Rapid identification method for salt tolerance of vegetables based on calea-dc3 expression product
By employing standardized experimental procedures and multiple validation methods, qRT-PCR and ELISA were used to detect the mRNA and protein of the CaLEA-Dc3 gene. This solved the problems of lag and subjectivity in the identification of salt tolerance in vegetables, and enabled rapid and accurate identification of salt tolerance. It is suitable for the screening and breeding research of varieties grown in saline-alkali soils.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HUANGHUAI UNIV
- Filing Date
- 2026-05-09
- Publication Date
- 2026-06-09
AI Technical Summary
Existing methods for identifying salt tolerance in vegetables suffer from lag and subjectivity, and lack complementary verification at the transcriptional and translational levels, leading to biased results and insufficient universality of application, making it difficult to achieve rapid and accurate salt tolerance screening.
Standardized experimental procedures were adopted, and specific primers were designed and antibodies were validated. The mRNA expression level of CaLEA-Dc3 gene was detected by qRT-PCR and the protein concentration of CaLEA-Dc3 was detected by ELISA. Complementary validation of transcription and translation levels was carried out, and a salt tolerance threshold was set and repeated tests were performed to ensure data reliability.
It significantly improves the accuracy and efficiency of salt tolerance identification in vegetables, provides quantifiable scientific classification basis, and is suitable for rapid high-throughput classification in the screening and breeding research of varieties grown in saline soils.
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Figure CN122168733A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a rapid identification method for salt tolerance of vegetables based on CaLEA-Dc3 expression products. Background Technology
[0002] Salt tolerance identification in vegetables is a core component of agriculture in saline-alkali soils and stress-resistance breeding. However, traditional methods mainly rely on plant phenotypic observations (such as growth inhibition and leaf wilting), which are significantly time-consuming and subjective. Because the effects of salt stress take a long time to manifest, the identification cycle is lengthy; furthermore, phenotypic characteristics are easily affected by environmental interference or human judgment bias, making accurate classification difficult, especially in the early screening stage where potential salt-tolerant varieties cannot be quickly identified. Therefore, developing rapid, objective, and quantitative methods based on molecular markers is a key requirement for improving the efficiency of vegetable salt tolerance identification. Existing methods for identifying vegetable salt tolerance based on CaLEA-Dc3 gene expression products mostly employ single-level detection, lacking complementary verification at the transcriptional and translational levels. This makes them prone to result deviations due to differences in translational regulation or experimental errors. Furthermore, insufficient standardization of experimental procedures, lack of RNA purity thresholds, unverified antibodies, and missing repeatability design make it difficult to guarantee data reliability. These issues directly lead to significant limitations in the accuracy, repeatability, and applicability of existing technologies. Summary of the Invention
[0003] In view of this, the present invention provides a rapid identification method for salt tolerance of vegetables based on CaLEA-Dc3 expression products. This method ensures data reliability through a standardized experimental procedure: specific primer design, antibody verification, and repeated verification. It overcomes the lag and subjectivity of traditional phenotypic observation, providing quantifiable scientific classification evidence. Through multiple verifications, complementary verification at the transcriptional and translational levels is achieved, avoiding errors from single detections and ensuring the reliability of salt tolerance assessment results. This significantly improves the efficiency and accuracy of salt tolerance identification in vegetables. Furthermore, the complementary detection at the transcriptional and translational levels enhances the method's universality, making it suitable for rapid, high-throughput classification needs in the screening and breeding research of varieties grown in saline-alkali soils.
[0004] To address the aforementioned technical problems, this invention provides a rapid identification method for salt tolerance of vegetables based on CaLEA-Dc3 expression products, comprising the following steps: Vegetable materials with consistent growth status were selected and divided into a control group and a salt stress treatment group. The control group was given water, while the salt stress treatment group was given a salt solution. The CaLEA-Dc3 expression product was used as the detection target. When the CaLEA-Dc3 expression product is mRNA transcribed from the CaLEA-Dc3 gene, it is detected by qRT-PCR. When the CaLEA-Dc3 expression product is the protein produced by the translation of the CaLEA-Dc3 gene, it is detected by ELISA. Verify the repeatability of the test results and determine the critical threshold for salt tolerance by combining salt-tolerant vegetable varieties; The test results of the sample to be identified are compared with the salt tolerance threshold to determine the salt tolerance level of the vegetable, and finally the identification result of the salt tolerance of the vegetable is obtained.
[0005] The vegetable material samples were leaves or root tissues, and the salt stress treatment group was treated with sodium chloride solution.
[0006] When the CaLEA-Dc3 expression product is mRNA transcribed from the CaLEA-Dc3 gene, it is detected using qRT-PCR, including the following steps: Total RNA was extracted from vegetable samples using the TRIzol method. After verifying the integrity and purity of the RNA, cDNA was synthesized by reverse transcription. Design specific primers for the CaLEA-Dc3 gene and internal reference primers for the housekeeping gene; The forward primer sequence for the specific primer is 5'-ATGGCTCCAAACGACAAGAC-3', and the reverse primer sequence is 5'-TCACATCCACGTTGGATCTC-3'. Using cDNA as a template, qRT-PCR was performed. The reaction system included real-time PCR premix, upstream and downstream primers, cDNA template, and enzyme-free water. The reaction program was a qRT-PCR program suitable for CaLEA-Dc3 gene detection, including pre-denaturation, denaturation, annealing and extension steps, and melting curve analysis was performed after the cycle was completed; The relative expression level of the CaLEA-Dc3 gene was calculated using the 2^-ΔΔCT method.
[0007] Total RNA was extracted from vegetable samples using the TRIzol method, including the following steps: RNA integrity was verified by gel electrophoresis; RNA purity was determined using a spectrophotometer.
[0008] The critical threshold for salt tolerance is: the relative expression level of the CaLEA-Dc3 gene in the salt stress group is ≥2 times that in the control group.
[0009] The criteria for determining salt tolerance level are as follows: relative expression level ≥ 2 times is salt-tolerant, 1-2 times is moderately salt-tolerant, and < 1 times is salt-sensitive.
[0010] When the CaLEA-Dc3 expression product is the protein produced by the translation of the CaLEA-Dc3 gene, it is detected using an ELISA method, including the following steps: A specific polyclonal antibody against CaLEA-Dc3 protein was prepared, and its specificity was verified after purification by affinity chromatography. Total protein was extracted from vegetable samples, and the total protein concentration was determined using the BCA method. To perform ELISA testing, the steps of coating, blocking, sample addition, secondary antibody incubation, and color development are completed sequentially, and the absorbance value is measured using a microplate reader. A standard curve was plotted based on the recombinant CaLEA-Dc3 protein standard, and the actual concentration of CaLEA-Dc3 protein in the sample was calculated.
[0011] The critical threshold for salt tolerance is: the concentration of CaLEA-Dc3 protein in vegetable samples is ≥50 ng / mL.
[0012] The criteria for determining salt tolerance levels are as follows: a protein concentration ≥ 50 ng / mL indicates a salt-tolerant variety, 30-50 ng / mL indicates a moderately salt-tolerant variety, and < 30 ng / mL indicates a salt-sensitive variety.
[0013] Each group of vegetable samples should have at least two biological replicates, and the coefficient of variation of the test results should be ≤10%.
[0014] The beneficial effects of the above-described technical solution of the present invention are as follows: 1. By dividing vegetable materials into a control group treated with water and a salt stress group treated with 200 mmol / L NaCl solution, the mRNA and protein of the CaLEA-Dc3 gene were used as detection targets. Quantitative detection methods were selected according to the expression product type. Total RNA could be extracted using the TRIzol method, or ELISA could be used. The expression level of CaLEA-Dc3 gene mRNA was detected by qRT-PCR, and the protein concentration was detected by ELISA. This achieved complementary verification at the transcriptional and translational levels, avoiding errors from single detection, ensuring the reliability of salt tolerance assessment results, significantly improving the efficiency and accuracy of vegetable salt tolerance identification, and enhancing the universality of the method through complementary detection at the transcriptional and translational levels. It is especially suitable for the rapid and high-throughput classification needs in the screening and breeding research of varieties grown in saline-alkali soils.
[0015] 2. Set salt tolerance thresholds: mRNA ≥ 2 times, protein ≥ 50 ng / mL and three-level classification criteria, and require three biological replicates for each sample group, with a coefficient of variation ≤ 10%. Through standardized experimental procedures: primer design, antibody verification, and replication verification, overcome the lag and subjectivity of traditional phenotypic observation and provide quantifiable scientific classification basis.
[0016] 3. The combination of 40-cycle melting curve analysis with ELISA microplate color development and standard curve calculation technology, along with preset thresholds, enables rapid processing of large numbers of samples. At the same time, the dual-dimensional detection based on the molecular marker CaLEA-Dc3 significantly improves the efficiency of salt tolerance identification. Attached Figure Description
[0017] Figure 1 This is a flowchart of the rapid identification method for salt tolerance of vegetables based on CaLEA-Dc3 expression products according to the present invention. Figure 2 This is a flowchart of the vegetable sample pretreatment method of the present invention; Figure 3 This is a flowchart of the qRT-PCR method for detecting CaLEA-Dc3 mRNA according to the present invention; Figure 4 This is a flowchart of the TRIzol method for extracting CaLEA-Dc3 protein according to the present invention. Detailed Implementation
[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will be described in conjunction with the accompanying drawings of the embodiments of the present invention. Figure 1-4 The technical solutions of the embodiments of the present invention will be clearly and completely described herein. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention are within the scope of protection of the present invention.
[0019] This embodiment provides a rapid method for identifying salt tolerance in vegetables based on the CaLEA-Dc3 expression product, such as... Figure 1-4 As shown: Includes the following steps: Vegetable materials with consistent growth status were screened and divided into a control group and a salt stress treatment group. The control group was given water, while the salt stress treatment group was given a salt solution. The vegetable material samples were leaves or root tissues. The salt stress treatment group was treated with sodium chloride solution. The CaLEA-Dc3 expression product was used as the detection target. When the CaLEA-Dc3 expression product is mRNA transcribed from the CaLEA-Dc3 gene, it is detected by qRT-PCR. When the CaLEA-Dc3 expression product is the protein produced by the translation of the CaLEA-Dc3 gene, it is detected by ELISA. Verify the repeatability of the test results and determine the critical threshold for salt tolerance by combining salt-tolerant vegetable varieties; The test results of the sample to be identified are compared with the salt tolerance threshold to determine the salt tolerance level of the vegetable, and finally the identification result of the salt tolerance of the vegetable is obtained.
[0020] Vegetable seedlings of uniform growth, such as tomatoes, cucumbers, and carrots, at the 3-4 leaf stage, or plump and uniform vegetable seeds, were selected and divided into a control group treated with water and a salt stress group. Each group had 3 biological replicates. A 200 mmol / L NaCl solution was used as the salt stress solution to irrigate the seedlings or to soak the seeds. The control group was treated with an equal amount of water. The seedlings were placed under the same environmental conditions, such as a temperature of 25±2℃ and a light / dark cycle of 16h and 8h for 24-48h. Functional leaves or root tissues of the vegetables after stress treatment were selected, with priority given to roots as they have a more direct salt stress response. The tissues were rinsed with sterile water, dried with filter paper, and quickly frozen in liquid nitrogen for subsequent nucleic acid or protein extraction.
[0021] like Figure 1-3 As shown, when the CaLEA-Dc3 expression product is mRNA transcribed from the CaLEA-Dc3 gene, it is detected using qRT-PCR, including the following steps: Total RNA was extracted from vegetable samples using the TRIzol method. After verifying the integrity and purity of the RNA, cDNA was synthesized by reverse transcription. Design specific primers for the CaLEA-Dc3 gene and internal reference primers for the housekeeping gene; The forward primer sequence for the specific primer is 5'-ATGGCTCCAAACGACAAGAC-3', and the reverse primer sequence is 5'-TCACATCCACGTTGGATCTC-3'. Using cDNA as a template, qRT-PCR was performed. The reaction system included real-time PCR premix, upstream and downstream primers, cDNA template, and enzyme-free water. The reaction program was a qRT-PCR program suitable for CaLEA-Dc3 gene detection, including pre-denaturation, denaturation, annealing and extension steps, and melting curve analysis was performed after the cycle was completed; The relative expression level of the CaLEA-Dc3 gene was calculated using the 2^-ΔΔCT method.
[0022] Take 0.1g of frozen sample and place it in a pre-chilled mortar. Grind it into powder and add 1mL of TRIzol reagent. Mix thoroughly and let stand at room temperature for 5min. Add chloroform and isopropanol according to the kit instructions for layering and precipitation. Wash with 75% ethanol and dissolve RNA in RNase-free water to obtain a total RNA solution. Then, check the RNA integrity by 1.5% agarose gel electrophoresis and check the RNA purity using a spectrophotometer. An OD260 / OD280 ratio between 1.8 and 2.0 is considered acceptable concentration. Using the acceptable total RNA as a template, add oligo(dT) primers, reverse transcriptase, dNTPs, and other reagents according to the reverse transcription kit instructions. Perform reverse transcription on a PCR instrument under the following conditions: 42℃ for 60min, 70℃ for 15min to terminate the reaction, and obtain cDNA product. Store at -20℃ for later use. Based on the conserved sequence of the CaLEA-Dc3 gene, use Premi Specific primers were designed using er5 software. Example of a forward primer: 5'-ATGGCTCCAAACGACAAGAC-3'; example of a reverse primer: 5'-TCACATCCACGTTGGATCTC-3'. An internal control primer for the housekeeping gene was also designed to correct for detection errors. A 20 μL reaction mixture was prepared, containing 10 μL of 2×SYBR GreenMix, 0.5 μL each of forward and reverse primers, 2 μL of cDNA template, and 7 μL of enzyme-free water. The reaction program was set as follows: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s; 60℃ annealing and extension for 30 s, for a total of 40 cycles. After cycling, melting curve analysis was performed at 65℃-95℃, with a 5 s interval of 0.5℃ to verify product specificity. The relative expression level of the CaLEA-Dc3 gene was calculated using the 2^-ΔΔCT method, where ΔCT = target gene CT value - internal control gene CT value, and ΔΔCT = stress group ΔCT - control group ΔCT.
[0023] like Figure 1-3 As shown, total RNA was extracted from vegetable samples using the TRIzol method, including the following steps: RNA integrity was verified by gel electrophoresis; RNA purity was determined using a spectrophotometer.
[0024] After extracting total RNA from vegetable leaves using the TRIzol method, the integrity was first verified by 1.2% agarose gel electrophoresis: 1 μg of RNA was mixed with RNA loading buffer, electrophoresed for 30 minutes, and then observed under a UV transilluminator. Qualified samples should clearly show 28S and 18S rRNA bands, and the gray ratio of the two should be ≥1.8. Subsequently, the purity was detected using a NanoDrop spectrophotometer, requiring an A260 / A280 ratio ≥1.9 and an A260 / A230 ratio ≥2.0. Samples that did not meet the standards needed to be repurified or discarded. Only RNA that passed both tests could be used for subsequent qRT-PCR. This process, through dual verification of physical separation and spectral analysis, ensured the reliability of downstream experimental data and avoided deviations in transcriptional level detection caused by RNA degradation or contamination.
[0025] like Figure 1-3 As shown, the critical threshold for salt tolerance is: the relative expression level of the CaLEA-Dc3 gene in the salt stress group is ≥2 times that of the control group. The criteria for judging the salt tolerance level are: relative expression level ≥2 times is a salt-tolerant variety, 1-2 times is a moderately salt-tolerant variety, and <1 times is a salt-sensitive variety.
[0026] like Figure 1-4 As shown, when the CaLEA-Dc3 expression product is the protein produced by the translation of the CaLEA-Dc3 gene, it is detected using the ELISA method, including the following steps: A specific polyclonal antibody against CaLEA-Dc3 protein was prepared, and its specificity was verified after purification by affinity chromatography. Total protein was extracted from vegetable samples, and the total protein concentration was determined using the BCA method. To perform ELISA testing, the steps of coating, blocking, sample addition, secondary antibody incubation, and color development are completed sequentially, and the absorbance value is measured using a microplate reader. A standard curve was plotted based on the recombinant CaLEA-Dc3 protein standard, and the actual concentration of CaLEA-Dc3 protein in the sample was calculated.
[0027] like Figure 1-4 As shown, the critical threshold for salt tolerance is: the concentration of CaLEA-Dc3 protein in vegetable samples is ≥50 ng / mL. The criteria for judging the salt tolerance level are: a protein concentration ≥50 ng / mL is a salt-tolerant variety, 30-50 ng / mL is a moderately salt-tolerant variety, and <30 ng / mL is a salt-sensitive variety.
[0028] Specific antigenic peptides of CaLEA-Dc3 protein were synthesized in advance and polyclonal antibodies were prepared by immunizing rabbits. After purification of the antibodies by affinity chromatography, Western blot was used to verify antibody specificity, ensuring that they only bind to CaLEA-Dc3 protein and have no cross-reactivity. 0.5g of frozen sample was taken and added to pre-chilled protein extraction buffer containing protease inhibitors to avoid protein degradation. The mixture was homogenized on ice. Centrifuged at 12000×g for 15 min at 4°C, and the supernatant was collected as the total protein extract. The BCA method was used to prepare a standard curve and reaction system according to the kit instructions. The absorbance value at 562nm was measured by an ELISA reader to calculate the total protein concentration in the sample, which was used for subsequent sample dilution and standardization. For ELISA testing, the purified CaLEA-Dc3 antibody was diluted to 10μg / mL with coating buffer, added to the ELISA plate, and incubated overnight at 4°C. The next day... Discard the liquid in the wells, wash three times with PBST buffer for 3 min each time, add 200 μL of 5% skim milk blocking buffer to each well, and incubate at room temperature for 2 h; discard the blocking buffer, wash three times with PBST, dilute the total protein extract of the sample 100-fold with the blocking buffer, and prepare a series of recombinant CaLEA-Dc3 protein standards at different concentrations; add 100 μL of diluted sample or standard to each well and incubate at 37°C for 1 h; wash three times with PBST, add 100 μL of HRP-labeled secondary antibody to each well, and incubate at 37°C for 30 min; wash three times with PBST, and perform color development and reading: add 100 μL of TMB colorimetric solution to each well and incubate at room temperature in the dark for 15 min; add 50 μL of stop solution to terminate the reaction, and measure the absorbance at 450 nm using a microplate reader. Plot a standard curve based on the absorbance values of the standards, substitute the absorbance values of the samples, and calculate the actual concentration of CaLEA-Dc3 protein in the sample.
[0029] like Figure 1-4 As shown, each group of vegetable samples was set up with at least two biological replicates, and the coefficient of variation of the test results was ≤10%.
[0030] By comparing the expression levels or concentrations between the salt stress group and the control group, a critical threshold for salt tolerance was set: mRNA must be ≥2 times that of the control group, and protein must be ≥50 ng / mL. Based on the threshold, the salt tolerance of the samples was divided into three levels: relative expression level ≥2 times or protein concentration ≥50 ng / mL for salt-tolerant varieties, 1-2 times or 30-50 ng / mL for moderately salt-tolerant varieties, and <1 times or <30 ng / mL for salt-sensitive varieties. Each group of samples was required to have three biological replicates, and the coefficient of variation of the test results was required to be ≤10%. A standardized experimental procedure was used: specific primer design, antibody verification, and replication verification to ensure data reliability. Ultimately, precise quantification based on the molecular marker CaLEA-Dc3 was achieved, overcoming the lag and subjective limitations of traditional phenotypic observation and providing quantifiable scientific classification evidence. If the results exceeded the range, retesting was required. Simultaneously, traditional physiological salt tolerance indicators of vegetables, such as relative conductivity and proline content, were tested to verify the consistency between the CaLEA-Dc3 expression product detection results and physiological indicators, improving the reliability of identification.
[0031] The working principle of the rapid identification method for salt tolerance of vegetables based on CaLEA-Dc3 expression products provided by this invention is as follows: Vegetable materials are divided into a control group treated with water and a salt stress group treated with 200 mmol / L NaCl solution. The mRNA and protein of the CaLEA-Dc3 gene are used as detection targets. A quantitative detection method is selected according to the type of expression product: When detecting mRNA, total RNA is extracted using the TRIzol method. Integrity is verified by 1.5% agarose gel electrophoresis, and purity is confirmed by spectrophotometer detection of the OD260 / OD280 ratio (1.8-2.0). The RNA is then reverse transcribed into cDNA, and specific primers are used for forward 5'-ATGGCTCCAAACGACAAGAC-3', reverse 5'-TCACATCCACGTTGGATCTC-3', and SYBR. qRT-PCR amplification was performed using the Green system. The reaction program included 40 cycles of 95℃ pre-denaturation, 95℃ denaturation for 5 seconds, and 60℃ annealing extension for 30 seconds. Specificity was ensured by melting curve analysis. Finally, the relative expression level of the CaLEA-Dc3 gene was calculated using the 2^-ΔΔCT method. When detecting protein, a CaLEA-Dc3 specific polyclonal antibody was prepared, purified by affinity chromatography, and the total protein concentration was determined by the BCA method. ELISA was then performed, sequentially completing the steps of antigen coating, blocking buffer blocking, sample addition, secondary antibody incubation, and color development. The absorbance was measured using a microplate reader, and the actual concentration was calculated using a standard curve plotted with recombinant protein standards. Subsequently, by comparing the expression levels or concentration differences between the salt stress group and the control group, a salt tolerance threshold was set: mRNA ≥ 2 times that of the control group, and protein ≥ 50 ng / mL. Based on this threshold, the salt tolerance of the samples was divided into three levels: relative expression level ≥ 2 times or protein concentration ≥ 50 ng / mL for salt-tolerant varieties, 1-2 times or 30 times for salt-tolerant varieties, and so on. -50 ng / mL indicates a moderately salt-tolerant variety, while <1 ng / mL or <30 ng / mL indicates a salt-sensitive variety. Each sample group must have three biological replicates, and the coefficient of variation of the test results must be ≤10%. A standardized experimental procedure—specific primer design, antibody validation, and replication validation—ensures data reliability. Ultimately, precise quantification based on the molecular marker CaLEA-Dc3 is achieved, overcoming the lag and subjective limitations of traditional phenotypic observation. This provides quantifiable scientific classification evidence, significantly improving the efficiency and accuracy of vegetable salt tolerance identification. Furthermore, the complementary detection methods at the transcriptional and translational levels enhance the method's universality, making it particularly suitable for rapid, high-throughput classification needs in variety screening and breeding research in saline-alkali soils.
[0032] Furthermore, it should be noted that, in the description of this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.
[0033] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A rapid method for identifying salt tolerance in vegetables based on CaLEA-Dc3 expression products, characterized in that: Including the following steps: Vegetable materials with consistent growth status were selected and divided into a control group and a salt stress treatment group. The control group was given water, while the salt stress treatment group was given a salt solution. The CaLEA-Dc3 expression product was used as the detection target. When the CaLEA-Dc3 expression product is mRNA transcribed from the CaLEA-Dc3 gene, it is detected by qRT-PCR. When the CaLEA-Dc3 expression product is the protein produced by the translation of the CaLEA-Dc3 gene, it is detected by ELISA. Verify the repeatability of the test results and determine the critical threshold for salt tolerance by combining salt-tolerant vegetable varieties; The test results of the sample to be identified are compared with the salt tolerance threshold to determine the salt tolerance level of the vegetable, and finally the identification result of the salt tolerance of the vegetable is obtained.
2. The rapid identification method for vegetable salt tolerance based on CaLEA-Dc3 expression product as described in claim 1, characterized in that: The vegetable material samples were leaves or root tissues, and the salt stress treatment group was treated with sodium chloride solution.
3. The rapid identification method for vegetable salt tolerance based on CaLEA-Dc3 expression product as described in claim 1, characterized in that: When the CaLEA-Dc3 expression product is mRNA transcribed from the CaLEA-Dc3 gene, it is detected using qRT-PCR, including the following steps: Total RNA was extracted from vegetable samples using the TRIzol method. After verifying the integrity and purity of the RNA, cDNA was synthesized by reverse transcription. Design specific primers for the CaLEA-Dc3 gene and internal reference primers for the housekeeping gene; The forward primer sequence for the specific primer is 5'-ATGGCTCCAAACGACAAGAC-3', and the reverse primer sequence is 5'-TCACATCCACGTTGGATCTC-3'. Using cDNA as a template, qRT-PCR was performed. The reaction system included real-time PCR premix, upstream and downstream primers, cDNA template, and enzyme-free water. The reaction program was a qRT-PCR program suitable for CaLEA-Dc3 gene detection, including pre-denaturation, denaturation, annealing and extension steps, and melting curve analysis was performed after the cycle was completed; The relative expression level of the CaLEA-Dc3 gene was calculated using the 2^-ΔΔCT method.
4. The rapid identification method for salt tolerance of vegetables based on CaLEA-Dc3 expression products as described in claim 3, characterized in that: Total RNA was extracted from vegetable samples using the TRIzol method, including the following steps: RNA integrity was verified by gel electrophoresis; RNA purity was determined using a spectrophotometer.
5. The rapid identification method for salt tolerance of vegetables based on CaLEA-Dc3 expression product as described in claim 3, characterized in that: The critical threshold for salt tolerance is: the relative expression level of the CaLEA-Dc3 gene in the salt stress group is ≥ 2 times that of the control group.
6. The rapid identification method for salt tolerance of vegetables based on CaLEA-Dc3 expression products as described in claim 5, characterized in that: The criteria for determining salt tolerance levels are as follows: relative expression levels ≥ 2 times indicate salt-tolerant varieties, 1-2 times indicate moderately salt-tolerant varieties, and < 1 times indicate salt-sensitive varieties.
7. The rapid identification method for salt tolerance of vegetables based on CaLEA-Dc3 expression products as described in claim 1, characterized in that: When the CaLEA-Dc3 expression product is the protein produced by the translation of the CaLEA-Dc3 gene, it is detected using an ELISA method, including the following steps: A specific polyclonal antibody against CaLEA-Dc3 protein was prepared, and its specificity was verified after purification by affinity chromatography. Total protein was extracted from vegetable samples, and the total protein concentration was determined using the BCA method. To perform ELISA testing, the steps of coating, blocking, sample addition, secondary antibody incubation, and color development are completed sequentially, and the absorbance value is measured using a microplate reader. A standard curve was plotted based on the recombinant CaLEA-Dc3 protein standard, and the actual concentration of CaLEA-Dc3 protein in the sample was calculated.
8. The rapid identification method for salt tolerance of vegetables based on CaLEA-Dc3 expression products as described in claim 7, characterized in that: The salt tolerance threshold is: the concentration of CaLEA-Dc3 protein in the vegetable sample is ≥50 ng / mL.
9. The rapid identification method for salt tolerance of vegetables based on CaLEA-Dc3 expression products as described in claim 8, characterized in that: The criteria for determining salt tolerance levels are as follows: a protein concentration ≥ 50 ng / mL indicates a salt-tolerant variety, 30-50 ng / mL indicates a moderately salt-tolerant variety, and < 30 ng / mL indicates a salt-sensitive variety.
10. The rapid identification method for salt tolerance of vegetables based on CaLEA-Dc3 expression products as described in claim 1, characterized in that: Each group of vegetable samples was set up with at least two biological replicates, and the coefficient of variation of the test results was ≤10%.