Application of PS gene in regulating tomato stem and leaf color, fruit color and maturity

Abstract

This invention utilizes tomato gene tissue expression screening to obtain genes that are induced to be expressed during the ripening period of tomato fruits. PS ( Solyc10g084600 The gene encodes a protein containing a plant-specific TIGR01589 conserved domain of 57 amino acids. Furthermore, overexpression and gene editing technologies were used to... PS Overexpression and knockout of the gene coding region were performed, and genotypic and phenotypic identification were conducted on the overexpressed and mutant materials. Finally, it was determined that the gene has the biological function of negatively regulating chlorophyll accumulation and fruit ripening, and can be used for the breeding of tomato varieties with different ripening periods.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a... PS The application of genes in regulating tomato stems, leaves, fruit color, and ripening. Background Technology

[0002] The color of tomato leaves is mainly composed of two major types of pigments: chlorophyll and carotenoids. Chlorophyll, the most abundant pigment in leaves, is primarily located within chloroplasts and is responsible for absorbing light energy and driving photosynthesis, thereby synthesizing organic matter to support the growth and development of the tomato plant. To date, several gene loci closely related to tomato leaf color have been identified, including but not limited to… au , hp-2 , glk1 , slbbx20 and yg-2 Carotenoids, another important pigment in tomato leaves, although present in much lower amounts than chlorophyll, play an indispensable role in assisting photosynthesis and regulating various physiological processes under stress. Carotenoids not only enhance the efficiency of light capture and utilization by leaves but also protect plant cells from oxidative damage under adverse conditions, thereby improving the tomato's resistance to stress. The presence of these pigments further enriches the color diversity of leaves and has a potential impact on the growth and development of tomatoes.

[0003] Tomatoes contain a rich and diverse array of pigments, primarily including carotenoids, chlorophyll, and other types of pigments (such as flavonoids). Carotenoids, a broad category of pigments, encompass various components such as lycopene, β-carotene, and phytoene. Lycopene, in particular, is the main pigment responsible for the bright red color of ripe tomatoes and possesses various biological activities, including antioxidant and anti-mutagenic properties, making it crucial for enhancing the nutritional value and health benefits of tomatoes. In the early stages of fruit development until color development, chlorophyll is the dominant pigment, abundant in fruit cells and giving the fruit its green appearance. As the fruit matures, the chlorophyll content gradually decreases, while the content of other pigments, such as carotenoids, gradually increases. This process determines the final color of the tomato fruit. These pigment changes not only affect the color of the tomato fruit but are also closely related to its nutritional value, flavor, and market acceptance.

[0004] The ripening of tomato fruit is a strictly regulated biological process, typically divided into four key stages: green ripening, color breaking, red ripening, and full ripening. As the fruit ripens, its internal plastids undergo corresponding changes. Based on the different pigments they contain, plastids can be divided into chloroplasts, chromoplasts (color-containing plastids), and leucoplasts. Chloroplasts are rich in chlorophyll and are primarily responsible for photosynthesis in plants; while chromoplasts contain pigments such as carotenoids.

[0005] Tomatoes are a typical example of climacteric fruits, with ethylene playing a crucial role in the respiratory climacteric process. Ethylene, through a series of biochemical reactions, causes a sharp increase in the respiration rate within the fruit, forming a respiratory peak. Ethylene synthesis begins with methionine, which is converted to S-adenosylmethionine by SAM synthase, and then catalyzed by ACC synthase (ACS) and ACC oxidase (ACO) to finally produce ethylene. ACS and ACO are the two core enzymes in the ethylene biosynthesis pathway. Ethylene regulates the ripening process of tomato fruits, including changes in color, flavor compounds, and aroma. During the green ripening stage, ethylene release is extremely low, almost nonexistent. As the fruit ripens, ethylene release gradually increases, peaking especially during the color-breaking stage. Subsequently, as the fruit further ripens, ethylene release gradually decreases. This process demonstrates the crucial role of ethylene in regulating tomato fruit ripening.

[0006] Recent years have seen significant progress in research on genes related to tomato leaf color, fruit color, and ripening. In 2020, a team from Zhejiang University used CRISPR / Cas9 gene editing technology to construct a gene knockout... RIN The mutant of the gene exhibits significantly reduced levels of ethylene and carotenoids, and the fruit fails to fully ripen. The GLK transcription factor family includes two types: SlGLK1 and SlGLK2. Although both are expressed in leaves, only a small percentage are expressed. SlGLK2 Expressed in the fruit. GLK Overexpression of these genes can enhance the expression of photosynthetic genes and chloroplast development in fruits, leading to increased carbohydrate and carotenoid content in mature fruits. In 2021, two genes related to chloroplast development and chlorophyll synthesis were discovered— L1 , L2 Decreased expression of both genes leads to a reduction in the number of thylakoid stacks in chloroplasts, resulting in decreased chlorophyll content in leaves and fruits during the green ripening stage. These studies not only reveal the crucial role of genes in regulating tomato growth and development but also provide new ideas and methods for tomato quality improvement and breeding.

[0007] tomato( Solanum lycopersicum L As a popular vegetable crop enjoyed by consumers worldwide, tomatoes are not only a key model organism for studying fruit development, but their chloroplast development and chlorophyll synthesis play a crucial role in both leaves and fruits. Genes related to tomato chloroplast development, fruit color changes, and ripening have been reported, laying a solid foundation for a deeper understanding of the molecular mechanisms regulating tomato color and ripening. However, despite significant progress in these individual aspects, systematic research reports on the complex mechanisms by which leaf color, fruit color, and fruit ripening synergistically regulate tomato growth and development remain lacking. Summary of the Invention

[0008] In view of this, this invention verifies and identifies the regulation of tomato chloroplast development, fruit color and ripening by PS. In the future, molecular biology, cell biology and multi-omics technologies will be used to analyze the regulatory mechanism of PS in regulating chloroplast development and fruit ripening, which will broaden the theoretical research on the synergistic regulation of leaf and fruit color and fruit ripening, and provide a theoretical basis for tomato breeding.

[0009] One objective of this invention is to provide a method for regulating the color of tomato stems and leaves, fruit color, and ripeness. PS protein, PS The amino acid sequence of the protein is shown in SEQ ID NO.1.

[0010] The second objective of this invention is to provide an encoding of the above. PS protein PS Genes, the ones mentioned PS The nucleotide sequence of the gene is shown in SEQ ID NO.2. PS The cDNA sequence of the gene is shown in SEQ ID NO.3. PS The promoter sequence of the gene is shown in SEQ ID NO.4.

[0011] The third objective of this invention is to provide a product containing the above-mentioned... PS Gene recombination vectors.

[0012] The fourth objective of this invention is to provide a product containing the above-mentioned... PS The host bacteria of the gene.

[0013] The fifth objective of this invention is to provide a product containing the above-mentioned... PS Gene expression cassettes.

[0014] The sixth objective of this invention is to provide the above-mentioned PS Protein, or the above PS Application of genes, or the above-mentioned recombinant vectors, or the above-mentioned host bacteria, or the above-mentioned expression cassettes in regulating the color of tomato stems and leaves, fruit color and ripeness.

[0015] Furthermore, by knocking out PS Genes cause tomato stems and leaves to darken in color, and at the same time cause the fruit to ripen earlier.

[0016] Furthermore, through overexpression PS Genes cause tomato stems and leaves to be lighter in color, while also delaying fruit ripening.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes overexpression and gene editing technology to... PSOverexpression and knockout of the gene coding region were performed, and genotypic and phenotypic identification were conducted on the overexpressed and mutant materials. Finally, it was determined that the gene has the biological function of negatively regulating chlorophyll accumulation and fruit ripening, and can be used for the breeding of tomato varieties with different ripening periods. Attached Figure Description

[0018] Figure 1 The gene induced to be expressed during fruit ripening in Example 1 of this invention. PS PS contains the plant-specific protein family domain TIGR01589.

[0019] Figure 2 In Embodiment 1 of the present invention ps Genotypic diagram of knockout mutant (A). PS Gene expression levels in the excess line (B).

[0020] Figure 3 AC in Embodiment 1 of the present invention PS Chlorophyll content in leaves (A) and stems (B) of the excess and knockout lines.

[0021] Figure 4 AC in Embodiment 1 of the present invention PS Pigment content of fruits with excess and knockout lines; Figure A shows fruit development at different stages, Figure B shows chlorophyll content of fruits at 30 dpa, and Figure C shows carotenoid content of fruits at 36 dpa.

[0022] Figure 5 For example, in Embodiment 1 of this invention, transmission electron microscopy was used to observe wild-type AC, PS Ultrastructure of leaves (A) and chloroplasts (B) in fruits of the excess and knockout lines.

[0023] Figure 6 AC in Embodiment 1 of the present invention PS Determination of fruit firmness (A) and ethylene content (B) at different developmental stages in excess and knockout lines.

[0024] Figure 7 In Embodiment 2 of the present invention PS The expression pattern; where Figure A is PS The GUS staining results are shown in Figure B, which shows the subcellular localization of PS, placing PS in the nucleus and vacuolar membrane.

[0025] Figure 8 In Embodiment 2 of the present invention PSpro Results of the RIN transcription factor LUC assay.

[0026] Figure 9The results of the PS and GLK2 protein interaction in Example 2 of the present invention are shown in Figure A, which is the yeast two-spot-to-spot verification (Y2H) experiment, Figure B is the luciferase complementation imaging (LCI) experiment, and Figure C is the bimolecular fluorescence complementation (BiFC) experiment. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0028] Example 1 This embodiment provides PS The specific applications of genes in regulating stem and leaf color, fruit color, and ripening are as follows: This invention screened out a gene that is induced to be expressed during fruit ripening— PS The gene, PS, contains a 57-amino acid plant-specific protein family domain, TIGR01589 (also known as A_thal_3526) (see results). Figure 1 (and further functional verification and identification) PS How genes regulate tomato chloroplast development, fruit color, and ripening.

[0029] 1.1 PS Obtaining excess and mutant materials PS Obtaining excess materials: PS The CDS sequence of the gene was ligated into the vector pHELLSGATE8, and genetic transformation was performed using AC as background material. Positive detection and gene expression level assays were then performed (results are shown in [link to results]). Figure 2 B), ultimately obtaining an excess of materials. PS -OE-1、 PS -OE-2.

[0030] in, PS The gene expression levels of the excess materials were detected as follows: Extracting AC, different systems PS RNA was extracted from fruits at the 36-day period of excess material (Trizol method, kit purchased from Invitrogen). The RNA was then reverse transcribed into cDNA using a reverse transcription kit (purchased from Novizan Biotechnology Co., Ltd.). Quantitative real-time PCR (qRT-PCR) was performed using the Light tCycler 480 SYBR Green I Master Kit (Roche, http: / / www.roche.com / ) according to the kit instructions. PSThe relative expression levels and the qRT-PCR primers used in the experiment are shown in Table 1.

[0031] Table 1 Detection PS Details of qRT-PCR primers used for gene expression in supercapacitors

[0032] PS Obtained by knocking out materials: PS A dual-target primer was designed on the first exon to construct a CRISPR / Cas9 vector (pTX). Genetic transformation was performed using AC as background material, and mutant plants were subjected to positive detection and genotyping analysis (results are shown in...). Figure 2 A), ultimately obtaining the knockout material. ps-cr-9 , ps-cr-126 .

[0033] Get PS The PCR primers used for excess and mutant materials are shown in Table 2: Table 2. Detailed information on PCR primers

[0034] 1.2 Determination of chlorophyll content in tomato plants, fruits, and carotene content Take AC respectively PS For excess and mutant tissue samples, tomato tissue samples were pulverized using liquid nitrogen. 0.1 g of sample was weighed and extracted with 2 mL of 95% ethanol in the dark for 5.5 h. The sample was centrifuged at 12000 rpm for 5 min, and the residue was centrifuged to the bottom of the tube. 100 µL of the supernatant was transferred to an ELISA plate. This technique was repeated three times, using 95% ethanol as a blank. Absorbance was measured at wavelengths of 775 nm, 749 nm, and 470 nm. The chlorophyll and carotenoid content per unit sample was calculated using the formula based on the absorbance values ​​(results are shown in [link to results]). Figure 3 , 4 ).

[0035] Depend on Figure 3 , 4 It can be seen that: the chlorophyll content of the leaves shows that, compared with the wild-type AC, PS -The chlorophyll content of the OE overexpression material decreased significantly ( P <0.0001) and PS The chlorophyll content in the leaves of the knockout mutant was significantly increased. P <0.0001) (Results are shown in the table below) Figure 3 A). Results of stem epidermal chlorophyll content showed that... PS -OE overexpression lines had significantly lower stem epidermal chlorophyll content compared to wild-type AC. PS -OE-1 decreased by 44.07%. PS -OE-2 decreased by 30.34%. PS Knockout mutants show a significant increase ( ps-cr-126 Up 7.33%, ps-cr-9 An increase of 14.92% (see results) Figure 3 B). The above results collectively indicate that, PS Overexpression of the gene significantly inhibits chlorophyll accumulation in the stem and leaf tissues of tomato plants, while PS The loss of gene function promotes chlorophyll accumulation, proving that... PS The gene has a negative biological function of regulating the accumulation of chlorophyll in the stems and leaves of tomato plants.

[0036] Based on observation of fruit phenotype and measurement of chlorophyll content in fruits at 30 dpa, it was found that compared to wild-type AC plants... PS The chlorophyll accumulation in the fruits of the gene overexpression lines showed a highly significant decreasing trend. PS -OE-1 decreased by 31.78%. PS -OE-2 decreased by 14.91%); while ps The chlorophyll content of the knockout mutant fruit was significantly higher than that of the wild-type control. ps-cr-126 Up 19.04%, ps-cr-9 (Up 9.33%) (See results) Figure 4 AB). This result indicates that PS Gene expression affects the accumulation of chlorophyll in immature tomato fruits, and changes in gene expression are closely related to the early color formation of the fruit.

[0037] Based on fruit phenotypic observation and quantitative analysis of the total carotenoid content in fruits at 36 dpa, the results showed that: PS Gene overexpression lines ( PS -OE-1 and PS The carotenoid content of the fruit of the OE-2 group was below the detection limit (nd), indicating that carotenoid synthesis and accumulation had not yet begun; while ps-cr-126 and ps-cr-9 The carotenoid content in the fruits of the two knockout mutants was significantly higher than that of the wild-type AC. P <0.01), reaching 2.4 times and 2.7 times AC respectively (see results). Figure 4 C). The above results indicate that PS Genes are key negative regulators of carotenoid biosynthesis in tomato fruits, and increased expression levels of these genes inhibit the synthesis and accumulation of carotenoids.

[0038] 1.3 Transmission electron microscopy observation of tomato leaves and fruits Take AC respectively PSFor excess and mutant materials, 2 mm square slices of leaf blades avoiding the veins were taken, and 1 mm thick, 2 mm square slices of pulp tissue from 30 dpa fruits were taken. All tissues were fixed with 2% glutaraldehyde solution, and then post-processed and observed on the electron microscopy platform of Huazhong Agricultural University (results are shown in...). Figure 5 ).

[0039] Depend on Figure 5 It can be seen that in leaf tissues, compared with wild-type AC, PS In gene-overexpressing lines, the number of chloroplasts in mesophyll cells was significantly reduced, and the cross-sectional area of ​​individual chloroplasts was significantly decreased; simultaneously, the number of stacked thylakoid lamellae was greatly reduced, and the overall developmental process of chloroplasts was significantly inhibited, exhibiting phenotypic characteristics of incomplete structure and abnormal development; while PS The knockout mutant showed a significant increase in the number of chloroplasts in leaf cells, an enlarged area of ​​individual chloroplasts, denser and more numerous thylakoid membrane stacking, and superior chloroplast development compared to the control (see results). Figure 5 A). Indicates PS Genes are highly correlated with the molecular mechanisms of leaf chloroplast development.

[0040] In 30 dpa fruit, compared to wild-type AC, PS In the overexpression strain, the number of chloroplasts in the fruit cells was reduced by nearly half, the area of ​​individual chloroplasts was significantly reduced, and there was almost no stacking of thylakoid membranes inside the chloroplasts, indicating severe abnormal chloroplast development and inability to perform normal functions; ps The knockout mutant 30dpa showed a significant increase in the number of chloroplasts in fruit cells, an enlarged area of ​​individual chloroplasts, a significant increase in the number of thylakoid membrane stacks, and a more complete chloroplast development (see results). Figure 5 B).

[0041] Based on the combined observations of the ultrastructure of chloroplasts in different tissues, it can be clearly determined that... PS Genes are involved in the development and regulation of chloroplasts in tomato leaves and fruits during the green ripening stage, and changes in their expression levels directly affect the number, morphology, and formation of internal thylakoid membrane structures of chloroplasts.

[0042] 1.4 Determination of fruit firmness and ethylene content at different developmental stages of tomato materials Fruit firmness test: Select AC, PS For fruits from excess and mutant materials, the equatorial region was measured using a fruit firmness tester. An 8mm diameter cylindrical probe was used, pressing down 5mm onto the equatorial surface of the fruit at a testing speed of 1mm / s. The maximum force applied to the probe was recorded as the fruit firmness (unit: N). Three uniformly sized, undamaged fruits were selected from each plant, and each fruit was measured three times. The average value was taken (see results below). Figure 6 A).

[0043] Determination of ethylene content in fruit: This experiment used gas chromatography to measure AC, PS To investigate ethylene release during fruit ripening in both excess and mutant materials, fruits of uniform size, free from pests, diseases, and mechanical damage were selected based on their flowering and labeling time. Fruits at different stages of ripening were harvested and brought back to the laboratory, where they were left to stand for one day to exclude ethylene release induced by harvesting damage. Three fruits were placed in a sealed container and sealed for 5 hours. Ethylene release was detected by gas chromatography (GC), with an injection volume of 1 mL and three replicates per sample group. The ethylene release was calculated using a standard curve (results are shown in [link to results]). Figure 6 B).

[0044] Fruit firmness testing results showed that, with wild-type AC as the control, the differences in softening and ripening of fruits from different genotypes at 36 dpa were already quite significant. PS The fruit firmness of the excess strain was significantly higher than that of AC, yet it still maintained typical firm fruit characteristics; while ps The fruit firmness of the knockout mutant was significantly lower than that of AC, and the softening process had begun. Subsequently, PS The fruit firmness of the super-yield strain remained at the highest level, significantly higher than that of AC at 42 dpa, 44 dpa and 48 dpa, with the lowest degree of fruit softening and the slowest ripening process. ps The fruit firmness of the knockout mutant was significantly lower than that of AC, and the softening rate was significantly faster than that of the control, with the most pronounced ripening and softening process (see results). Figure 6 A). The results show that, PS Genes play a negative regulatory role in the softening process of tomato fruit.

[0045] The results of ethylene release measurements showed that the ethylene release rate of wild-type AC tomatoes showed a significant upward trend at 36 dpa (approximately the color breaking stage), reached a peak at 44 dpa, and then showed a sharp downward trend, which is consistent with the ethylene release pattern of climacteric fruits. PS The peak ethylene release from the excess fruit was significantly lower than that of the wild-type AC, while ps The peak ethylene release from the knockout mutant fruit was significantly higher than that of the wild-type AC (see results). Figure 6 B). This result indicates that... PS Increased gene expression levels can also negatively regulate the ethylene release process in tomato fruits.

[0046] Example 2 Further exploration in this embodiment PS The mechanisms by which genes regulate tomato chloroplast development, fruit color, and ripening are as follows: 2.1 PS Gene expression pattern analysis Amplification PSA fragment of the gene promoter region (as shown in SEQ ID NO:4) was ligated into the pMV2-GUS vector to construct... PS The pro::GUS vector was used to genetically transform tomatoes, resulting in transgenic plants. PS Stems, leaves, flowers, and fruits of pro::GUS transgenic plants at the seedling and mature stages were stained with GUS for 24 hours and then washed with ethanol for 48 hours. The degree of tissue staining reflects the staining intensity. PS The degree of expression (see results) Figure 7 A).

[0047] Depend on Figure 7 A indicates that staining results show that GUS activity in tomato seedlings is mainly concentrated in leaf and stem tissues. Further microscopic observation of stem cross-sections revealed that GUS signals are primarily concentrated in epidermal cells, cortical cells, and vascular bundles. During fruit development, significant and stable GUS signals were observed in the pulp tissue. In summary, the above experimental results indicate that… PS The gene exhibits a significant tissue-specific expression pattern, mainly expressed in vegetative organs such as leaves and stems, as well as fruit tissues.

[0048] 2.2 PS Subcellular localization analysis The amplification does not contain a stop codon. PS The CDS sequence was ligated into the 101YFP vector. The recombinant plasmid was transformed into Agrobacterium GV3101 competent cells using liquid nitrogen. Agrobacterium bacterial suspensions labeled with nuclear and vacuolar membrane proteins, along with 35S::PS-YFP Agrobacterium bacterial suspensions, were co-injected into tobacco leaves. After 40 hours of growth in low-light conditions, lower epidermal cells were harvested, and fluorescence signals were observed under a laser confocal microscope. (Results are shown in...) Figure 7 B).

[0049] Depend on Figure 7 B indicates that the detection results show that the fluorescence signal of the empty YFP carrier is diffusely distributed in the cytoplasm and nucleus, and the green fluorescence signal of the PS-YFP fusion protein completely overlaps with the red fluorescence signals of the vacuolar membrane marker protein CBL6-RFP and the nuclear marker protein EFR-RFP, exhibiting good co-localization characteristics. These results confirm that the PS protein is mainly located in the vacuolar membrane and nucleus.

[0050] 2.3 PS Promoter and RIN dual-luciferase assay Will RIN ( Solyc05g012020 The gene CDS was inserted into the pGreenII62-SK vector. PSThe upstream 3000 bp promoter sequence (as shown in SEQ ID NO:4) was divided into three segments: the first segment (pro1) was from 0 bp to 1075 bp; the second segment (pro2) was from 1002 bp to 2235 bp; and the third segment (pro3) was from 2170 bp to 3000 bp. These three promoter segments were inserted into pGreenII 0800-LUC and transformed into Agrobacterium tumefaciens (GV3101) with the pSoup helper plasmid. Tobacco leaves were infected with Agrobacterium strain, and manipulation was performed three days later. The activities of firefly (LUC) and Renilla luciferase (RLU) were quantified using a dual-luciferase reporter assay system, and the LUC / RLU ratio was calculated (results are shown in [link to results]). Figure 8 ).

[0051] Depend on Figure 8 It can be seen that the luciferase reporter gene assay indicates that the RIN transcription factor can bind to... PS The RIN transcription factor inhibits the first and third regions of the gene promoter and suppresses its promoter activity. However, the luciferase activity in the RIN-62SK and pro2-PS-0800 co-injection group showed no significant difference compared to the control, indicating that the RIN transcription factor does not bind to the gene promoter. PS The second region of the promoter also does not have a regulatory effect on it.

[0052] 2.4 PS Interacting with GLK2 protein Amplification PS and GLK2 Gene( Solyc10g008160 The CDS of the constructed AD and BD vectors were ligated into PGADT7 and PGBKT7 vectors, respectively, to serve as AD and BD vectors. The plastids of the constructed AD and BD vectors were mixed and transformed into AH109 strain using a small-scale transfection method, and then plated on SD / -trp-leu plates. After yeast growth, a small amount of bacterial plaque was scraped off, resuspended in ddH2O, and then spotted onto SD / -trp-leu and SD / -trp-leu-his-ade plates. After incubation at 30℃ for 48 h, the yeast growth on both media was observed to verify the interaction between the two proteins.

[0053] target protein PS and GLK2 Construct bimolecular fluorescent complementary supports 35S:: PS -NE、35S:: GLK2 -CE was transferred into Agrobacterium GV3101 strain and injected into leaves of Tobacco Benzovia seedlings approximately one month old. After about 48 hours, fluorescence was observed using a confocal fluorescence microscope to verify the protein-protein interaction.

[0054] target protein PS and GLK2 Construct complementary vectors for luciferase fragments, 35S:: PS -LUC-N、35S:: PS -LUC-C、35S:: GLK2 -LUC-N、35S:: GLK2 -LUC-C was transferred into Agrobacterium GV3101 strain and injected into leaves of approximately one-month-old Nicotiana benthamiana seedlings. Approximately 48 hours later, fluorescence was observed using a plant in vivo imaging system to verify protein-protein interactions (results are shown in...). Figure 9 ).

[0055] Depend on Figure 9 The results of the Y2H point-to-point experiment show that the yeast strains co-transformed with AD-PS and BD-GLK2 can survive and proliferate normally on a four-deficient medium. Preliminary experimental support was provided for the interaction between PS and GLK2 in the in vitro yeast system (see results). Figure 9 A).

[0056] Fluorescence detection results showed that strong fluorescence signals were detected in both experimental groups co-expressing PS-nLUC with GLK1-cLUC and GLK2-cLUC, while no fluorescence signals were produced in any control group. This indicates that PS protein can interact with GLK1 and GLK2 proteins in plants (see results). Figure 9 B).

[0057] BiFC experiments, using confocal microscopy at an excitation wavelength of 512 nm, detected significantly specific YFP fluorescence signals for PS-cYFP+GLK1-nYFP and PS-cYFP+GLK2-nYFP, while no fluorescence signals were observed in any control combinations. This further demonstrates the interaction between PS protein and GLK1 and GLK2 proteins in plants (see results). Figure 9 C).

[0058] In summary, the results of the three experimental methods, Y2H, BiFC, and LCI, corroborate each other, preliminarily demonstrating that PS protein and GLK2 protein have a stable interaction in both in vitro yeast systems and in plants.

[0059] Based on the above series of investigations, this invention has verified the role of PS in regulating tomato chloroplast development, fruit color, and ripening through functional verification. Further research will utilize molecular biology, cell biology, and multi-omics technologies to elucidate the regulatory mechanisms of PS in regulating chloroplast development and fruit ripening, thus broadening the theoretical research on the synergistic regulation of leaf and fruit color and fruit ripening, and providing a theoretical basis for tomato breeding.

[0060] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0061] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for regulating the color of tomato stems and leaves, fruit color, and ripeness. PS Protein, characterized by, PS The amino acid sequence of the protein is shown in SEQ ID NO.

1.

2. An encoding method as described in claim 1 PS protein PS Genes, characterized by, The PS The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. Containing the contents of claim 2 PS Gene recombination vectors.

4. Containing the contents of claim 2 PS The host bacteria of the gene.

5. Containing the contents of claim 2 PS Gene expression cassettes.

6. The claim 1 PS Protein, or as described in claim 2 PS The application of the gene, or the recombinant vector of claim 3, or the host bacterium of claim 4, or the expression cassette of claim 5 in regulating the color of tomato stems and leaves, fruit color and ripeness.

7. The application according to claim 6, characterized in that, By knocking PS Genes cause tomato stems and leaves to darken in color, and at the same time cause the fruit to ripen earlier.

8. The application according to claim 6, characterized in that, Through overexpression PS Genes cause tomato stems and leaves to be lighter in color, while also delaying fruit ripening.

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