This recombinant plasmid was then transformed into the Escherichia coli expression strain Rosetta(DE3).

[0096] Protein expression induction: Select positive clones and expand the culture to OD. 600 When the concentration was 0.6, isopropyl-β-D-thiogalactoside was added to a final concentration of 1 mM, and His-SlNAC078 fusion protein was induced to be expressed overnight at 16 °C.

[0097] Protein purification: Bacterial cells were collected and sonicated. The His-tagged protein in the supernatant was purified using a resin affinity chromatography column filled with Ni-NTAResin. Gradient elution was performed using elution buffers containing 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 400 mM, and 500 mM imidazole, and the filtrate was collected. 20 µL of the filtrate was added to an equal volume of protein loading buffer, and the mixture was heated at 99 °C for 10 min before SDS-PAGE analysis.

[0098] Protein validation and concentration: The purity and size of the target protein were confirmed by detecting the elution fraction using SDS-PAGE gel electrophoresis. The eluent containing the target protein was concentrated using an ultrafiltration concentrator (Millipore). After centrifugation to approximately 1 mL, 10 mL of imidazole-free Tris-HCl buffer was added, and the mixture was centrifuged again to 0.5 mL. The concentrate was then aliquoted and stored at -80°C for later use.

[0099] S2. Preparation of biotin-labeled DNA probes.

[0100] Specific oligonucleotides were designed and synthesized targeting the core sequences (approximately 40 bp) containing predicted NAC binding elements in the promoter regions of the SlCHS1, SlCHS2, and SlFLS genes. Double-stranded DNA probes were synthesized by biotin labeling the 5' end of the forward primers using a professional company (Shanghai Jereh Biotechnology Co., Ltd.). Simultaneously, identical unlabeled DNA fragments were synthesized as competing probes.

[0101] S3, EMSA binding reaction and detection.

[0102] Reaction: Performed in a 20 μL reaction system according to the Thermo Fisher Scientific LightShift Chemiluminescent EMSA Kit instructions. The following reaction groups were set up.

[0103] Negative control: Contains only biotin-labeled probes.

[0104] Experimental group: Biotin-labeled probe + purified His-SlNAC078 protein.

[0105] Competitive experimental group: Biotin-labeled probe + His-SlNAC078 protein + excess (50× and 100× molar ratio) of unlabeled competitive probe.

[0106] The reaction system consisted of 1× binding buffer, 2.5% glycerol, 1 mmol / L MgCl2, 0.5% NP-40, and 1 μg / μL poly(dI-dC). Incubate at room temperature for 20-30 minutes.

[0107] S4. Non-denaturing gel electrophoresis and transfer: The reaction products were added to the wells of a 6% non-denaturing polyacrylamide gel (prepared with 0.5×TBE and containing 2.5% glycerol) and electrophoresed at a constant voltage (80V) for approximately 90 minutes at 4°C in 0.5×TBE buffer. Subsequently, the DNA probes and protein-DNA complexes in the gel were transferred to a positively charged nylon membrane using capillary blotting.

[0108] S5. Crosslinking and Chemiluminescence Detection: The nylon membrane was placed in a UV crosslinker and crosslinked at 1200J. Subsequently, the membrane was sealed, incubated with horseradish peroxidase-labeled streptavidin, and washed. Finally, a chemiluminescent substrate was added, and the signal was acquired using a chemiluminescence imaging system.

[0109] The results are as follows Figure 8As shown in Figure B, in the experimental group, with the increase of His-SlNAC078 protein amount, the free biotin-labeled probe band decreased, while a significant, dose-dependent migration lag band (protein-DNA complex) appeared. Most importantly, when an excess of unlabeled competing probe was added, this lag band significantly weakened or disappeared. This conclusively demonstrates that the SlNAC078 protein can directly and specifically bind to the promoter probes of SlCHS1, SlCHS2, and SlFLS in vitro, and that this binding can be effectively competed for by homologous sequences.

[0110] 4) Dual-luciferase reporter gene detection.

[0111] To verify whether SlNAC078 can activate the transcriptional activity of target gene promoters in plant cells that are closer to their natural state, a dual-luciferase reporter gene assay was performed.

[0112] S1. Construction of effect carriers and report carriers.

[0113] Effector vector construction: The full-length coding sequence of the SlNAC078 gene (including the stop codon) was cloned downstream of the CaMV 35S promoter in the plant binary vector pGreenII 62-SK to construct the effector vector 62SK-35S::SlNAC078. An empty pGreenII 62-SK vector was used as a negative control.

[0114] Report platform construction: Using AC tomato genomic DNA as a template, the promoter sequences approximately 2000 bp upstream of the translation start sites of the SlCHS1, SlCHS2, and SlFLS genes were amplified by PCR. Each promoter fragment was then cloned upstream of the firefly luciferase (LUC) gene in the reporter vector pGreenII0800-LUC to construct reporter vectors 0800-ProSlCHS1::LUC, 0800-ProSlCHS2::LUC, and 0800-ProSlFLS::LUC.

[0115] S2, Preparation of Agrobacterium-engineered bacteria and instantaneous conversion of tobacco.

[0116] The correctly constructed effect vector and reporter vector plasmids were transformed into Agrobacterium GV3101 strain to obtain the corresponding engineered bacteria.

[0117] Agrobacterium clones containing effector vectors and reporter vectors were activated separately and cultured to OD200. 600 =1.0. Collect bacterial cells by centrifugation and resuspend in Agrobacterium resuspension solution (containing 10 mM MES, 10 mM MgCl2, 200 μM acetylsylgenone, pH 5.6).

[0118] The effector vector engineered bacteria and the reporter vector engineered bacteria were mixed at a ratio of 9:1 (volume ratio) and left to stand at room temperature in the dark for 3 hours to promote the induction of the Vir gene.

[0119] The mixed bacterial solution was injected into healthy leaves of *Nicotiana benthamiana* using a sterile syringe. For internal control, the same leaf was divided into sections along the main vein, and each section was injected with either a mixed bacterial solution containing the SlNAC078 effector vector or a mixed bacterial solution containing an empty effector vector. The injected plants were then incubated in the dark for 8 hours, followed by 48 hours of normal photoperiod culture.

[0120] S3. Dual-luciferase activity assay and data analysis.

[0121] Sample preparation: Samples were taken from the injection area using a punch, flash-frozen in liquid nitrogen, and then ground into powder. Passive lysis buffer from the LightShift Chemiluminescent EMSA Kit was added, and the mixture was vortexed, repeatedly frozen and thawed, and centrifuged. The supernatant was then used as the sample to be tested.

[0122] Activity assay: Using the GloMax® 20 / 20 luminescence detector, LAR II reagent was first added to the sample, and the firefly luciferase (LUC) activity was immediately measured; then Stop&Glo® reagent was added to quench the LUC reaction and initiate the Renilla luciferase (REN) reaction, and the REN activity was measured.

[0123] Data processing: The LUC / REN activity ratio was calculated for each sample point to correct for differences in transformation efficiency. The average ratio of the experimental group containing the SlNAC078 effector vector was compared with the average ratio of the control group containing the empty effector vector on the same leaf to calculate the relative activation fold.

[0124] The results are as follows Figure 9 As shown, compared with the control group co-transformed with the empty effector vector, co-expression of SlNAC078 significantly activated the activity of the LUC reporter gene driven by the SlCHS1, SlCHS2, and SlFLS promoters. The relative activation folds were approximately 1.9-fold, 1.7-fold, and 1.9-fold, respectively. This result directly demonstrates in living plant cells that SlNAC078 has a strong transcriptional activation function on the promoters of these three target genes.

[0125] In summary, this invention, through systematic molecular biology, genetics, and metabolomics experiments, reveals and confirms for the first time the core role and molecular mechanism of the tomato transcription factor SlNAC078 in the synergistic regulation of fruit yield and nutritional quality. Specifically: In terms of functional validation, overexpression of the SlNAC078 gene significantly increased the root dry weight, single fruit weight, and flavonoid content (especially flavonols such as naringenin and quercetin) in tomato plants; while knockout resulted in the opposite phenotype. In terms of mechanism analysis, the SlNAC078 protein is located in the cell nucleus and can directly bind to and activate the promoters of two key nodes in the flavonoid biosynthesis pathway—the upstream genes SlCHS1 and SlCHS2, and the downstream branching gene SlFLS—thereby precisely driving metabolic flux and promoting flavonoid accumulation. In terms of application value, this invention not only identified a novel synergistic regulator of quality and yield but also provided a clear technical solution and stable genetic material for cultivating high-flavonoid, high-yield tomato varieties by overexpressing the SlNAC078 gene. This provides crucial genetic resources and theoretical basis for the simultaneous genetic improvement of nutritional quality and yield in tomatoes and other crops.

[0126] This invention protects sequence variants containing the core function of the SlNAC078 gene. The SlNAC078 gene, obtained from different strains or through artificial optimization, may have sequences that differ somewhat from the sequence shown in SEQ ID NO.1. However, as long as it has a high sequence identity with SEQ ID NO.1 (at least 80%, 85%, 90%, 95%, 98%, or 99% identity) and retains the biological function of regulating flavonoid synthesis discovered in this invention, it falls within the scope of protection of this invention. The functions include, but are not limited to: It positively regulates the accumulation of flavonoids in tomato fruits; activates the expression of key genes for flavonoid synthesis (such as SlCHS1, SlCHS2, and SlFLS); and directly binds to the promoter regions of the aforementioned key genes.

[0127] By creating transgenic tomato lines with SlNAC078 gene knockout (KO) and overexpression (OE), the inventors have for the first time revealed and confirmed the following novel application values ​​of SlNAC078, which are specifically reflected in three interrelated aspects.

[0128] In the first aspect, it is applied in molecular breeding of tomatoes: the SlNAC078 gene is used as a key target to increase the flavonoid content in tomato fruits, thereby cultivating new functional tomato varieties with better nutritional quality.

[0129] In the second aspect, it is applied in the regulation of metabolic pathways: the SlNAC078 gene and its encoded protein are the core switches for regulating the flavonoid synthesis pathway in tomatoes, and its function is specifically reflected in regulating the expression of key genes such as SlCHS1, SlCHS2 and SlFLS.

[0130] In the third aspect, the invention relates to applications in breeding methods and products: it provides a reliable method for overexpressing the SlNAC078 gene to breed tomato varieties with high flavonoid content, and the resulting new tomato varieties or seeds that are morphologically stable and nutritionally fortified.

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A transcription factor SlNAC078, characterized in that, The CDS sequence of the transcription factor SlNAC078 gene is shown in SEQ ID NO.

1.

2. A carrier, characterized in that, It contains the transcription factor SlNAC078 as described in claim 1.

3. The carrier according to claim 2, characterized in that, The vector is the plant expression vector pCAMBIA1305-Flag.

4. A recombinant bacterial strain, characterized in that, It contains the transcription factor SlNAC078 as described in claim 1.

5. The recombinant strain according to claim 4, characterized in that, The recombinant strain was prepared from Agrobacterium GV3101.

6. The application of a transcription factor SlNAC078 in regulating the synthesis of flavonoids in tomatoes, characterized in that, The CDS sequence of the transcription factor SlNAC078 gene is shown in SEQ ID NO.

1.

7. The application according to claim 6, characterized in that, The regulation is positive regulation, which involves overexpressing the SlNAC078 gene, activating the promoters of the SlCHS1, SlCHS2 and SlFLS genes, and increasing the content of flavonoids.

8. The application of a transcription factor SlNAC078 in increasing the root dry weight of tomato plants, characterized in that, The CDS sequence of the transcription factor SlNAC078 gene is shown in SEQ ID NO.

1.

9. The application of a transcription factor SlNAC078 in increasing the single fruit weight of tomato plants, characterized in that, The CDS sequence of the transcription factor SlNAC078 gene is shown in SEQ ID NO.

1.

10. A method for cultivating tomatoes with high flavonoid content, characterized in that, Includes the following steps: The nucleotide sequence of the SlNAC078 gene was amplified, an overexpression vector of the SlNAC078 gene was constructed, Agrobacterium was transformed, and tomato explants were infected to obtain a new tomato variety.

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