Tomato slcas protein and its coding gene in regulating plant tolerance to low night temperature stress

Abstract

The application discloses a tomato SlCAS protein and application of an encoding gene thereof in regulation of plant tolerance to low night temperature stress. An amino acid sequence of the SlCAS 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 proves that the SlCAS positively regulates chlorophyll accumulation, calcium signal transduction and active oxygen homeostasis under low night temperature (less than or equal to 6 DEG C), and maintains cell membrane integrity by constructing SlCAS overexpression and CRISPR / Cas9 gene editing tomato plants. Overexpression of the SlCAS can significantly improve chlorophyll content of the tomato under low night temperature, reduce chlorophyllase activity and oxidative damage, and enhance plant resistance. Knockout of the SlCAS leads to significant enhancement of low night temperature sensitivity. Exogenous calcium pretreatment can synergistically enhance the low night temperature resistance mediated by the SlCAS. The application provides a key gene resource and technical scheme for tomato low temperature tolerance breeding, and can be applied to improve the tolerance of horticultural crops to low temperature stress.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and molecular biology, specifically to the application of the tomato calcium-sensing receptor SlCAS and its encoding gene in regulating plant tolerance to low night temperature stress, particularly the method and application of improving tomato resistance to low night temperature (≤6℃) stress by regulating SlCAS gene expression. Background Technology

[0002] Tomato (Solanum lycopersicum) is a widely cultivated vegetable crop globally and one of the main vegetable varieties grown in greenhouses in my country. During winter and spring greenhouse cultivation, tomato plants are highly susceptible to low night temperature (LNT) stress. LNT stress significantly inhibits tomato photosynthesis, leading to reduced chlorophyll content in leaves, damage to the photosystem, and impaired photosynthetic electron transport, thus severely impacting tomato yield and fruit quality. Therefore, elucidating the molecular mechanisms of tomato response to LNT stress and identifying key genes for LNT tolerance is of great significance for breeding new cold-resistant tomato varieties.

[0003] Photosynthesis is one of the physiological processes in plants most sensitive to low-temperature stress. Under low-temperature stress, the chlorophyll content in plant leaves decreases, and the activities of photosystem II (PSII) and photosystem I (PSI) are inhibited. Excessive light energy leads to a large accumulation of reactive oxygen species (ROS), causing oxidative damage to the photosynthetic apparatus. Previous studies have shown that exogenous calcium ions (Ca... 2+ Pretreatment can increase the chlorophyll content and electron transport rate of tomato leaves under low night temperatures, thereby enhancing photosynthetic capacity, but the specific molecular mechanism by which exogenous calcium exerts its effect is still unclear.

[0004] Calcium ions (Ca 2+ Ca2+ is an important second messenger in plants, participating in the regulation of plant growth and development and responses to various abiotic stresses. When plants sense abiotic stress signals, intracellular Ca2+... 2+ The concentration increases rapidly, generating a calcium signal, which is then recognized and transduced by downstream calcium-sensing proteins, ultimately activating the expression of stress-response genes. Currently, the calcium-sensing proteins identified in plants mainly include calmodulin (CaM), calcineurin B-like proteins (CBLs), calcium-dependent protein kinases (CDPKs), and calcium-sensing receptors (CAS) located on chloroplasts.

[0005] CAS proteins are calcium-sensing proteins located on the thylakoid membrane of chloroplasts. Initially identified in Arabidopsis thaliana, they are involved in regulating stomatal movement and CO2 concentration mechanisms. Current research indicates that CAS plays an important role in photophosphorylation and photosynthetic electron transport. However, the regulatory role of CAS proteins on chlorophyll metabolism under low nighttime temperature stress, and their mediating effects on calcium... 2+ The molecular mechanism by which signaling alleviates photosynthetic damage caused by low night temperatures has not yet been reported. In particular, the function of the SlCAS protein in tomatoes and its application in resistance to low night temperatures remain a blank.

[0006] Therefore, identifying the key gene SlCAS in tomatoes that responds to low night temperature stress, elucidating its function in regulating chlorophyll homeostasis and photosynthesis, and developing technical solutions based on the SlCAS gene to improve plant resistance to low night temperature are of great theoretical significance and application value for molecular breeding of cold-resistant tomato varieties. Summary of the Invention

[0007] The present invention aims to solve the problem of the lack of specific technical solutions in the existing technology for effectively improving the resistance of plants to low night temperatures by utilizing the tomato calcium-sensing protein SlCAS (or its encoding gene).

[0008] Therefore, this invention provides the application of the tomato SlCAS protein and its encoding gene in regulating plant tolerance to low night temperature stress. Specifically, this invention utilizes various techniques, including gene overexpression, gene editing (knockout), and exogenous calcium synergistic treatment, to demonstrate that the SlCAS protein is a key positive regulator of plant response to low night temperature stress. Based on this, breeding strategies to improve plant cold tolerance, molecular markers for screening sensitive germplasm, and related biological materials can be developed.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention protects an isolated protein (SlCAS).

[0011] The protein is derived from tomato (Solanum lycopersicum), and its amino acid sequence is shown in SEQ ID NO:1. This protein is located in the thylakoid membrane of chloroplasts and can sense and transmit calcium signals, positively regulating chlorophyll homeostasis under low nighttime temperature stress.

[0012] Secondly, the present invention protects the nucleic acid molecule encoding the protein.

[0013] The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:2, and contains the complete coding region (CDS) sequence of the SlCAS gene.

[0014] Thirdly, the present invention protects the use of the protein or nucleic acid molecule in improving plant resistance to low night temperatures.

[0015] The low night temperature refers to a nighttime temperature of ≤6℃.

[0016] Specifically, by overexpressing the aforementioned nucleic acid molecules in plants, the chlorophyll content (including chlorophyll a and chlorophyll b) can be significantly increased, the accumulation of reactive oxygen species (especially hydrogen peroxide H2O2) can be reduced, and cell membrane integrity can be maintained (reducing malondialdehyde (MDA) content and relative conductivity REC), thereby improving the overall tolerance of plants to low night temperature stress.

[0017] Alternatively, by using CRISPR / Cas9 gene editing technology to knock out the nucleic acid molecules at specific points, mutant plants that are highly sensitive to low night temperature stress can be obtained. These mutants can serve as ideal screening models for low night temperature resistance studies or for identifying low-temperature tolerant germplasm resources.

[0018] Fourthly, the present invention provides a method for improving the resistance of plants to low night temperatures.

[0019] The method includes the following steps: operably linking the above-mentioned nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:2 to a promoter (e.g., a constitutive promoter CaMV 35S or a stress-inducible promoter) to construct a plant overexpression vector; transforming the overexpression vector into plant cells using Agrobacterium-mediated transformation or gene gun method; obtaining transgenic plants overexpressing the SlCAS gene through tissue culture and resistance screening; the transgenic plants exhibit significantly enhanced resistance under low night temperature stress.

[0020] Furthermore, the method further includes applying a CaCl2 aqueous solution at a concentration of 5-50 mmol / L (preferably 27 mmol / L) to the plants before or during low night temperature stress. Exogenous calcium pretreatment can synergistically enhance the plant's resistance to low night temperature by overexpressing endogenous SlCAS.

[0021] Fifthly, the present invention provides a molecular marker for screening plants sensitive to low night temperatures.

[0022] The molecular marker is the CRISPR / Cas9 gene editing target sequence of the SlCAS gene. Mutant plants containing this target sequence and with lost SlCAS gene function (Slcas mutants) exhibit high sensitivity to low night temperatures, with leaf yellowing, significantly reduced chlorophyll content, and severely impaired photosynthetic capacity. This molecular marker can be used for rapid identification or screening of germplasm resources sensitive to low night temperatures.

[0023] Sixthly, the present invention protects a recombinant expression vector that enhances plant resistance to low night temperatures.

[0024] The recombinant expression vector contains a nucleic acid molecule with the nucleotide sequence shown in SEQ ID NO:2, and the nucleic acid molecule is operatively linked to a constitutive promoter (such as the CaMV 35S promoter) or a low-night-temperature-inducible promoter. The recombinant expression vector can be used to transform plants to obtain transgenic plants with enhanced resistance to low night temperatures.

[0025] Seventhly, the present invention protects the use of the protein or nucleic acid molecule in the preparation of a plant low night temperature resistance identification kit or a breeding kit.

[0026] The kit contains primer pairs (as shown in SEQ ID NO:3 and SEQ ID NO:4) that specifically detect SlCAS gene expression levels, or contains antibodies that specifically bind to the SlCAS protein. By detecting the expression levels of the SlCAS gene or protein in the test plant, its resistance to low night temperatures can be predicted.

[0027] Compared with the prior art, the present invention has the following outstanding advantages:

[0028] 1. This invention reveals for the first time the positive regulatory function of the tomato SlCAS protein under low night temperature stress. By constructing tomato plants with SlCAS gene overexpression and CRISPR / Cas9 gene knockout, combined with low night temperature (6℃) treatment, this invention systematically identified the core role of SlCAS in regulating chlorophyll metabolism, maintaining calcium signaling, and controlling reactive oxygen species homeostasis. No prior art has reported linking SlCAS to plant resistance to low night temperature.

[0029] 2. This invention provides a clear and industrially applicable technical solution. It not only identifies the sequence of the SlCAS protein and its encoding gene, but also provides specific methods for constructing overexpression vectors, screening transgenic plants, synergistic treatment with exogenous calcium, and applying molecular markers. These technical solutions are highly operable and reproducible, and can be directly applied to low-temperature tolerance breeding of tomatoes and other horticultural crops.

[0030] 3. The technical effects are significant and verifiable. Experimental data show that, compared with the wild type, SlCAS overexpressing plants, after low night temperature treatment, exhibited a 19-21% increase in chlorophyll a content, a 21-23% increase in chlorophyll b content, a significant decrease in chlorophyllase activity, reduced H2O2 accumulation, increased CAT activity, and decreased MDA and REC. In contrast, SlCAS knockout plants exhibited the completely opposite phenotype, which could be partially restored by exogenous calcium. These data fully demonstrate the significant effect of SlCAS in improving resistance to low night temperatures.

[0031] 4. Wide applicability and comprehensive protection. This invention simultaneously protects multiple aspects, including isolated proteins / nucleic acids, uses, methods, expression vectors, molecular markers, and reagent kits, forming a complete chain of protection from gene resources to technological applications. This provides strong legal safeguards for subsequent commercial development and infringement protection.

[0032] 5. This study lays the foundation for further elucidation of the molecular mechanism by which calcium signaling regulates photosynthesis. As a chloroplast-localized calcium-sensing protein, the elucidation of the function of SlCAS provides a basis for understanding the role of calcium in photosynthesis. 2+ This provides a new perspective on how signals are transduced from the cytoplasm to the chloroplasts, thereby regulating chlorophyll metabolism and photosynthesis, and has significant theoretical value. Attached Figure Description

[0033] Figure 1 Effects of exogenous calcium and EGTA pretreatment on chlorophyll content in tomato leaves under low night temperature stress;

[0034] Where A represents chlorophyll a content; B represents chlorophyll b content; C represents the ratio of chlorophyll a to chlorophyll b; and D represents chlorophyllase activity.

[0035] Figure 2 Expression analysis and subcellular localization of the tomato SlCAS gene;

[0036] In this table, A represents the relative expression level of the SlCAS gene in leaves at different developmental stages; B represents the immunoblotting localization results of the SlCAS protein on the thylakoid membrane of chloroplasts, with OEC33 as the thylakoid marker protein.

[0037] Figure 3 : Obtaining Slcas gene-edited mutants and Slcas overexpression plants in tomatoes;

[0038] In this diagram, A shows the CRISPR / Cas9 editing site and protein domain of the SlCAS gene; B shows the relative expression level of the SlCAS gene in the overexpression lines (qRT-PCR); C shows the Western blot identification results of the SlCAS-GFP fusion protein in the overexpression lines, with Actin as an internal control; and D shows the phenotypic images of wild-type (WT), Slcas mutant, and SlCAS-OE overexpression lines.

[0039] Figure 4 Effects of SlCAS on chlorophyll content in tomato leaves under low night temperatures;

[0040] Wherein, AD represents the chlorophyll a content (A), chlorophyll b content (B), Chl a / b ratio (C), and chlorophyllase activity (D) of wild type and Slcas mutant under low night temperature treatment; EH represents the chlorophyll a content (E), chlorophyll b content (F), Chl a / b ratio (G), and chlorophyllase activity (H) of wild type and SlCAS-OE overexpression line under low night temperature treatment.

[0041] Figure 5 Effects of SlCAS on calcium signaling in tomato chloroplasts under low night temperatures;

[0042] Where A represents the net calcium content of chloroplasts in wild-type and SlCAS-OE plants under low nighttime temperatures. 2+ Real-time flux recording curve; B represents the net Ca of chloroplasts measured using non-destructive microelectrode technology. 2+ Internal flow statistics; C is Fluo-4 AM calcium ion fluorescence staining image of leaves of wild-type, Slcas mutant and SlCAS-OE plants under low night temperature;

[0043] Figure 6 Effects of SlCAS on reactive oxygen species signaling in tomato chloroplasts under low night temperatures;

[0044] In this image, A shows H2DCF-DA staining images of leaves from wild-type, Slcas mutant, and SlCAS-OE plants under low night temperature; B shows the catalase (CAT) activity of wild-type and Slcas mutant plants under low night temperature treatment; and C shows the catalase (CAT) activity of wild-type and SlCAS-OE overexpression lines under low night temperature treatment.

[0045] Figure 7 The effect of SlCAS on the resistance of tomatoes to low night temperatures;

[0046] Where A represents the malondialdehyde (MDA) content of wild-type and Slcas mutant under low night temperature treatment; B represents the relative conductivity (REC) of wild-type and Slcas mutant under low night temperature treatment; C represents the MDA content of wild-type and SlCAS-OE overexpression lines under low night temperature treatment; and D represents the REC of wild-type and SlCAS-OE overexpression lines under low night temperature treatment. Detailed Implementation

[0047] 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.

[0048] 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.

[0049] The amino acid sequence of SlCAS: SEQ ID NO.1 = MALRASATAKSPLPLPPPPPSSSSSTSSPPKVFSLPKLTQKPVSVSFSTSTALFLFPLFTATHEARAINL

[0050] PKEDIVSSLNQVESVVNQAQEVGSNIFDTVSGVIGPVIEFVKPGIDAALPLVKQAGEEVLKNASPVISDA

[0051] TKKAQEAMQSAGMDSEPVMTAAKTVVDAAQQTSKVIEGAKPIATSTVETISSTDPAVIAVAGGTLFLAYL

[0052] LLPPVFSALSFSFRGYKGELTPAQTLDNMCSKNYVLIDIRTEKDKDKAGIPRLPSSAKNKMIQIPLEDLP

[0053] SKVKSLVRNPKKVEAEIVALKISFLKKINKGSNIVIMDSYSDSAKTVAKSLTSFGFNNCWIMTDGFSGGK

[0054] GWLQSRLGTDSYNFSFAEILSPSRVIPGRRFGTTGTVKLLSD.

[0055] The nucleic acid sequence of SlCAS is: SEQ ID NO.2=ATGGCACTTAGAGCTTCAGCCACCGCTAAATCACCTCTTCCTCTGCCCCCTCCTCCTCCTTCTTCTTCTT

[0056] CTTCTACTTCATCACCACCTAAAGTTTTTAGTCTTCCTAAACTTACTCAAAAACCTGTATCAGTATCATT

[0057] CTCTACATCCACTGCACTTTTCCTCTTTCCACTTTTCACTGCGACCCATGAAGCAAGAGCAATCAACTTA

[0058] CCCAAGGAAGACATCGTCTCTTCCCTTAATCAGGTAGAATCGGTAGTTAATCAAGCGCAAGAGGTTGGTT

[0059] CGAACATCTTTGATACTGTAAGCGGAGTGATTGGGCCCGTGATTGAATTTGTGAAACCCGGGATTGATGC

[0060] AGCATTACCTTTAGTAAAGCAGGCAGGAGAGGAAGTTTTAAAGAATGCTTCTCCTGTCATATCGGATGCC

[0061] ACTAAGAAGGCCCAAGAGGCAATGCAGAGTGCTGGCATGGACTCTGAGCCAGTGATGACTGCAGCTAAGA

[0062] CAGTAGTTGATGCAGCTCAACAGACATCCAAGGTGATCGAAGGGGCCAAACCAATTGCCACATCCACAGT

[0063] TGAAACCATTTCATCAACTGATCCAGCTGTTATTGCAGTGGCTGGTGGCACATTATTCCTGGCATATCTT

[0064] CTACTTCCCCCTGTCTTCTCCGCTCTCTCTTTCAGCTTTCGTGGTTACAAGGGTGAACTAACTCCTGCTC

[0065] AAACACTGGACAATATGTGTTCTAAGAATTATGTCTTGATTGATATTAGAACTGAGAAGGACAAGGATAA

[0066] GGCTGGAATCCCTCGTCTTCCATCTAGTGCTAAAAACAAGATGATTCAAATCCCTTTGGAAGATTTGCCG

[0067] AGTAAAGTGAAGAGTCTTGTGAGGAATCCGAAGAAAGTGGAAGCTGAAATAGTGGCTCTGAAGATATCAT

[0068] TCCTCAAGAAATCAACAAAGGGTCTAACATCGTGATAATGGACTCGTACTCTGATTCAGCTAAAACAGT

[0069] TGCTAAATCGCTGACGAGCTTTGGCTTCAATAACTGCTGGATCATGACTGATGGCTTCTCCGGAGGCAAG

[0070] GGGTGGTTGCAGAGTCGGCTAGGAACAGATTCATACAACTTTTCTTTTGCAGAAATTTTATCACCATCAA

[0071] GAGTCATTCCAGGTAGACGTTTTGGTACAACAGGCACTGTCAAATTGCTTTCAGATTAA.

[0072] The main primer sequences used in this embodiment are shown in Table 1.

[0073] Table 1 Primer sequences used in this invention

[0074] Example 1

[0075] Cloning of the tomato SlCAS gene and construction of its overexpression vector.

[0076] 1.1 Total RNA extraction and cDNA synthesis from tomatoes

[0077] Mature leaves from wild-type tomato seedlings ('Ailsa Craig', AC) were used to extract total RNA using a plant total RNA extraction kit (Vazyme). Following the instructions of the ReverTra Ace qRT-PCR kit (Vazyme), 1 μg of total RNA was reverse transcribed into first-strand cDNA.

[0078] Subsequently, the expression level of the SlCAS gene in leaves at different developmental stages was detected by qRT-PCR, using primers shown in SEQ ID NO:3 and SEQ ID NO:4. The results are as follows: Figure 2 As shown in Figure A, SlCAS expression was highest in mature leaves and lowest in senescent leaves. Subcellular chloroplast isolation and Western blot analysis, using SlCAS antibody detection, indicated that the SlCAS protein is localized on the thylakoid membrane. Figure 2 B).

[0079] 1.2 Amplification of the full-length coding region of the SlCAS gene

[0080] Using tomato cDNA as a template, the complete coding region of the SlCAS gene was amplified using specific primers (SEQ ID NO:7 and SEQ ID NO:8). The PCR reaction system consisted of 25 μL of 2×PhantaMax Master Mix, 2 μL each of forward and reverse primers (10 μM), 2 μL of cDNA template, and ddH2O to a final volume of 50 μL. Reaction conditions were as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, and 72℃ extension for 1 min, for a total of 35 cycles; and a final extension at 72℃ for 5 min. The PCR product was purified by 1% agarose gel electrophoresis, ligated into the pMD19-T vector, transformed into *E. coli* DH5α competent cells, and positive clones were screened and sequenced for verification. Sequencing results showed that the full-length coding region of the SlCAS gene is 1164 bp, encoding 387 amino acids. Its nucleotide sequence is shown in SEQ ID NO:2, and its amino acid sequence is shown in SEQ ID NO:1.

[0081] 1.3 Construction of overexpression vectors

[0082] The SlCAS coding region sequence (excluding the stop codon), which was verified by sequencing, was cloned into the plant expression vector pCAMBIA1300-GFP using homologous recombination. This fused SlCAS with the GFP tag and placed the vector under the control of the CaMV 35S constitutive promoter. The recombinant plasmid was then verified by enzyme digestion and sequencing to obtain the recombinant expression vector pCAMBIA1300-35S::SlCAS-GFP.

[0083] Example 2

[0084] Obtaining tomato plants overexpressing the SlCAS gene.

[0085] 2.1 Agrobacterium-mediated genetic transformation

[0086] The recombinant expression vector pCAMBIA1300-35S::SlCAS-GFP was transformed into Agrobacterium tumefaciens strain GV3101 using electroporation. Positive single colonies were picked and inoculated into YEB liquid medium containing 50 mg / L kanamycin and 25 mg / L rifampin, and cultured at 28°C with shaking until OD... 600 =0.6-0.8. The bacterial culture was centrifuged at 4℃ and 5000 rpm for 10 min, and the bacterial cells were collected and resuspended in MS liquid medium to OD0.6. 600 =0.5, used as the inoculum.

[0087] Seeds of wild-type tomato 'Ailsa Craig' were disinfected with 75% ethanol for 1 min and 20% sodium hypochlorite for 15 min, then rinsed 5 times with sterile water. They were then sown on MS solid medium and incubated in the dark at 28°C for 2 days to promote germination. After germination, the seeds were transferred to a 16-hour light / 8-hour dark photoperiod at 25°C. Cotyledons from 5-7 day old sterile seedlings were harvested, with both ends removed, and used as explants.

[0088] Immerse the explants in Agrobacterium infection solution for 15 min, gently shaking them during the process. Blot off excess bacterial solution with sterile filter paper and transfer to co-culture medium (MS + 2.0 mg / L 6-BA + 0.2 mg / L IAA + 100 μmol / L AS), incubate in the dark at 25°C for 2 days. Then transfer the explants to selection medium (MS + 2.0 mg / L 6-BA + 0.2 mg / L IAA + 50 mg / L hygromycin + 200 mg / L termethin), subculturing every 2 weeks to induce callus and adventitious shoot differentiation. When resistant shoots reach 2-3 cm in length, cut them off and transfer them to rooting medium (1 / 2 MS + 0.1 mg / L IAA + 25 mg / L hygromycin + 100 mg / L termethin) to induce rooting. Once the root system is well-developed, the regenerated plants are transplanted into nutrient pots containing a mixture of vermiculite and peat moss (1:3, v / v) and placed in an artificial climate chamber (25 / 15℃, 16 h light / 8 h dark, light intensity 600 μmol·m). -2 ·s -1 It grows in the environment.

[0089] 2.2 Molecular identification of transgenic plants overexpressing the gene

[0090] Genomic DNA and total RNA were extracted from the leaves of transgenic tomato plants (T0 generation) and detected by PCR and qRT-PCR, respectively.

[0091] Genomic PCR detection: T-DNA insertion was verified using hygromycin resistance gene-specific primers and SlCAS gene-specific primers (SEQ ID NO:7 and SEQ ID NO:8). PCR conditions were the same as in Example 1.

[0092] qRT-PCR detection: Total RNA was extracted from leaves and reverse transcribed into cDNA. Real-time quantitative PCR was performed using the ChamQ SYBR® qPCR MasterMix kit, with SlACTIN as an internal reference gene. The qRT-PCR primer sequences for SlCAS are SEQ ID NO:3 and SEQ ID NO:4. Reaction conditions: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 40 cycles.

[0093] Three overexpression lines (SlCAS-OE1, OE2, and OE3) were obtained through screening, and their SlCAS transcription levels were upregulated by approximately 6-fold, 6-fold, and 5-fold, respectively, compared to the wild-type (WT). Figure 3 (As shown in B). Western blot analysis using an anti-GFP antibody detected a band of approximately 68 kDa SlCAS-GFP fusion protein in the overexpression lines, while no band was observed in the wild-type (as shown in B). Figure 3 (as shown in C).

[0094] 2.3 Obtaining the gene-edited mutant Slcas

[0095] The tomato endogenous SlCAS gene was edited at specific sites using CRISPR / Cas9 technology. An sgRNA targeting exon 1 of the SlCAS gene was designed, with the target sequence being SEQ ID NO:9 (e.g., ...). Figure 3 (As shown in A). The sgRNA expression cassette and Cas9 expression cassette were assembled into the pCAMBIA1300 vector to obtain the CRISPR / Cas9 editing vector. Tomato explants were transformed using Agrobacterium-mediated transformation to obtain T0 generation regenerated plants. Genomic DNA was extracted from the T0 generation plants, and the target region was amplified by PCR and sequenced. Two homozygous mutant lines (*Slcas-1* and *Slcas-2*) were screened, both of which had base deletions leading to premature appearance of stop codons (e.g., ...). Figure 3 As shown in Figure A), the mutant plant could not translate the SlCAS protein with its complete functional domain. The mutant plant's leaves were pale green (as shown in Figure A). Figure 3 (as shown in D).

[0096] Example 3

[0097] Low nighttime temperature stress treatment and exogenous calcium treatment.

[0098] Tomato seedlings (wild type WT, *Slcas-1*, *Slcas-2*, SlCAS-OE1, SlCAS-OE2) that were 4 weeks old after sowing and had uniform growth were selected and subjected to low night temperature treatment.

[0099] 3.1 Low Nighttime Temperature Treatment

[0100] The plants were placed in an artificial climate chamber with a daytime temperature of 25℃ for 14 hours and a light intensity of 600 μmol·m⁻¹. -2 ·s -1 The nighttime temperature was 6℃ (10 h), and the treatment was continued for 5-7 days. Plants grown at normal temperature (25 / 15℃) were used as the control (RT).

[0101] 3.2 Pretreatment with exogenous calcium and EGTA

[0102] Before the low nighttime temperature treatment, the plant leaves were sprayed with either a CaCl2 solution (27 mmol / L) or an EGTA solution (5 mmol / L) once every morning for three consecutive days. Plants sprayed with distilled water served as a control. Each spray was applied until both sides of the leaves were evenly moistened.

[0103] Test Example 1

[0104] The effect of SlCAS on chlorophyll content in tomatoes at low night temperatures.

[0105] 1.1 Chlorophyll Extraction and Content Determination

[0106] Tomato leaves (from the same leaf position) treated with low nighttime temperature for 5 days were chopped, mixed thoroughly, and 0.1 g of sample was weighed. 10 mL of 95% (v / v) anhydrous ethanol was added, and the mixture was extracted overnight at 4°C in the dark. The extract was centrifuged at 4000 rpm for 5 min, and the supernatant was collected. The absorbance values ​​at 663 nm and 645 nm were measured using a spectrophotometer. The chlorophyll a and chlorophyll b contents and the Chl a / b ratio were calculated using the following formula:

[0107] Chlorophyll a (mg / g) = (12.7 × OD) 663 - 2.69×OD 645) × Extraction liquid volume / Sample fresh weight

[0108] Chlorophyll b (mg / g) = (22.9 × OD) 645 - 4.68×OD 663) × Extraction liquid volume / Sample fresh weight

[0109] 1.2 Chlorophyllase Activity Assay

[0110] The chlorophyllase ELISA kit (Mlbio; YJ620360) was used to determine the chlorophyllase activity according to the instructions. 0.1 g of fresh leaves were weighed, ground in liquid nitrogen, and then added to PBS buffer. After centrifugation, the supernatant was collected and added to an ELISA plate. The plate was reacted with the enzyme-labeled antibody, and the absorbance was measured at 450 nm after color development. Chlorophyllase activity (U / mg protein) was calculated based on the standard curve.

[0111] 1.3 Experimental Results

[0112] like Figure 1As shown in the AD diagram, compared with the normal temperature control (RT), wild-type (WT) plants exhibited significantly decreased chlorophyll a and chlorophyll b contents, a significantly increased Chl a / b ratio, and significantly enhanced chlorophyllase activity after low night temperature (LNT) treatment. Exogenous CaCl2 (27 mmol / L) pretreatment significantly alleviated the LNT-induced decrease in chlorophyll content and increase in chlorophyllase activity, while EGTA (5 mmol / L) pretreatment further exacerbated the LNT-induced disruption of chlorophyll metabolism. These results indicate that exogenous calcium can positively regulate chlorophyll homeostasis in tomato leaves under low night temperatures.

[0113] Further comparison of chlorophyll accumulation differences among different genotypes under LNT treatment yielded the following results: Figure 4 As shown in Figure AH, compared to the wild type, the Slcas mutant plants exhibited significantly reduced chlorophyll a content (approximately 18%), significantly reduced chlorophyll b content (approximately 48%), significantly increased Chl a / b ratio (approximately 65%), and significantly increased chlorophyllase activity. Exogenous CaCl2 pretreatment partially restored the chlorophyll content of the Slcas mutant. Conversely, SlCAS-OE overexpressing plants maintained high chlorophyll levels even after LNT treatment: chlorophyll a content increased by approximately 19-21% compared to the wild type, chlorophyll b content increased by approximately 21-23%, the Chl a / b ratio remained unchanged, and chlorophyllase activity was significantly reduced. EGTA pretreatment significantly reduced the chlorophyll levels of the overexpressing plants. These results indicate that SlCAS is a key positive regulator for maintaining chlorophyll homeostasis in tomato leaves under low night temperatures.

[0114] Test Example 2

[0115] Effects of SlCAS on calcium signaling in tomato chloroplasts under low night temperatures.

[0116] 2.1 Chloroplast Extraction

[0117] Weigh 3 g of fresh tomato leaves, cut them into small pieces, add the extraction solution (100 mM KCl, 250 mM mannitol, 0.2 mM HEPES, pH 7.0), and juice for 15 seconds. Filter through 6 layers of gauze. Centrifuge the filtrate at 1000 rpm for 2 minutes and discard the precipitate. Centrifuge the supernatant at 3000 rpm for 5 minutes; the precipitate is the chloroplast. Resuspend the chloroplast in the extraction solution and adjust the OD value to [value missing]. 600 =1.0, reserved.

[0118] 2.2 Non-destructive microelectrode technique for determining chloroplast Ca 2+ flux

[0119] The isolated intact chloroplasts (10 μg chlorophyll / mL) were suspended in buffer (0.33 M sorbitol, 50 mM methyl methacrylate-NaOH pH 8.0, 5 mM MgCl2, 100 μM CaCl2), and 100 μL aliquots were fixed onto a 0.22 μm MCE filter membrane. CaCl2 levels were recorded over 5 min using a non-destructive microelectrode system (NMT-YG-100, Younger USA LLC). 2+ Flux. In Ca 2+ Outflow is positive, inflow is negative.

[0120] 2.3 Fluo-4 AM staining

[0121] Tomato leaves were cut into strips approximately 0.2 mm thick and 2-3 mm long, and placed in phosphate buffer containing 5 μM Fluo-4 AM (Beyotime; S1060) and incubated at room temperature in the dark for 30 min. Excess dye was removed by washing three times with phosphate buffer. Fluorescence was observed using a confocal laser scanning microscope (TCS SP8, Leica) with an excitation wavelength of 488 nm and an emission wavelength of 520 nm.

[0122] 2.4 Experimental Results

[0123] Non-destructive micro-measurement results show ( Figure 5 A, B), low nighttime temperature treatment induces wild-type chloroplast Ca 2+ Inflow, net Ca2+ in SlCAS-OE plant chloroplasts 2+ The internal flow was significantly greater than that of the wild type. Fluo-4 AM staining results ( Figure 5 C) Further confirmation: Under low nighttime temperatures, the chloroplasts of SlCAS-OE plants show Ca... 2+ The signal was significantly enhanced, while Ca in Slcas plants 2+ The signal is significantly weakened.

[0124] Test Example 3

[0125] The effect of SlCAS on reactive oxygen species signaling in tomatoes at low night temperatures.

[0126] 3.1 H2DCF-DA staining for the detection of reactive oxygen species

[0127] Tomato leaves were incubated with 10 μM H2DCF-DA (MCE; HY-D0940) in the dark for 30 min, and then washed three times with distilled water. Fluorescence was detected using a confocal laser scanning microscope (TCS SP8, Leica) with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

[0128] 3.2 Assay of catalase activity

[0129] The catalase ELISA kit (Enzyme-linked Biotechnology; ML095171) was used according to the instructions. 0.1 g of leaf was weighed, ground in liquid nitrogen, added to PBS buffer, centrifuged, and the supernatant was collected. The supernatant was reacted with the enzyme-labeled antibody, and the absorbance was measured at 450 nm after color development. The CAT activity (U / mg protein) was calculated.

[0130] 3.3 Experimental Results

[0131] H2DCF-DA staining results showed ( Figure 6 A) After low nighttime temperature treatment, the fluorescence intensity of H2O2 in the leaves of Slcas plants was significantly higher than that of the wild type, while exogenous CaCl2 pretreatment significantly reduced the fluorescence intensity in Slcas plants. The fluorescence intensity of SlCAS-OE plants was significantly lower than that of the wild type. CAT activity assay results ( Figure 6 B and C show that the CAT activity of Slcas plants was significantly lower than that of the wild type; the CAT activity of SlCAS-OE plants was significantly higher than that of the wild type.

[0132] Test Example 4

[0133] Effects of SlCAS on tomato cell membrane damage at low night temperatures.

[0134] 4.1 Determination of malondialdehyde (MDA) content

[0135] Take 0.5 g of tomato leaves treated with low nighttime temperature for 7 days, add 5 mL of 10% trichloroacetic acid (TCA), and grind into a homogenate in an ice bath. Centrifuge at 10,000 g for 10 min at 4℃. Take 2 mL of the supernatant, add 2 mL of 0.6% thiobarbituric acid (TBA) solution (prepared with 10% TCA), mix well, heat in a boiling water bath for 15 min, cool rapidly, and centrifuge at 5,000 g for 10 min. Use distilled water instead of the supernatant as a blank control. Measure the absorbance of the supernatant at 450 nm, 532 nm, and 600 nm. Calculate the MDA content using the following formula:

[0136] MDA (nmol·g) -1 DW) = [6.452 × (OD) 532 – OD 600 - 0.559 × OD 450 × Total volume of extract (mL) / (dry weight of sample × 1.5)

[0137] 4.2 Relative Conductivity (REC) Measurement

[0138] Ten round slices of tomato leaves treated with low nighttime temperature were taken using a punch and placed in 20 mL of distilled water. The slices were shaken on a shaker at 28°C and 200 rpm for 1 h. The initial conductivity (S1) was measured using a conductivity meter. The samples were then boiled for 30 min, cooled to room temperature, and the final conductivity (S2) was measured. Distilled water was used as a blank control to measure the conductivity (S0). The relative conductivity was calculated using the following formula:

[0139] REC (%) = (S1 - S0) / (S2 - S0) × 100

[0140] 4.3 Experimental Results

[0141] like Figure 7 As shown in Figures A and B, after low night temperature treatment, compared with the wild type, the MDA and REC levels in the Slcas mutant plants were significantly increased, indicating exacerbated cell membrane damage and weakened tolerance to low night temperatures. Exogenous CaCl2 pretreatment can partially reduce the MDA and REC levels in Slcas plants. Figure 7 As shown in C and D, the MDA content and REC of SlCAS-OE overexpressing plants were significantly lower than those of wild type after low night temperature treatment, indicating that cell membrane integrity was better maintained.

[0142] This invention verifies the function of SlCAS in improving the resistance of tomatoes to low night temperatures through the above test examples:

[0143] Test Example 1 (Chlorophyll Content Determination): After low nighttime temperature treatment, compared with the wild type, the Slcas mutant plants showed a significant decrease in chlorophyll a content of approximately 18%, a significant decrease in chlorophyll b content of approximately 48%, a significant increase in the Chl a / b ratio of approximately 65%, and a significant increase in chlorophyllase activity. Exogenous calcium pretreatment could partially restore the mutant chlorophyll levels. SlCAS overexpression plants showed an increase in chlorophyll a and b contents of approximately 19-21% and 21-23%, respectively, and a significant decrease in chlorophyllase activity. These results indicate that SlCAS positively regulates chlorophyll accumulation under low nighttime temperatures.

[0144] Test Example 2 (Chloroplast Calcium Signal Detection): Non-destructive microelectrometry and Fluo-4 AM staining showed that low nighttime temperature treatment induced wild-type chloroplast calcium... 2+ Influx, Ca in SlCAS-overexpressing plants 2+ Influx was further enhanced, while Ca in the Slcas mutant was significantly increased. 2+ The signal was significantly weakened. This demonstrates that SlCAS mediates chloroplast calcium signaling induced by low night temperatures.

[0145] Test Example 3 (Detection of Reactive Oxygen Species Signal): H2DCF-DA staining and CAT activity assays showed that under low nighttime temperatures, the Slcas mutant exhibited significantly increased H2O2 accumulation and decreased CAT activity; overexpression plants showed decreased H2O2 accumulation and increased CAT activity. Exogenous calcium can alleviate oxidative stress in the mutant. This indicates that SlCAS enhances resistance by regulating reactive oxygen species homeostasis.

[0146] Test Example 4 (Cell Membrane Damage Detection): After low night temperature treatment, the MDA and REC levels in the Slcas mutant were significantly increased, indicating aggravated membrane damage; the MDA and REC levels in the overexpressing plants were significantly lower than those in the wild type, and the membrane integrity was better. Exogenous calcium treatment could partially reduce the MDA and REC levels in the mutant. This confirms that SlCAS is a positive regulator of low night temperature resistance.

[0147] In summary, SlCAS positively regulates the tolerance of tomatoes to low night temperature stress by maintaining chloroplast calcium signaling, inhibiting reactive oxygen species accumulation, and protecting chlorophyll metabolism and cell membrane integrity.

[0148] 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 tomato calcium-sensing protein SlCAS, 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. Use of the protein of claim 1 or the nucleic acid molecule of claim 2 in improving plant resistance to low night temperatures, wherein the low night temperature is a night temperature ≤6°C.

4. The use according to claim 3, characterized in that, The plant in question is tomato (Solanum lycopersicum).

5. The use according to claim 3, characterized in that, By overexpressing the nucleic acid molecule of claim 2, at least one of the following effects can be achieved: (a) Increase the content of chlorophyll a and / or chlorophyll b in plant leaves under low night temperatures; (b) Reduce the activity of chlorophyllase in plant leaves under low night temperatures; (c) Reduce the accumulation of hydrogen peroxide (H2O2) in plant leaves under low night temperatures; (d) Increase the activity of catalase CAT in plant leaves under low night temperatures; (e) Reduce the content of malondialdehyde (MDA) and / or relative conductivity (REC) in plant leaves at low night temperatures.

6. A method for improving plant resistance to low night temperatures, characterized in that, This includes operably linking the nucleic acid molecule of claim 2 to a promoter, constructing an overexpression vector, transforming plants, and screening to obtain transgenic plants that overexpress the protein of claim 1.

7. The method according to claim 6, characterized in that, This also includes applying a 5-50 mmol / L CaCl2 aqueous solution to the plants during periods of low nighttime temperature stress.

8. A molecular marker for screening plants sensitive to low night temperatures, characterized in that, The molecular marker is the CRISPR / Cas9 gene editing target sequence of the SlCAS gene. Mutant plants containing this target sequence and with loss of SlCAS gene function exhibit increased sensitivity to low night temperatures.

9. Use of the protein of claim 1 or the nucleic acid molecule of claim 2 in the preparation of a plant low night temperature resistance identification kit or breeding kit.

10. A recombinant expression vector for enhancing plant resistance to low night temperatures, characterized in that, The recombinant expression vector contains the nucleic acid molecule of claim 2, and the nucleic acid molecule is operatively linked to a constitutive promoter or an inducible promoter.

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