Application of slpml1 protein and coding gene in improving low night temperature resistance of plants

Abstract

The application discloses a tomato SlPML1 protein and application of an encoding gene thereof in improving plant tolerance to low night temperature stress. An amino acid sequence of the SlPML1 protein is shown as SEQ ID NO:1, and a nucleotide sequence of the encoding gene is shown as SEQ ID NO:2. The application first separates and identifies a UPF0016 family member SlPML1 from the tomato, proves that the SlPML1 is located in a chloroplast thylakoid membrane and has a calcium ion transport function. Low night temperature stress can significantly induce accumulation of the SlPML1 protein, overexpression of the SlPML1 can significantly reduce an electrolyte permeability and a malondialdehyde content of plant leaves, reduce active oxygen accumulation, improve photosynthetic capacity (including Fv / Fm, Pn, Y(I), Y(II) and the like) and an oxygen release rate, thereby enhancing the tolerance of the plant to the low night temperature stress. The application provides an important gene resource and an application method for cultivating the tomato and other solanaceous crops with low night temperature resistance.

Description

Technical Field

[0001] This invention belongs to the fields of plant genetic engineering and molecular biology, specifically relating to the application of a tomato SlPML1 protein and its encoding gene in improving plant tolerance to low night temperature stress. Background Technology

[0002] Tomato (Solanum lycopersicum) is an important economic crop widely cultivated globally. In winter and spring greenhouse cultivation in northern my country, nighttime temperatures often drop below the optimal temperature for tomato growth and development, causing frequent low night temperature stress. Low night temperature stress inhibits tomato photosynthetic efficiency, damages thylakoid membrane structure, and induces reactive oxygen species bursts, thus severely restricting tomato yield and quality.

[0003] Photosynthesis is the most important energy source for plants, and its normal operation depends on calcium ions (Ca) in the thylakoid lumen. 2+ ) and manganese ions (Mn 2+ The steady-state maintenance of plasma is crucial. Calcium ions, as an essential cofactor for the structural stability of the oxygen-evolving complex (OEC), directly participate in the assembly and repair of the PSII. However, our understanding of the calcium ion transport mechanism within the thylakoid lumen of plants remains very limited.

[0004] UPF0016 (Uncharacterized Protein Family 0016) is an evolutionarily conserved family of cation transporters found in both prokaryotes and eukaryotes. In Arabidopsis thaliana, the UPF0016 family has five members, among which AtPAM71 / CCHA1 / BICAT1 has been confirmed to be located on the thylakoid membrane of chloroplasts and possesses Ca2+. 2+ / H + The UPF0016 family plays a crucial role in antitransportation, maintaining calcium and manganese ion homeostasis within chloroplasts. Mutations in this family lead to decreased PSII activity, abnormal chloroplast structure, and stunted plant growth. Other UPF0016 family members in Arabidopsis, such as BICAT2 / CMT1, are located in the inner chloroplast membrane, while PML3, PML4, and PML5 are located in the Golgi apparatus and endoplasmic reticulum, respectively, participating in ion transport and cell wall synthesis. However, the composition, structural characteristics, tissue expression patterns, and functions of the UPF0016 family in tomato under abiotic stresses (especially low night temperature stress) have not been systematically studied.

[0005] While the functions of UPF0016 family members in model plants such as Arabidopsis thaliana have been reported in the prior art, functional differentiation of these family members may occur in different species. As an important warm-season horticultural crop, the location of UPF0016 family members in tomatoes—whether they are located in the thylakoid membrane, possess calcium ion transport functions, or participate in responses to low night temperature stress—has not been reported. Furthermore, the molecular link between chloroplast calcium ion homeostasis and photosynthesis under low night temperature stress remains unclear, and there is a lack of effective gene resources and molecular markers that can be used to improve the resistance of tomatoes to low night temperatures.

[0006] Therefore, there is an urgent need in this field to identify and isolate members of the UPF0016 family from tomatoes, and to analyze their functions in the response to low night temperature stress, so as to provide a theoretical basis and genetic resources for the genetic improvement of greenhouse tomato varieties tolerant to low night temperatures. This invention is based on this need. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention discloses the application of the tomato SlPML1 protein and its encoding gene in improving plant tolerance to low night temperature stress.

[0008] This invention includes the following specific technical solutions:

[0009] An isolated SlPML1 protein, the amino acid sequence of which is shown in SEQ ID NO:1.

[0010] This invention marks the first isolation and identification of SlPML1, a member of the UPF0016 family, from tomato, and confirms its location on the thylakoid membrane of chloroplasts, where it possesses calcium ion transport function. This protein consists of 362 amino acids, has a molecular weight of approximately 38.4 kDa, an isoelectric point of 5.59, and is a hydrophobic protein. Under low night temperature stress, this protein is significantly induced to accumulate, enhancing plant tolerance to low night temperature stress by maintaining the integrity of chloroplast ultrastructure, protecting the abundance of photosynthetic protein complexes (especially PsbP, PsbQ, and PsbO), and promoting the activity of the oxygen-evolving complex.

[0011] Furthermore, the present invention also provides a nucleic acid molecule encoding the above-mentioned SlPML1 protein, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0012] Furthermore, the present invention also provides a recombinant expression vector comprising the above-mentioned nucleic acid molecules.

[0013] Furthermore, the present invention also provides a host cell comprising the above-mentioned nucleic acid molecules or recombinant expression vectors. The host cell may be Agrobacterium cells, Escherichia coli cells, or plant cells.

[0014] In some embodiments, it has been demonstrated that the SlPML1 protein and its encoding gene described in this invention can enhance plant tolerance to low night temperature stress through the following mechanisms:

[0015] It significantly improves the net photosynthetic rate and PSII maximum photochemical efficiency (Fv / Fm) of plants under low night temperature stress.

[0016] Effectively maintain the effective photochemical efficiencies Y(I), Y(II) and relative electron transport rates ETR(I), ETR(II) of PSI and PSII under low night temperature stress;

[0017] Maintaining the integrity of the thylakoid membrane structure of chloroplasts and the normal stacking of grana lamellae, preventing chloroplast swelling and disintegration;

[0018] To increase the oxygen release rate of plants under low night temperature stress and protect the activity of the oxygen-evolving complex (OEC);

[0019] It reduces leaf electrolyte permeability and malondialdehyde (MDA) content, and increases leaf relative water content;

[0020] Reduce superoxide anions (O2) - The production rate and hydrogen peroxide (H2O2) content are controlled to suppress reactive oxygen species (ROS) bursts.

[0021] It interacts with chloroplast photosynthetic proteins SlPsbP, SlPsbQ, and SlPsbO, maintaining the stability of these proteins under low night temperature stress.

[0022] Furthermore, this invention also discloses the application of the gene encoding the SlPML1 protein in improving plant tolerance to low night temperature stress, the application comprising overexpressing the nucleic acid molecule in plants by the following method:

[0023] (a) Construction of recombinant expression vector: The nucleic acid molecule shown in SEQ ID NO:2 is operatively linked downstream of a promoter (e.g., CaMV 35S promoter) to construct a plant expression vector;

[0024] (b) Genetic transformation: The recombinant expression vector obtained in step (a) was introduced into Agrobacterium and plant explants were transformed using Agrobacterium-mediated transformation.

[0025] (c) Regeneration and selection: Transformed explants are induced to form callus and differentiate into seedlings on a medium containing selection markers (e.g., kanamycin) to obtain transgenic plants;

[0026] (d) Identification: SlPML1 overexpressing positive plants were screened by PCR, qRT-PCR and / or immunoblotting.

[0027] Furthermore, in the above application, the plant described in step (b) is a Solanaceae plant, preferably tomato (Solanum lycopersicum).

[0028] Furthermore, in the above applications, the method for overexpressing SlPML1 may also include integrating the SlPML1 gene into the plant genome via CRISPR / Cas9-mediated gene knock-in, or overexpressing it through a transient expression system.

[0029] More preferably, the SlPML1 overexpressing plant of the present invention can be obtained by the following preferred method, which is simple to operate, genetically stable, and can effectively improve the expression level of SlPML1 protein:

[0030] (1) Amplification of the target gene: Using leaf cDNA of tomato variety 'Ailsa Craig' as a template, the full-length coding sequence of SlPML1 (SEQ ID NO:2) was amplified using specific primers, and the PCR product was recovered and purified.

[0031] (2) Expression vector construction: The SlPML1 coding sequence was inserted downstream of the CaMV 35S promoter of the pCAMBIA1300-GFP vector via homologous recombination or enzyme digestion ligation to obtain the p35S:SlPML1-GFP recombinant plasmid. Sequencing verification was performed to ensure the correct reading frame.

[0032] (3) Agrobacterium transformation: Agrobacterium GV3101 strain was transformed by electroporation or freeze-thaw method with recombinant plasmid, and positive clones were identified by PCR.

[0033] (4) Tomato genetic transformation: Agrobacterium-mediated cotyledon transformation was used. Tomato 'Ailsa Craig' seeds were sterilized and sown on MS medium. Seven-day-old cotyledon segments were used as explants and infected with Agrobacterium tumefaciens solution with OD600=0.8 for 10 minutes. After co-culturing for 2 days, they were transferred to selection medium containing 50 mg / L kanamycin and 200 mg / L termethin to induce callus and shoot differentiation.

[0034] (5) Plant regeneration and screening: When the resistant shoots reach 2-3 cm in length, they are cut off and transferred to rooting medium. After rooting, the plants are hardened off and then transplanted to a greenhouse. DNA is extracted from the leaves for PCR positive identification, RNA is extracted for qRT-PCR to detect expression levels, and total protein is extracted for immunoblotting analysis to confirm the abundance of SlPML1 protein.

[0035] (6) Homozygous lines were obtained: T1 generation seeds were screened for kanamycin to obtain homozygous overexpression lines for low night temperature resistance function analysis.

[0036] The present invention also discloses the application of the above-mentioned SlPML1 protein or its encoding gene in the preparation of formulations or kits for improving plant tolerance to low night temperature stress.

[0037] This invention also discloses a method for improving plant tolerance to low night temperature stress, characterized by overexpressing the nucleic acid molecule shown in SEQ ID NO:2 in the plant. The plant exhibits enhanced photosynthetic capacity, reduced reactive oxygen species levels, and improved chloroplast structure under low night temperature stress.

[0038] This invention also discloses a method for cultivating transgenic plants tolerant to low night temperatures. The method includes operably linking the nucleic acid molecule shown in SEQ ID NO:2 to a promoter and then transferring it into the plant genome, followed by screening to obtain transgenic plants that, compared to the wild type, exhibit reduced electrolyte osmotic pressure, reduced malondialdehyde content, increased relative water content, improved photosynthetic capacity, and / or reduced reactive oxygen species accumulation under low night temperature stress. Preferably, the plant is a tomato.

[0039] To make the technical solution of the present invention easier to understand, the amino acid sequence of the SlPML1 protein is as follows:

[0040] MRSLVLSCESSSFLFKPSFALSNFSPVNPSLTAISRFSRRHSLCKQHGWSEDFPKVVRTRNYKRCHMTRSAYCDEECSITNKTALSKRKNQSTSVLQVLDMPENNYLKSIVLSGLFTLLFTQQASAASEVATGLQSFPFFGDLGDLSTGFASAFLLIFFSELGDKTFFIAALLAARNSAVVTF LGTFGALGVMTIISVVLGRTFHYVDDVLPFRLGGNDLPVDDIAAVCLLVYFGVSTLLDASSSDGMKAEEEQKEAELAVSEFSGNGAGLLSAASTIVSTFALVFVAEWGDKSFFSTIALAAASSPLGVIGGALAGHGAATLLAVLGGSLLGTFLSEKVIAYIGGALFLVFAAVTVIEIVS (SEQ ID NO:1).

[0041] The nucleotide sequence of the SlPML1 encoding gene is as follows:

[0042]

[0043] Compared with the prior art, the present invention has the following outstanding advantages:

[0044] 1. Providing a novel low-temperature resistance gene resource: This invention is the first to isolate and identify SlPML1, a member of the UPF0016 family, from tomato, and confirms that it is induced to express under low-temperature stress, positively regulating plant resistance to low-temperature stress by maintaining chloroplast structure and photosynthetic function. Although there are reports on genome identification and expression analysis of the tomato UPF0016 family in the prior art, the functional characterization of SlPML1 has not been performed. This invention fills this gap.

[0045] 2. Clarified Mechanism of Action: This invention reveals for the first time that SlPML1 is located in the thylakoid membrane of chloroplasts and possesses Ca 2+ This transport mechanism allows the calcium ion to interact with the oxygen-evolving complex subunits PsbP, PsbQ, and PsbO, maintaining the stability of these proteins under low nighttime temperature stress and thus protecting the photosynthetic system. This mechanism has never been reported before and provides new insights into the relationship between calcium ion transport and photosynthesis in plants.

[0046] 3. Sufficient Functional Validation: This invention systematically validated the function of SlPML1 under low nighttime temperature stress using CRISPR / Cas9 knockout and overexpression transgenic plants, including physiological indicators (electrolyte osmotic pressure, MDA, relative water content) and reactive oxygen species levels (DAB / NBT staining, O2). - The data are complete and reliable, including the production rate, H2O2 content, photosynthetic parameters (Fv / Fm, Pn, Y(I), Y(II), ETR(I), ETR(II)), chloroplast ultrastructure (transmission electron microscopy), and oxygen release rate.

[0047] 4. Clear application value: The SlPML1 gene provided by this invention can be used for the genetic improvement of low-temperature tolerance in Solanaceae plants (such as tomatoes, peppers, and eggplants). Through transgenic or molecular marker-assisted selection breeding, it can improve the tolerance of greenhouse-grown crops to low night temperature stress and provide new germplasm resources for greenhouse agricultural production in winter and spring.

[0048] 5. Expanding the research on photosynthetic regulation: This invention extends the function of the UPF0016 family from the model plant Arabidopsis thaliana to the important economic crop tomato, and for the first time directly links it to the response to low night temperature stress, providing a new molecular target for the study of plant calcium ion signaling and stress adaptation. Attached Figure Description

[0049] Figure 1 This is a diagram showing the subcellular localization results of SlPML1 in Example 5 of the present invention, wherein... Figure 1A is a transient colocalization image of SlPML1-GFP expression in tobacco leaves. The GFP channel shows green fluorescence, and the Chlorophyll channel shows auto-red fluorescence of chloroplasts. Merge is an overlay image with a scale bar of 50 μm. Figure 1 B shows the results of immunoblotting analysis of various subcellular components of tomato leaves, detecting the distribution of SlPML1 protein in total protein, nucleus, cytoplasm, chloroplast, and thylakoid membrane, with Actin (cytoplasmic marker), H3 (nuclear marker), and OEC33 (thylakoid membrane marker) as controls;

[0050] Figure 2 This is a graph showing the analysis results of the calcium ion transport capacity of SlPML1 in Example 6 of the present invention, wherein... Figure 2 A shows the growth of yeast strain K667 expressing the empty vector (EV) or SlPML1 on YPD medium containing 0 or 150 mmol / L CaCl2. Figure 2 B represents the growth curve of the above yeast strain in high-calcium liquid culture medium; Figure 2 C represents the result of intracellular calcium ion content determination in yeast strains (**p<0.01).

[0051] Figure 3 This is a diagram showing the identification results of SlPML1 knockout and overexpression plants in Example 4 of the present invention. Figure 3 A represents the sequencing results of target site editing in SlPML1 knockout plants, where slpml1-1 is a 2 bp deletion and slpml1-2 is a 1 bp insertion. Figure 3 B represents the qRT-PCR identification results of SlPML1 overexpressing plants (p<0.01, *p<0.001). Figure 3 C represents the immunoblotting results of SlPML1 overexpressing plants;

[0052] Figure 4 This is a diagram showing the phenotypic analysis results of SlPML1 knockout and overexpression plants in Example 7 of the present invention. Figure 4 A shows phenotypic photographs of wild-type (WT), knockout (slpml1-1, slpml1-2), and overexpression (SlPML1-OE-1, SlPML1-OE-2) plants, with a scale bar of 10 cm. Figure 4 B represents the statistical results of plant height; Figure 4 C represents the statistical result of stem diameter; Figure 4 D represents the leaf area statistics; Figure 4 E represents the fresh weight statistical result; Figure 4F represents the statistical results of relative chlorophyll content (SPAD value) (p<0.05, p<0.01, p<0.001, ****p<0.0001).

[0053] Figure 5 This is an immunoblot analysis result of SlPML1 protein accumulation under low night temperature stress in Example 8 of the present invention. The changes in SlPML1 protein abundance were detected in wild-type plants after low night temperature treatment for 0, 0.5, 1, 2, 4 and 12 h.

[0054] Figure 6 This is a graph showing the phenotypic and physiological results of the effect of SlPML1 on low night temperature resistance in Example 9 of the present invention. Figure 6 A shows phenotypic images of wild-type, knockout, and overexpression plants after 7 days of low nighttime temperature treatment, with a scale bar of 10 cm. Figure 6 B represents the statistical results of electrolyte permeability (REL); Figure 6 C represents the statistical results of malondialdehyde (MDA) content; Figure 6 D represents the statistical results of relative water content (RWC) (p<0.05, p<0.01, p<0.001, ****p<0.0001);

[0055] Figure 7 This is a graph showing the effect of SlPML1 on reactive oxygen species content under low nighttime temperature stress in Example 10 of the present invention. Figure 7 A represents DAB staining (for detecting H2O2) and NBT staining (for detecting O2). - )result; Figure 7 B is superoxide anion (O2) - Generate rate statistics; Figure 7 C represents the statistical results of hydrogen peroxide (H2O2) content (p<0.05, p<0.01, p<0.001, ****p<0.0001); Figure 7 D represents the H2DCFDA reactive oxygen species fluorescence staining result, with a scale bar of 50 μm;

[0056] Figure 8 This is a graph showing the effect of SlPML1 on photosynthetic capacity under low night temperature stress in Example 11 of the present invention. Figure 8 A represents the statistical results of the maximum photochemical efficiency (Fv / Fm) of PSII; Figure 8 B represents the statistical results of net photosynthetic rate (Pn); Figure 8 C represents the statistical result of the effective photochemical efficiency Y(I) of PSI; Figure 8 D represents the statistical result of the PSI relative electron transport rate ETR(I); Figure 8 E represents the statistical result of the effective photochemical efficiency Y(II) of PSII; Figure 8F represents the statistical results of the relative electron transport rate ETR(II) of PSII (p<0.05, p<0.01, p<0.001, ****p<0.0001).

[0057] Figure 9 The image shows the transmission electron microscopy results of chloroplast ultrastructure under low night temperature stress in Example 12 of this invention. The image shows the chloroplast structure of wild-type, SlPML1 knockout and overexpression plants under normal conditions and after low night temperature treatment, respectively. The scale bar is 0.5 μm.

[0058] Figure 10 This is a diagram showing the results of the SlPML1-chloroplast protein interaction analysis in Example 13 of the present invention. Figure 10 A represents the result of a yeast two-hybrid (Y2H) experiment; Figure 10 B represents the luciferase complementation (LCI) result; Figure 10 C represents the bimolecular fluorescence complementation (BiFC) results, with a scale bar of 50 μm;

[0059] Figure 11 This is a graph showing the effect of calcium ions on the interaction strength between SlPML1 and chloroplast proteins in Example 13 of the present invention. Figure 11 A is for adding Ca 2+ The results of the yeast two-hybrid experiment; Figure 11 B represents the result of the pull-down test; Figure 11 C is for adding Ca 2+ The results of subsequent LCI fluorescence intensity quantification;

[0060] Figure 12 This is a graph showing the immunoblotting results of the abundance of SlPsbP, SlPsbQ, and SlPsbO proteins in each strain under low night temperature stress in Example 13 of the present invention.

[0061] Figure 13 The figure shows the effect of SlPML1 on oxygen release rate under low night temperature stress in Example 14 of the present invention (p<0.05, p<0.01, p<0.001, ****p<0.0001).

[0062] Figure 14 This is a diagram showing the phenotypic results of SlPML1 knockout plants recombined with exogenous sucrose in Example 15 of this invention. Figure 14 A shows phenotypic photographs of wild-type and knockout plants after treatment with exogenous sucrose, with a scale bar of 10 cm; Figure 14 B represents the statistical results of plant height; Figure 14 C represents the statistical result of stem diameter; Figure 14 D represents the leaf area statistics; Figure 14 E represents the fresh weight statistical result; Figure 14F represents the statistical results of chlorophyll content (different letters indicate p<0.05);

[0063] Figure 15 This is a graph showing the effect of exogenous sucrose on the low night temperature resistance of SlPML1 knockout plants in Example 15 of the present invention. Figure 15 A is a phenotypic photograph after low night temperature stress, with a scale bar of 10 cm; Figure 15 B represents the statistical results of electrolyte permeability (REL); Figure 15 C represents the statistical results of malondialdehyde (MDA) content; Figure 15 D represents the statistical results of relative water content (RWC) (different letters indicate p<0.05);

[0064] Figure 16 This is a qRT-PCR result of the relative expression level of SlPML1 in different tomato tissues in test example 2 of the present invention. The data are expressed as mean ± SD, n=3;

[0065] Figure 17 This is an immunoblot analysis result (non-reducing SDS-PAGE) of the redox state of SlPML1 under different concentrations of DTT treatment in Test Example 3 of the present invention. Detailed Implementation

[0066] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0067] Unless otherwise specified, all materials, reagents, and instruments used in the embodiments of this invention are commercially available. Experimental methods without specific conditions are generally performed under standard conditions or as recommended by the reagent manufacturer.

[0068] The main primer sequences used in this embodiment are shown in Table 1.

[0069] Table 1 Primer sequences used in this invention

[0070] Example 1

[0071] Cloning and sequence analysis of the tomato SlPML1 gene.

[0072] 1.1 Plant materials

[0073] Using the tomato (Solanum lycopersicum) variety 'Ailsa Craig' as the experimental material, seeds were sown in an artificial climate chamber under the following conditions: daytime temperature 28℃, nighttime temperature 18℃, and light intensity 600 μmol·m⁻¹. -2 ·s -1 The photoperiod was 16 hours of light / 8 hours of darkness. Tender leaves from seedlings at the six-leaf-one-heart stage were used for RNA extraction.

[0074] 1.2 RNA extraction and cDNA synthesis

[0075] Total RNA was extracted from tomato leaves using a plant RNA extraction kit (CWBIO, China). The specific steps were as follows: Approximately 100 mg of fresh leaves were ground into powder using liquid nitrogen. 1 mL of TRlzon Reagent was added, and the mixture was thoroughly vortexed and allowed to stand at room temperature for 5 min. 200 μL of chloroform was added, and the mixture was vigorously vortexed for 15 s and allowed to stand at room temperature for 2 min. The mixture was then centrifuged at 12,000 rpm at 4°C for 10 min. The supernatant was transferred to a centrifuge tube containing an equal volume of 70% ethanol, mixed, and transferred to an adsorption column. The column was centrifuged at 12,000 rpm for 1 min. The leaves were washed sequentially with 700 μL of RW1 and 500 μL of RW2 (twice). Finally, RNA was eluted with 50 μL of RNase-Free Water. RNA concentration and purity (A260 / A280 ratio 1.8–2.0) were determined using a NanoDrop One spectrophotometer.

[0076] cDNA was synthesized using 1 μg of total RNA as a template via the Evo M-MLV reverse transcription kit (Accurate Biology, China). The reaction mixture consisted of 1 μg total RNA, 4 μL of 5×RT Master Mix, and RNase-free dH2O to a final volume of 20 μL. The reaction program was 37℃ for 15 min followed by 85℃ for 5 s. The resulting cDNA was stored at -20℃ for later use.

[0077] 1.3 SlPML1 gene amplification

[0078] Based on the CDS sequence of the SlPML1 gene (Solyc08g081770) in the Tomato Genome Database (ITAG version 4.0), specific amplification primers were designed (sequences shown in Table 1). Using tomato leaf cDNA as a template, PCR amplification was performed using PrimeSTAR HS high-fidelity enzyme (TaKaRa, Japan). The reaction mixture consisted of 25 μL of 2×PrimeSTAR HS Premix, 2 μL each of forward and reverse primers (10 μM), 50 ng of cDNA, and ddH2O to a final volume of 50 μL. The reaction program was as follows: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 30 cycles; and a final extension at 72℃ for 10 min.

[0079] The PCR product was detected by 1% agarose gel electrophoresis, and a specific band appeared at approximately 1100 bp. The target fragment was recovered, ligated into the pMD19-T vector, transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing ampicillin (100 μg / mL), and incubated overnight at 37°C. Positive clones were picked for sequencing verification. Sequencing results showed that the cloned SlPML1 gene CDS length was 1089 bp (including the stop codon), encoding 362 amino acids. The nucleotide sequence is shown in SEQ ID NO:2, and the amino acid sequence is shown in SEQ ID NO:1.

[0080] 1.4 Bioinformatics Analysis

[0081] Physicochemical properties of SlPML1 protein were analyzed using the ExPASY ProtParam online tool: molecular weight approximately 38.4 kDa, isoelectric point pI 5.59, instability index 43.59, and hydrophilicity coefficient 0.51, indicating it is a hydrophobic protein. SignalP 4.1 prediction showed that SlPML1 has no signal peptide. Protein structure prediction using AlphaFold2 showed that SlPML1 contains a typical UPF0016 family conserved domain, including the ExGD-(KR)-(TS) motif.

[0082] Example 2

[0083] Construction of SlPML1 overexpression plant expression vector.

[0084] 2.1 Vector digestion

[0085] The pCAMBIA1300-GFP vector plasmid was double-digested with restriction endonucleases BamHI and SalI. The reaction mixture consisted of 5 μL 10× Green Buffer, 1 μL BamHI, 1 μL SalI, 500 ng of the vector plasmid, and ddH2O to a final volume of 50 μL. Digestion was carried out at 37℃ for 1 h. The digestion products were identified by 1% agarose gel electrophoresis, and the linearized vector was recovered.

[0086] 2.2 Homologous recombination linkage

[0087] The target fragment was ligated to the linearized vector using a seamless cloning kit (Biosharp, China). The reaction mixture consisted of 5 μL of 2×Seamless Cloning Mix, 3 μL of enzyme digestion product, 1 μL of purified SlPML1 PCR product, and ddH2O to a final volume of 10 μL. The reaction was carried out at 50°C for 1 h.

[0088] 2.3 Transformation and Identification

[0089] The ligation product was transformed into TOP10 competent E. coli cells and plated on LB agar plates containing kanamycin (50 μg / mL). Single colonies were picked for colony PCR identification (using primers in Table 1), and positive clones were sent for sequencing verification. The recombinant plasmid with correct sequencing was named p35S:SlPML1-GFP and used for subsequent Agrobacterium transformation.

[0090] Example 3

[0091] Construction of SlPML1 gene knockout vector

[0092] A SlPML1 gene knockout vector was constructed using CRISPR / Cas9 technology. Based on the first exon sequence of the SlPML1 gene, an sgRNA sequence targeting the editing site was designed: 5'-GGTCTGAGGACTTCCCCAAG-3' (Table 1). The sgRNA sequence was cloned into the pCAMBIA1300-Cas9 vector (containing a Cas9 expression cassette) to obtain the pCas9-SlPML1 recombinant plasmid. Sequencing verified the correct insertion sequence.

[0093] Example 4

[0094] Tomato genetic transformation and acquisition of transgenic plants

[0095] 4.1 Agrobacterium-mediated transformation

[0096] The p35S:SlPML1-GFP and pCas9-SlPML1 plasmids were introduced into Agrobacterium GV3101 competent cells by electroporation, respectively. The cells were then plated on YEP plates containing kanamycin (50 μg / mL) and rifampin (25 μg / mL) and incubated at 28°C for 48 h. Single colonies were picked for PCR verification of positive clones.

[0097] 4.2 Tomato cotyledon transformation

[0098] Referring to the conventional Agrobacterium-mediated tomato cotyledon transformation method, the simplified steps are as follows:

[0099] (1) Seed disinfection: Tomato 'Ailsa Craig' seeds were soaked in 70% ethanol for 1 min and rinsed 3 times with sterile water; then soaked in 2% sodium hypochlorite solution for 15 min and rinsed 5 times with sterile water; sown on MS solid medium and cultured in the dark at 28℃ for 2-3 days, and then transferred to light for culture until the cotyledons were fully expanded (about 7 days).

[0100] (2) Explant preparation: cut off the cotyledons, remove both ends, and place the cotyledon segments on the pre-culture medium for 2 days.

[0101] (3) Agrobacterium infection: A single positive Agrobacterium colony was inoculated into YEP liquid medium (containing 50 μg / mL Kan and 25 μg / mL Rif), cultured at 28℃ with shaking until OD600=0.8, centrifuged at 5,000 rpm for 10 min to collect the cells, and resuspended in MS liquid medium until OD600=0.5.

[0102] (4) Co-culture: Immerse the cotyledon explants in Agrobacterium bacterial solution for 10 min, remove them and blot dry the residual bacterial solution with sterile filter paper, transfer them to co-culture medium, and incubate in the dark at 25°C for 2 days.

[0103] (5) Screening culture: The co-cultured explants were transferred to screening medium (MS + 2.0 mg / L 6-BA + 0.2 mg / L IAA + 50 mg / L Kan + 200 mg / L Timentin) and subcultured every 2 weeks until the resistant callus differentiated into shoots.

[0104] (6) Rooting and transplanting: When the resistant buds grow to 2-3 cm, cut them off and transfer them to rooting medium (1 / 2 MS + 0.1 mg / L IAA + 25 mg / L Kan). After rooting, harden the seedlings and transplant them to the greenhouse.

[0105] 4.3 Identification of transgenic plants

[0106] (1) PCR identification: DNA was extracted from the leaves of transgenic plants and PCR amplified using SlPML1 specific primers (Table 1) to detect the integration of the target gene. The target site editing status of the knockout plants was confirmed by amplifying the target site and sequencing.

[0107] (2) qRT-PCR identification: Total RNA was extracted and reverse transcribed into cDNA. The transcription level of SlPML1 was detected by qRT-PCR. Reaction system: 10 μL of 2×SYBR qPCR MasterMix, 0.4 μL each of forward and reverse primers (10 μM), 2 μL of cDNA, and ddH2O to 20 μL. Reaction program: 95℃ for 15 min; 95℃ for 30 s, 60℃ for 30 s, 40 cycles. Actin was used as an internal control (primers are shown in Table 1), and the relative expression level was calculated by the 2^-ΔΔCt method. Two lines with significantly higher expression levels than wild type were screened and named SlPML1-OE-1 and SlPML1-OE-2. Figure 3 B).

[0108] (3) Immunoblot identification: Total protein was extracted from the leaves and quantified by BCA method. An equal amount of protein (30 μg) was added to 5×SDS loading buffer and denatured at 95℃ for 10 min. After separation by 4%-20% SDS-PAGE, it was wet-transferred to a PVDF membrane. Blocked with 5% skim milk powder for 1 h, and incubated overnight at 4℃ with SlPML1 antibody (1:1000, Sangon Biotech, which was prepared based on a specific polypeptide fragment of SEQ ID NO:1) and Actin antibody (1:1000, CWBIO), respectively. After washing the membrane 3 times with TBST, it was incubated at room temperature with HRP-labeled secondary antibody for 1 h, and then developed by ECL chemiluminescence. The results showed that the abundance of SlPML1 protein in SlPML1-OE-1 and SlPML1-OE-2 was significantly higher than that in wild type ( Figure 3 C).

[0109] Sequencing analysis of the knockout plants revealed two independent lineages: slpml1-1 with a 2 bp deletion and slpml1-2 with a 1 bp insertion. Figure 3 A), all of which lead to frameshift mutations and premature termination of protein translation.

[0110] Example 5

[0111] SlPML1 subcellular localization analysis

[0112] 5.1 Transient expression in tobacco

[0113] The p35S:SlPML1-GFP plasmid was transformed into Agrobacterium GV3101. Positive Agrobacterium strains were inoculated into YEP liquid medium and cultured at 28°C with shaking until OD600 = 0.8. The culture was then centrifuged at 5,000 rpm for 10 min and resuspended in infection buffer (10 mM MgCl2, 10 mM MES-KOH pH 5.6, 100 μM AS) until OD600 = 0.5. Tobacco (Nicotiana abenthamiana) leaves that had grown for 4-5 weeks were selected, and the bacterial solution was injected into the mesophyll tissue from the underside of the leaf using a 1 mL syringe (needle removed). The injected tobacco leaves were cultured in the dark for 24 h, then transferred to normal light for 24-48 h. Fluorescence signals were observed using a laser confocal microscope (LSM880NLO, Zeiss). GFP excitation wavelength was 488 nm, and emission wavelength was 520-540 nm; chloroplast autofluorescence excitation wavelength was 488 nm, and emission wavelength was 670-690 nm.

[0114] The results showed that the green fluorescence signal of SlPML1-GFP completely overlapped with the auto-red fluorescence of chloroplasts. Figure 1 A), indicating that SlPML1 is located in chloroplasts.

[0115] 5.2 Localization of chloroplasts and thylakoid membranes

[0116] Subcellular components were extracted from wild-type tomato leaves: Total protein, cytoplasmic protein, chloroplast protein, and thylakoid membrane protein were extracted using a plant chloroplast extraction kit (Zhongke Ruitai, China) and a plant thylakoid membrane extraction kit (Zhongke Ruitai, China). Western blot analysis was performed using Actin (cytoplasmic marker), H3 (nuclear marker), and OEC33 (thylakoid membrane marker) as controls. Results showed that SlPML1 was only present in chloroplast and thylakoid membrane components, consistent with the localization pattern of OEC33. Figure 1 B), confirming that SlPML1 is specifically located on the thylakoid membrane.

[0117] Example 6

[0118] Validation of SlPML1 calcium ion transport function

[0119] 6.1 Construction of yeast expression vector

[0120] The SlPML1 coding sequence was cloned into the pYES2 vector (Invitrogen) to construct the pYES2-SlPML1 recombinant plasmid. This plasmid was then transformed into the Saccharomyces cerevisiae calcium-sensitive mutant K667 (Δgdt1, sensitive to high concentrations of Ca). 2+ (Sensitive). Use empty vector pYES2 as a control.

[0121] 6.2 Plate Spot Test

[0122] The transformed yeast strain was inoculated into SD / -Ura liquid medium and cultured at 30°C with shaking until the logarithmic growth phase. The cells were collected, washed with sterile water, and resuspended to OD600 = 1.0. The cells were then serially diluted 10-fold (10... 0 10 -1 10 -2 10 -3 Take 5 μL of each sample and spot it onto YPD solid medium containing 0 or 150 mM CaCl2. Incubate at 30℃ upside down for 3-4 days and observe the colony growth.

[0123] The results are as follows Figure 2 As shown in Figure A: All strains grew well on normal YPD medium; on medium containing 150 mM CaCl2, the growth of K667 strain transformed with empty vector was severely inhibited, while the growth of strain expressing SlPML1 was significantly restored, indicating that SlPML1 can compensate for the calcium sensitivity defect of K667.

[0124] 6.3 Determination of growth curve in liquid culture

[0125] The above-mentioned strains were inoculated into SD / -Ura induction medium containing 0 or 150 mM CaCl2, with an initial OD600 of 0.05, and cultured at 30°C with shaking. OD600 values ​​were measured every 2 h, and growth curves were plotted. Results are as follows: Figure 2 As shown in B, under high calcium conditions, the yeast strain expressing SlPML1 grew significantly faster than the empty vector control.

[0126] 6.4 Determination of Intracellular Calcium Content in Yeast

[0127] Yeast cells cultured to the stationary phase were collected and washed three times with sterile deionized water. The cell pellet was transferred to a quartz crucible and dried at 105°C to constant weight. The dry weight was recorded. The cells were digested with concentrated nitric acid and brought to a final volume of 5 mL with ultrapure water. The calcium ion concentration was determined using atomic absorption spectrometry. The results are as follows: Figure 2 As shown in Figure C, the intracellular calcium content of the yeast strain expressing SlPML1 was significantly higher than that of the empty vector control (p<0.01), further confirming that SlPML1 has calcium ion transport activity.

[0128] Example 7

[0129] The effects of SlPML1 on tomato growth and development

[0130] Wild-type (WT), SlPML1 knockout plants (slpml1-1, slpml1-2), and SlPML1 overexpressing plants (SlPML1-OE-1, SlPML1-OE-2) at the six-leaf-one-heart stage were collected for morphological observation and physiological index measurement.

[0131] 7.1 Phenotypic Observation

[0132] like Figure 4 As shown in A, slpml1-1 and slpml1-2 plants were significantly dwarfed, with lighter leaf color than the wild type and weaker growth; while slPML1-OE-1 and slPML1-OE-2 showed no significant difference compared to the wild type.

[0133] 7.2 Growth index determination

[0134] Plant height (height from cotyledon to growing point), stem diameter (base diameter), leaf area (portable leaf area meter), fresh weight (total fresh weight of aboveground and underground parts), and relative chlorophyll content (SPAD value) were measured. Ten plants were measured for each line, with three replicates. The results are summarized in Table 2 and... Figure 4 BF.

[0135] Table 2. Statistics of growth indicators in SlPML1 knockout and overexpression plants

[0136] Note: ** indicates p < 0.01 compared to WT (One-way ANOVA, Tukey's test); data are mean ± SD, n = 10.

[0137] The above results indicate that the loss of SlPML1 function seriously affects the growth and development of tomatoes and chlorophyll accumulation.

[0138] Example 8

[0139] Analysis of SlPML1 protein accumulation under low night temperature stress

[0140] Wild-type tomato plants were subjected to low nighttime temperature stress (daytime temperature 28℃, nighttime temperature 4℃, light intensity 600 μmol·m⁻¹). -2 ·s -1 Leaves were collected at 0, 0.5, 1, 2, 4, and 12 h after treatment, and total protein was extracted. Western blot analysis was used to detect the abundance of SlPML1 protein. Figure 5 ).

[0141] The results showed that SlPML1 protein began to accumulate 0.5 h after low night temperature stress; it increased significantly 1-2 h; and remained at a high level 4-12 h. This indicates that SlPML1 protein expression is induced by low night temperature stress.

[0142] Example 9

[0143] Phenotypic and physiological parameters of the effect of SlPML1 on resistance to low night temperatures

[0144] Wild-type, slpml1-1, slpml1-2, SlPML1-OE-1, and SlPML1-OE-2 plants (six-leaf-one-heart stage) were subjected to low night temperature stress (28°C daytime, 4°C nighttime, for 7 days), while a normal temperature control (28°C daytime, 18°C ​​nighttime) was set up. Phenotypic results were observed and relevant indicators were measured after the treatment.

[0145] 9.1 Phenotypic Observation

[0146] like Figure 6 As shown in A, all strains grew well under normal conditions. After 7 days of low night temperature stress, some leaves of the wild-type plants wilted slightly; the slpml1-1 and slpml1-2 plants wilted severely, with leaves drooping, losing chlorophyll, and drying out; the slPML1-OE-1 and slPML1-OE-2 plants remained basically normal, with only a few leaves having slightly curled edges.

[0147] 9.2 Electrolyte Permeability Measurement

[0148] Take 0.2 g of fresh leaves, rinse with deionized water, cut into small pieces, place in 10 mL of deionized water, shake at room temperature for 4 h, and measure the initial conductivity E1; then boil in water for 30 min, and measure the maximum conductivity E2 after cooling. Electrolyte permeability (REL) = (E1 / E2) × 100%. The results are summarized in Table 3.

[0149] Table 3 Electrolyte permeability (REL, %) of each strain before and after low night temperature stress

[0150] Note: p < 0.05 compared to WT, ** indicates p < 0.001; data are mean ± SD, n = 6.

[0151] 9.3 Determination of malondialdehyde (MDA) content

[0152] The thiobarbituric acid (TBA) method was used. 0.2 g of leaf samples were mixed with 5 mL of 10% trichloroacetic acid (TCA), ground into a homogenate, and centrifuged at 5,000 rpm for 10 min. 2 mL of the supernatant was collected, and 2 mL of 0.6% TBA solution was added. The mixture was incubated in a boiling water bath for 30 min, cooled, and then the absorbance at 450 nm, 532 nm, and 600 nm was measured. MDA concentration (μmol / L) = 6.45 × (OD532 - OD600) - 0.56 × OD450. MDA content (μmol / g FW) = (C × V total) / (W × 1000). The results are summarized in Table 4.

[0153] Table 4. MDA content (μmol / g FW) of each strain before and after low night temperature stress

[0154] Note: ** indicates p < 0.05, ** indicates p < 0.001; data are mean ± SD, n = 6.

[0155] 9.4 Determination of Relative Moisture Content (RWC)

[0156] Leaves were collected and their fresh weight (FW) was recorded. The leaves were then soaked in deionized water at 4°C in the dark until constant weight was achieved, and their saturated fresh weight (TW) was recorded. The leaves were then dried at 75°C until constant weight was achieved, and their dry weight (DW) was recorded. RWC (%) = (FW - DW) / (TW - DW) × 100%. The results are summarized in Table 5.

[0157] Table 5. Relative water content (RWC, %) of each strain before and after low night temperature stress

[0158] Note: ** indicates p < 0.05, ** indicates p < 0.001; data are mean ± SD, n = 6.

[0159] The above results demonstrate that SlPML1 positively regulates the low night temperature resistance of tomatoes.

[0160] Example 10

[0161] Effects of SlPML1 on reactive oxygen species content under low night temperature stress

[0162] 10.1 DAB staining (for H2O2 detection)

[0163] Leaves from each strain before and after low nighttime temperature treatment were collected and immersed in 10 μg / mL DAB staining solution (pH 3.8) and incubated at room temperature in the dark for 12 h. The staining solution was discarded, and 95% ethanol was added. The leaves were then destained in an 80℃ water bath until they were completely decolorized, and the results were observed by scanning. Figure 7 As shown in Figure A.

[0164] 10.2 NBT staining (for O2 detection) - )

[0165] 0.2% (w / v) NBT staining solution was used, and the remaining steps were the same as for DAB staining. Results are as follows: Figure 7 As shown in Figure A.

[0166] 10.3 O2 - Generation rate determination

[0167] Take 0.5 g of leaves, add 5 mL of 50 mM phosphate buffer (pH 7.8), grind into a homogenate, and centrifuge at 12,000 rpm for 20 min. Take 0.5 mL of the supernatant, add 0.1 mL of 10 mM hydroxylamine hydrochloride and 0.4 mL of PBS, and incubate at 25°C for 20 min. Then add 1 mL of 58 mM sulfonamide and 1 mL of 7 mM α-naphthylamine, and develop in the dark at 25°C for 20 min. Extract with an equal volume of chloroform, and measure the absorbance at 530 nm of the upper aqueous phase. Calculate O2 according to the standard curve. - Generation rate. The results are summarized in Table 6.

[0168] Table 6 O2 of each strain before and after low night temperature stress - Production rate (nmol·g) -1 FW·min -1 )

[0169] Note: ** indicates p < 0.05, ** indicates p < 0.001; data are mean ± SD, n = 6.

[0170] 10.4 Determination of H2O2 content

[0171] Take 0.3 g of leaf material, add 3 mL of 0.1% TCA and grind into a homogenate. Centrifuge at 12,000 rpm for 15 min. Take 1 mL of the supernatant, add 2 mL of 100 mM phosphate buffer and 1 mL of 1 M KI, mix well, and react in the dark for 30 min. Measure the absorbance at 390 nm and calculate the H2O2 content according to the standard curve. The results are summarized in Table 7.

[0172] Table 7. H2O2 content (μmol·g) of each strain before and after low night temperature stress -1 FW)

[0173] 10.5 Reactive oxygen species fluorescent staining (H2DCFDA)

[0174] Leaf samples were soaked in 0.01 M PBS for 30 min, dried, and transferred to a staining solution containing 10 μM H2DCFDA. Incubation was performed at room temperature in the dark for 30 min. The samples were rinsed three times with PBS and observed using a laser confocal microscope (excitation wavelength 488 nm, emission wavelength 515-535 nm). Figure 7 As shown in D, strong green fluorescence signals appeared in the chloroplast regions of slpml1-1 leaves after low night temperature stress, while the wild type showed weaker signals, and overexpressing plants showed almost no signal.

[0175] In summary, SlPML1 can significantly reduce reactive oxygen species accumulation induced by low nighttime temperature stress.

[0176] Example 11

[0177] Effects of SlPML1 on photosynthetic capacity under low night temperature stress

[0178] 11.1 Determination of PSII maximum photochemical efficiency (Fv / Fm) and net photosynthetic rate (Pn)

[0179] The photosynthetic rate was measured using a GFS-3000 portable photosynthesis system coupled with a Dual-PAM-100 fluorescence analyzer. Initial fluorescence (Fo) and maximum fluorescence (Fm) were measured 30 min after dark adaptation of the plants, and Fv / Fm = (Fm-Fo) / Fm was calculated. Net photosynthetic rate (Pn) was also measured simultaneously under the following conditions: CO2 concentration 600 μmol / mol, light intensity 600 μmol·m⁻¹. -2 ·s -1 The leaf chamber temperature was 25℃. The results are summarized in Table 8. Figure 8 A, 8B).

[0180] Table 8. Fv / Fm and Pn of each strain before and after low night temperature stress

[0181] Note: ** indicates p < 0.05, ** indicates p < 0.001; data are mean ± SD, n = 6.

[0182] 11.2 Measurement of PSI and PSII chlorophyll fluorescence parameters

[0183] The effective photochemical efficiency Y(I), relative electron transport rate ETR(I) of PSI, and effective photochemical efficiency Y(II) and relative electron transport rate ETR(II) of PSII were measured using a Dual-PAM-100 dual-channel synchronous measurement system. Parameter calculation formulas:

[0184] Y(I) = 1 - Y(ND) - Y(NA)

[0185] ETR(I) = Y(I) × PAR × 0.5 × 0.84

[0186] Y(II)=(Fm'-Fs) / Fm'

[0187] ETR(II) = Y(II) × PAR × 0.5 × 0.84

[0188] The results are as follows Figure 8 As shown in CF. Quantitative data are summarized in Table 9.

[0189] Table 9. Fluorescence parameters of PSI and PSII for each strain after low night temperature stress.

[0190] Note: ** indicates p < 0.05, ** indicates p < 0.001; data are mean ± SD, n = 6.

[0191] This indicates that SlPML1 overexpression can protect the activity of PSI and PSII under low night temperature stress.

[0192] Example 12

[0193] Chloroplast ultrastructure observation

[0194] Leaves from each strain before and after low-temperature treatment were cut into 1 mm × 2 mm pieces and immediately placed in 2.5% glutaraldehyde fixative (prepared with 0.1 M phosphate buffer, pH 7.2) at 4°C overnight. After fixation with 1% osmium tetroxide, graded ethanol dehydration, epoxy resin embedding, ultrathin sectioning (70 nm), and double staining with uranium acetate and lead citrate, the ultrastructure of chloroplasts was observed under a transmission electron microscope (HT7700, Hitachi). Results are as follows: Figure 9 As shown.

[0195] Example 13

[0196] Interaction analysis of SlPML1 with chloroplast proteins

[0197] 13.1 Yeast two-hybrid (Y2H) screening for interacting proteins

[0198] Using SlPML1 as bait, the pGBKT7-SlPML1 recombinant plasmid was constructed. It was transformed into Y2H Gold yeast strain and plated on SD / -Trp medium. The constructed tomato yeast cDNA library was mixed with the bait yeast strain in 2×YPDA medium and cultured with gentle shaking at 30℃ for 24 h. The cells were collected and plated on DDO / X / A solid medium (SD / -Leu / -Trp supplemented with X-α-Gal and Aureobasidin A), and cultured at 30℃ for 3-4 days. Blue single colonies were picked and further screened on QDO / X / A medium (SD / -Leu / -Trp / -His / -Ade supplemented with X-α-Gal and Aureobasidin A). Positive clone plasmids were extracted, sequenced, and compared with the tomato genome database.

[0199] Candidate proteins that interact with SlPML1 were screened, including SlPsbP, SlPsbQ, and SlPsbO (Table 10).

[0200] Table 10 SlPML1 interacting proteins obtained from yeast two-hybrid screening

[0201] 13.2 Yeast two-hybrid verification of interactions

[0202] SlPML1 was cloned into pGADT7(AD), and SlPsbP, SlPsbQ, and SlPsbO were cloned into pGBKT7(BK), respectively. All were co-transformed into Y2H Gold yeast strain and plated on DDO medium. Positive clones were spotted onto QDO / X / A medium. Results are as follows: Figure 10 As shown in Figure A.

[0203] 13.3 Validation of luciferase complementation (LCI)

[0204] SlPML1 was cloned into pCAMBIA1300-nLuc (NLuc), and SlPsbP, SlPsbQ, and SlPsbO were cloned into pCAMBIA1300-cLuc (CLuc), respectively. These were co-expressed in tobacco leaves, and D-luciferin potassium salt was sprayed after 48 h. Fluorescence was detected using an in vivo imaging system. Results are as follows: Figure 10 As shown in B.

[0205] 13.4 Validation of Bimolecular Fluorescence Complementary (BiFC)

[0206] SlPML1 was cloned into pSPYNE-35S (nYFP), and SlPsbP, SlPsbQ, and SlPsbO were cloned into pSPYCE-35S (cYFP), respectively. These were co-expressed in tobacco leaves, and YFP fluorescence (excitation 488 nm, emission 520-540 nm) was observed by laser confocal microscopy after 48 h. Results are as follows: Figure 10 As shown in C.

[0207] 13.5 Calcium ion-enhanced interaction strength

[0208] Add 5 mM CaCl2 to Y2H medium and observe yeast growth. Figure 11 A). Pull-down experiments also confirmed that increasing the calcium ion concentration enhanced the binding of SlPML1-His to GST-PsbP, PsbQ, and PsbO ( Figure 11 B). The LCI quantification results also showed that exogenous calcium ion treatment significantly increased fluorescence intensity (B). Figure 11 C).

[0209] 13.6 Detection of SlPsbP, SlPsbQ, and SlPsbO protein abundance

[0210] Leaf proteins of wild-type, slpml1-1, slpml1-2, SlPML1-OE-1, and SlPML1-OE-2 were extracted before and after low night temperature treatment. The abundance of SlPsbP, SlPsbQ, and SlPsbO was detected by Western blotting. Figure 12 The results of the quantitative analysis are summarized in Table 11.

[0211] Table 11. Relative abundance of photosynthetic proteins in each strain after low night temperature stress (with wild type under normal conditions as 1).

[0212] Note: p < 0.05 compared to WT (stress), ** indicates p < 0.001; data are mean ± SD, n = 3.

[0213] This indicates that SlPML1 inhibits the degradation of the aforementioned proteins under low nighttime temperature stress by interacting with them.

[0214] Example 14

[0215] Effect of SlPML1 on oxygen evolution rate

[0216] Intact chloroplasts were extracted from leaves of each strain, and chlorophyll content was determined by extraction with 80% acetone. The photosynthetic oxygen evolution rate was measured using an oxygen electrode (Hansatech, UK). Measurement conditions: temperature 25℃, saturated light intensity 1000 μmol·m⁻¹. -2 ·s -1 The oxygen release rate is expressed in μmol O2·mg -1 Chl·h -1 The results are summarized in Table 12 and... Figure 13 .

[0217] Table 12 Oxygen release rates (μmol O2·mg) of different strains before and after low night temperature stress -1 Chl·h -1 )

[0218] Note: p < 0.05 compared to WT, ** indicates p < 0.001; data are mean ± SD, n = 6.

[0219] Example 15

[0220] Exogenous sucrose supplementation of SlPML1 knockout plant phenotype

[0221] 15.1 Processing Method

[0222] Wild-type plants and slpml1-1 and slpml1-2 plants were treated with ordinary nutrient solution (control) and nutrient solution supplemented with 2% (w / v) sucrose, respectively, for one month. Phenotypic observation and physiological index measurement were then performed. Results are as follows: Figure 14 and Figure 15 As shown.

[0223] 15.2 Phenotypic Data Recovery

[0224] The quantitative determination results are summarized in Table 13.

[0225] Table 13 Effects of exogenous sucrose on growth parameters of the slpml1 mutant

[0226] Note: Different letters indicate p < 0.05 (One-way ANOVA, Tukey's test); data are mean ± SD, n = 10.

[0227] The above results indicate that exogenous sucrose can partially compensate for the growth defects and low night temperature sensitivity caused by SlPML1 deficiency, indirectly proving that SlPML1 plays a role by affecting the synthesis of photosynthetic products.

[0228] Test Example 1

[0229] SlPML1 overexpression shows specificity in response to different stress types.

[0230] Wild-type and SlPML1-OE-1 plants were subjected to low temperature (6℃, constant day / night temperature), high temperature (35℃ / 25℃), drought (15% PEG6000) and salt stress (100 mM NaCl), respectively. The expression level of SlPML1 was detected by qRT-PCR, and the electrolyte permeability of the plants after stress was measured.

[0231] The results showed that SlPML1 was significantly induced after one day of low-temperature treatment and responded to varying degrees under other stresses. However, overexpression only significantly enhanced resistance to low night temperature (4°C) and low temperature (6°C) stresses, without significantly improving resistance to high temperature, drought, or salt stresses. This indicates that SlPML1 has a relatively specific protective effect against low-temperature stress (especially low night temperature).

[0232] Test Example 2

[0233] Expression analysis of SlPML1 in different tissues

[0234] RNA was extracted from roots, stems, young leaves, senescent leaves, flowers, and fruits of wild-type tomato, and the expression level of SlPML1 was detected by qRT-PCR. The relative expression level in each tissue was calculated using the expression level in roots as a baseline (set as 1). The results are summarized in Table 14. Figure 16.

[0235] Table 14. Relative expression levels of SlPML1 in different tomato tissues

[0236] The data are mean ± SD, n=3.

[0237] The results showed that SlPML1 was expressed at the highest level in young leaves and at the lowest level in roots, indicating that it mainly plays a role in photosynthetic tissues.

[0238] Test Example 3

[0239] Preliminary Study on the Redox State of SlPML1 Protein

[0240] To investigate whether SlPML1 is subject to redox regulation, wild-type leaves were treated with different concentrations of DTT (0, 5, 10, 20 mM). Protein extraction was performed, and the migration changes of SlPML1 were detected by non-reducing SDS-PAGE and Western blotting. The results showed that with increasing DTT concentration, the proportion of reduced SlPML1 gradually increased. Figure 17 This suggests that SlPML1 undergoes reversible redox modification.

[0241] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0242] The foregoing embodiments are merely illustrative examples of preferred embodiments of the present invention and do not constitute any limitation on the scope of protection of the present invention. For those skilled in the art, any modifications, equivalent variations, and alterations made to the present invention without departing from the principles and spirit of the invention should be considered to fall within the scope of protection defined by the appended claims.

Claims

1. An isolated protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the protein of claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

2.

3. A recombinant expression vector comprising the nucleic acid molecule of claim 2.

4. A host cell comprising the nucleic acid molecule of claim 2 or the recombinant expression vector of claim 3.

5. The application of the protein of claim 1 or the nucleic acid molecule of claim 2 in improving plant tolerance to low night temperature stress.

6. The application according to claim 5, characterized in that, The improvement of plant tolerance to low night temperature stress is manifested in at least one of the following: (a) Increase net photosynthetic rate Pn; (b) Improve the maximum photochemical efficiency of PSII, Fv / Fm; (c) Improve the effective photochemical efficiency Y(I) of PSI and / or the effective photochemical efficiency Y(II) of PSII; (d) Improve the relative electron transport rate ETR(I) of PSI and / or the relative electron transport rate ETR(II) of PSII; (e) Increase the oxygen release rate; (f) Reduce the electrolyte permeability of the leaves; (g) Reduce malondialdehyde (MDA) content; (h) Increase the relative water content of the leaves; (i) Reduce superoxide anion O2 - Production rate and / or hydrogen peroxide (H2O2) content; (j) Maintain the integrity of the thylakoid membrane structure of chloroplasts and the stacking of grana lamellae.

7. A method for improving plant tolerance to low night temperature stress, characterized in that, This includes overexpressing the nucleic acid molecule of claim 2 in plants.

8. The method according to claim 7, characterized in that, The overexpression includes introducing the recombinant expression vector of claim 3 into plant cells and regenerating a complete plant.

9. The method according to claim 7 or 8, characterized in that, The plant is a member of the Solanaceae family, preferably tomato (Solanum lycopersicum).

10. A method for cultivating transgenic plants tolerant to low night temperatures, characterized in that, After the nucleic acid molecule described in claim 2 is operably linked to the promoter and transferred into the plant genome, transgenic plants with reduced electrolyte permeability, reduced malondialdehyde content, increased relative water content, improved photosynthetic capacity and / or reduced reactive oxygen species accumulation under low night temperature stress are screened to obtain transgenic plants compared with wild type.

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