Tomato hy5 gene low temperature response state comprehensive evaluation method based on time series analysis and application thereof

Abstract

The application discloses a kind of tomato HY5 gene low temperature response state comprehensive evaluation method based on time series analysis and application thereof, belong to plant molecular biology and agricultural cultivation technical field;The application is handled by low temperature stress to tomato seedling, and the expression amount of HY5 gene is detected by collecting leaf sample at multiple continuous time points, expression amount dynamic change curve is drawn, response starting point and peak point of HY5 gene starting low temperature response are determined, and critical period of low temperature response is defined;In the critical period, the indexes of four dimensions of dynamic characteristics, ratio characteristics, recovery characteristics and integral characteristics are detected respectively, and the low temperature response state of tomato plant is comprehensively evaluated.The application first combines time series analysis with multi-dimensional evaluation index, systematically evaluates the low temperature response process mediated by tomato HY5 gene, and can be applied to early screening of tomato low temperature tolerance germplasm and low temperature early warning in facility cultivation, to provide scientific basis and technical support for tomato stress resistance evaluation and cultivation management.

Description

Technical Field

[0001] This invention relates to the fields of plant molecular biology and agricultural cultivation technology, specifically to a comprehensive evaluation method for the low-temperature response status of the tomato HY5 gene based on time series analysis and its application. Background Technology

[0002] Tomato (Solanum lycopersicum L.) is an important vegetable crop widely cultivated globally. Originally from tropical South America, it prefers warm temperatures and is intolerant of cold. In early spring and autumn-winter greenhouse cultivation, low-temperature stress is one of the main environmental factors limiting tomato growth and development, and affecting yield and quality. Therefore, elucidating the molecular mechanisms of tomato's response to low temperatures and establishing precise methods for assessing low-temperature responses are of great significance for screening and identifying cold-resistant germplasm and optimizing cold-resistant cultivation techniques.

[0003] The HY5 (ELONGATED HYPOCOTYL 5) gene is a core transcription factor in plant photomorphogenesis and belongs to the bZIP transcription factor family. Recent studies have shown that HY5 not only participates in light signal transduction but also plays a wide role in plant responses to various abiotic stresses. In tomatoes, low temperature can induce transcription and protein accumulation of the SlHY5 gene; SlHY5 expression shows a gradually upregulated trend under low temperature stress; overexpression of SlHY5 significantly enhances the low temperature resistance of tomatoes, while silencing SlHY5 increases the plant's cold sensitivity. At the molecular level, the molecular mechanism by which the SlHY5-SlBBX31-SlCBFs molecular module regulates low-temperature resistance has been elucidated. SlHY5 can directly bind to and activate the transcription of the ABA synthesis gene SlNCED6 and the GA inactivation gene SlGA2ox4, thereby enhancing low-temperature resistance by regulating hormone balance. SlHY5 can also physically interact with SlFHY3 to promote the transcriptional activity of SlHY5 on SlMIPS3, thereby enhancing cold resistance through the synergistic effect of inositol and light signals. In addition, SlHY5 plays an important role in the low-temperature response of tomatoes through the HY5-MYB15-CBFs transcriptional cascade.

[0004] However, current research on the response characteristics of the tomato HY5 gene under low temperature stress still has the following shortcomings: First, there is a lack of detailed time series analysis. Although existing studies have reported that low temperature can induce the upregulation of SlHY5 expression, most of them use detection at a single time point or a few time points, and have not systematically analyzed the temporal expression characteristics of the HY5 gene throughout the entire process of low temperature treatment, nor have they accurately defined the time point of response initiation, the time point of reaching the peak, and the start and end range of the entire response window.

[0005] Second, there is a lack of a multi-dimensional comprehensive evaluation system. Current technologies only focus on the expression changes of the HY5 gene itself, and have not yet integrated multi-dimensional indicators such as the temporal expression characteristics of HY5, its paired expression relationship with downstream target genes, the expression recovery characteristics after stress relief, and the time integral expression level to form a complete method for evaluating the low-temperature response status.

[0006] Third, there is a lack of practical application technologies. Although the molecular mechanism by which HY5 regulates low-temperature resistance has been extensively studied, existing technologies remain at the level of basic research and have not yet been transformed into technical solutions applicable to germplasm screening and cultivation management.

[0007] Therefore, developing a method based on time series analysis that can systematically evaluate the low-temperature response status of tomatoes and applying it to the screening of low-temperature tolerant germplasm and low-temperature early warning in greenhouse cultivation has important theoretical significance and industrial application value. Summary of the Invention

[0008] To address the aforementioned problems, the present invention aims to provide a comprehensive evaluation method for the low-temperature response status of the tomato HY5 gene based on time series analysis and its application, thereby overcoming the shortcomings of existing technologies, such as the lack of a systematic analysis method for the time-series characteristics of the low-temperature response of the tomato HY5 gene, the lack of a multi-dimensional comprehensive evaluation system, and the lack of operable application technology solutions.

[0009] The objective of this invention is achieved through the following technical solution: First, this invention provides a comprehensive assessment method for the low-temperature response status of the tomato HY5 gene based on time series analysis, comprising the following steps: (1) Low temperature stress treatment was applied to tomato seedlings; (2) Tomato leaf samples were collected at multiple consecutive time points after low-temperature treatment; (3) Detect the expression level of HY5 gene in samples at each time point, plot the dynamic curve of the expression level of HY5 gene with the low temperature treatment time based on the expression level of HY5 gene at each time point, determine the response initiation point and peak point of HY5 gene to initiate low temperature response based on the dynamic curve, and determine the continuous interval from the response initiation point to the peak point as the key period for HY5 gene to initiate low temperature response. (4) During the aforementioned critical period, the following four dimensions of indicators will be monitored: a) Dynamic characteristics: Expression levels of the HY5 gene at the response initiation and peak points; b) Ratio characteristics: The expression ratio of the HY5 gene to its downstream target genes; c) Recovery characteristics: The rate of decline in HY5 expression levels during the recovery process after the low-temperature treatment and subsequent return to room temperature culture; d) Integral characteristics: time-integrated expression level of the HY5 gene during low-temperature treatment; (5) Compare the four indicators in step (4) with the reference benchmark respectively, and comprehensively determine the low temperature response status of the tomato plant based on the number of indicators that meet the standards in each dimension.

[0010] Preferably, the temperature of the low-temperature stress treatment is 2-6°C.

[0011] Preferably, the plurality of consecutive time points include at least 1 hour, 3 hours, 6 hours, 12 hours and 24 hours after the low temperature treatment.

[0012] Preferably, in step (3), the expression level of the HY5 gene is detected by real-time PCR.

[0013] Preferably, in step (3), the expression level at the early time point of low-temperature treatment is used as the benchmark value.

[0014] Preferably, in step (3), the significant increase is defined as a statistical significance test, where the probability of significance (P) is less than 0.05.

[0015] Preferably, the downstream target gene is at least one of SlCBF1, SlNCED6, or SlMIPS3.

[0016] Preferably, the time integral expression is calculated according to the following formula: AUC=Σ[(E i + E i+1 ) / 2×Δt i ] Where E i Let Δt be the relative expression level at the i-th time point. i This represents the time interval between two adjacent time points.

[0017] Secondly, this invention provides the application of the above method in the screening of low-temperature resistant tomato germplasm.

[0018] Finally, this invention provides the application of the above method in low-temperature early warning for tomato greenhouse cultivation.

[0019] The beneficial effects of this invention are as follows: (1) This invention, through systematic sampling and expression level detection at multiple consecutive time points, accurately plotted the time-series expression dynamic curve of the tomato HY5 gene under low temperature stress for the first time, clarified the time position of the response initiation point and peak point, defined the key period for HY5 to initiate the low temperature response, and provided accurate time basis for subsequent evaluation.

[0020] (2) This invention integrates four dimensions—dynamic features (expression levels at the start and peak points), ratio features (HY5 / target gene expression ratio), recovery features (recovery rate after stress relief) and integral features (time integral expression level AUC)—into a complete evaluation system for the first time. This overcomes the limitations of single-indicator evaluation and realizes a multi-angle and all-round evaluation of the low-temperature response status of tomatoes.

[0021] (3) Existing technologies only focus on the expression level of HY5 itself. This invention introduces the pairing detection of transcription factors and their downstream target genes into the assessment of low temperature response for the first time. The expression ratio of HY5 / target gene can reflect the actual working efficiency of transcription factors, providing a new quantitative indicator for assessing the quality of low temperature response.

[0022] (4) Existing technologies only study the expression of HY5 during low temperature stress. This invention reveals for the first time that the rate of decline of HY5 expression after stress relief can be used as an effective indicator for assessing the degree of low temperature damage and the plant's recovery status.

[0023] (5) This invention integrates multi-time point expression data into the area under the curve (AUC) for the first time, overcoming the defect that single-point detection is easily affected by sampling time deviation, and providing a more stable and reliable means of quantitative evaluation of response intensity.

[0024] (6) The comprehensive evaluation method provided by the present invention can be directly applied to the early screening of low-temperature resistant tomato germplasm and low-temperature early warning in facility cultivation, providing a scientific basis for breeding selection and cultivation decisions. Attached Figure Description

[0025] Figure 1 This is a graph showing the dynamic changes in the relative expression level of the tomato HY5 gene at different time points under 4℃ low-temperature stress in Example 1 of the present invention.

[0026] Figure 2 This is a flowchart illustrating the comprehensive evaluation method for the low-temperature response status of the tomato HY5 gene based on time series analysis, as proposed in this invention. Detailed Implementation

[0027] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The described embodiments are merely 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. Example 1

[0028] This embodiment provides a method for identifying the critical period of low-temperature response of the tomato HY5 gene, including the following steps: (1) Preparation of experimental materials The tomato variety used in the experiment was Moneymaker. Seeds were sown in seedling trays and cultured in an artificial climate chamber. Culture conditions: temperature 25℃ / 18℃ (day / night), light intensity 200 μmol·m⁻². -2 ·s -1 The photoperiod was 16 hours of light / 8 hours of darkness, with a relative humidity of 70%. Low-temperature treatment was carried out when the seedlings reached the four-leaf-one-heart stage.

[0029] (2) Low temperature treatment and sampling Tomato seedlings of uniform growth were transferred to a 4°C artificial climate chamber for low-temperature stress treatment, while other environmental conditions remained unchanged. The third true leaf of each plant was collected at 1 h, 3 h, 6 h, 12 h, and 24 h after treatment. Leaves from three seedlings at each time point were combined to form a single sample, with three biological replicates. The collected samples were immediately flash-frozen in liquid nitrogen and then transferred to a -80°C ultra-low temperature freezer for storage.

[0030] (3) RNA extraction and cDNA synthesis Approximately 100 mg of frozen leaf tissue was collected and ground into powder in liquid nitrogen. Total RNA was extracted using the Trizol method. RNA integrity was assessed by 1% agarose gel electrophoresis, and RNA concentration and purity (A) were determined using a UV spectrophotometer. 260 / A 280 (The ratio should be between 1.8 and 2.0). Take 1 μg of total RNA and synthesize the first strand of cDNA using a reverse transcription kit. Store the product at -20°C for later use.

[0031] (4) qRT-PCR detection Using cDNA as a template and the tomato Actin gene as an internal control, quantitative real-time PCR was performed. The reaction mixture (20 μL) consisted of 2 μL cDNA template, 0.4 μL each of forward and reverse primers, 10 μL SYBR Green premix, and ddH2O to a final volume of 20 μL. The reaction program was: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 10 s, 58℃ annealing for 20 s, and 72℃ extension for 20 s, for a total of 40 cycles. Melting curve analysis (65-95℃) was performed to verify the specificity of the amplified products. The relative expression level of the HY5 gene was calculated using the 2^-ΔΔCt method, with the expression level at 1 hour as the baseline. Three technical replicates were performed for each sample.

[0032] (5) Experimental results The relative expression levels of the tomato HY5 gene at different time points under 4℃ low-temperature stress are shown in Table 1 and 2. Figure 1 As shown.

[0033] Table 1. Relative expression levels of the HY5 gene in tomato at different time points under low temperature stress.

[0034] Note: The expression level at 1 hour was set as the baseline value 1, and the data are the mean ± standard deviation of 3 biological replicates.

[0035] (6) Results Analysis like Figure 1 As shown in Table 1, in the early stages of low-temperature treatment (1h and 3h), the expression level of the HY5 gene remained at a low level (approximately 1.2-fold), with no significant difference between the two periods (P>0.05), indicating that HY5 had not been significantly activated by the low-temperature signal before 3h. At 6h of treatment, the expression level increased sharply to 5.60-fold, approximately 4.4-fold above the baseline level, a statistically significant difference (P<0.05), indicating that the HY5 gene was significantly induced by the low-temperature signal between 3 and 6h. At 12h of treatment, the expression level further increased to 7.04-fold, reaching the maximum value (peak point) within the entire detection time range. At 24h of treatment, the expression level rapidly decreased to 1.38-fold, close to the baseline level.

[0036] The above results indicate that the expression of the tomato HY5 gene under low temperature stress exhibits a typical single-peak curve characteristic of "baseline maintenance - rapid rise - peak - rapid decline". Specifically, the time point at which the expression level first shows a significant increase is between 3 and 6 hours (response initiation point), the time point at which the expression level reaches its maximum value is 12 hours (peak point), and the continuous interval from the response initiation point to the peak point is 3-12 hours (critical period for the HY5 gene to initiate a low temperature response). Example 2

[0037] Based on the time series analysis results of Example 1, this example provides a specific method for comprehensive evaluation of the low-temperature response status of tomatoes, including the following steps: (1) Low temperature treatment Following the method in Example 1, the tested tomato seedlings were subjected to a 4°C low-temperature stress treatment.

[0038] (2) Time series sampling Leaf samples were collected at 1h, 3h, 6h, 12h and 24h after treatment.

[0039] (3) Expression level detection and key period determination The expression level of the HY5 gene at each time point was detected according to the method in Example 1, and the dynamic curve of expression level was plotted to determine the response initiation point (between 3-6h) and the peak point (12h), and the critical period was determined to be 3-12h.

[0040] (4) Four-dimensional indicator detection and evaluation Within the defined critical period (3-12 hours), the following four dimensions of indicators will be measured: a) Dynamic characteristics: The expression levels of the HY5 gene were recorded at the response initiation point (approximately 6 hours) and the peak point (12 hours). The expression level at 6 hours was 5.60, and the expression level at 12 hours was 7.04, indicating that the plant could quickly reach a high expression level after low-temperature response initiation, and the response initiation efficiency was high.

[0041] b) Ratio characteristics: The expression levels of HY5 and its downstream target gene SlCBF1 were measured simultaneously. The HY5 / CBF1 expression ratios at each time point are shown in Table 2.

[0042] Table 2. HY5 / CBF1 expression ratio at different time points under low temperature stress

[0043] As shown in Table 2, the ratio significantly increased to 1.75 at 6 h, indicating that the transcriptional activation efficiency of HY5 was highest during the response initiation phase; at 12 h, the ratio dropped back to 1.21, indicating that although HY5 expression reached its peak, the expression of the target gene SlCBF1 increased synchronously, maintaining a good ratio between transcription factors and target genes. If the ratio is abnormal (too high or too low) at a certain time point, it may indicate an imbalance in transcriptional regulation.

[0044] c) Recovery characteristics: Tomato plants treated at 4℃ for 12h were transferred back to 25℃ for recovery culture, and the expression level of HY5 was detected at 0h, 3h, 6h, 12h and 24h after recovery (Table 3).

[0045] Table 3. Relative expression levels of the HY5 gene at different time points during recovery culture.

[0046] As shown in Table 3, the expression level of HY5 showed a continuous downward trend after recovery, reaching near the baseline level (approximately 1.2) at 24 hours, indicating that the low-temperature response of the plant had essentially ceased and the physiological effects caused by low temperature were being eliminated. If the recovery rate of a plant was significantly slower than that of normal plants (e.g., it remained above 3.0 at 24 hours), it suggested that the plant may have suffered more severe low-temperature damage and required a longer recovery time.

[0047] d) Integral characteristics: Based on the data from 5 time points in Example 1, the time integral expression (AUC) of HY5 within 0-24h was calculated using the following formula: AUC=Σ[(E i + E i+1 ) / 2×Δt i ] Where E i Let Δt be the relative expression level at the i-th time point. i This represents the time interval between two adjacent time points.

[0048] The specific calculations are as follows: AUC=(1.27+1.19) / 2×2+(1.19+5.60) / 2×3+(5.60+7.04) / 2×6+(7.04+1.38) / 2×12 =2.46 + 10.185 + 37.92 + 50.52 =101.085 The AUC value can be used as the integral value of HY5 low-temperature response of this variety (or this plant) under this low-temperature treatment. The higher the AUC value, the higher the cumulative expression level of HY5 within the entire response window, and the stronger the low-temperature response.

[0049] (5) Comprehensive evaluation The indicators of the above four dimensions are compared with the reference benchmark. Based on the number of indicators that meet the standards for each dimension, the low-temperature response status of the tomato plants is comprehensively determined. Specifically, each dimension is evaluated according to the standards shown in Table 4. The low-temperature response status of the tomato plants is determined by combining the evaluation results of the four dimensions according to the following logic: If three or more dimensions are positive assessment conclusions (i.e., "rapid response initiation / high peak level", "high transcriptional activation efficiency", "strong recovery ability", "high response intensity"), the low-temperature response status is judged to be good.

[0050] If both dimensions yield positive assessment results, the low-temperature response status is determined to be moderate.

[0051] If one or zero dimensions yield a positive assessment result, the low-temperature response status is judged to be poor.

[0052] Table 4 Evaluation Criteria for Each Dimension

[0053] Based on the standards described in Table 4, the tomato plants tested in this embodiment were comprehensively evaluated across four dimensions: Dynamic characteristics: 6h expression level was 5.60 (≥4.0), 12h expression level was 7.04 (≥6.0), and the evaluation conclusion was "rapid response initiation and high peak level".

[0054] Ratio characteristics: The HY5 / CBF1 ratio at 6h was 1.75 (≥1.5), and the assessment conclusion was "high transcriptional activation efficiency".

[0055] Recovery characteristics: After 24 hours of recovery culture, the HY5 expression level was 1.20 (≤1.5), and the assessment conclusion was "strong recovery ability and mild low temperature damage".

[0056] Integral characteristic: AUC value is 101.085.

[0057] All four dimensions yielded positive assessment results, indicating that the tomato plant's low-temperature response was good. The four dimensions combined demonstrate that the tomato plant initiated an effective low-temperature defense response under this low-temperature stress, with high response efficiency and good recovery ability.

[0058] The thresholds mentioned in the above-mentioned criteria (such as 6h expression level ≥ 4.0, 12h expression level ≥ 6.0, ratio ≥ 1.5, etc.) are all set based on the experimental data of the Moneymaker variety in Example 1 of this invention. For different tomato varieties or different experimental conditions, those skilled in the art can refer to the above method to determine the corresponding thresholds through conventional experiments. This is an equivalent substitution of this invention and does not depart from the protection scope of this invention. Example 3

[0059] The application of the evaluation method described in this invention in the screening of cold-resistant tomato germplasm is as follows: Two tomato germplasms (germplasm A and germplasm B) were taken and subjected to low-temperature treatment at 4℃ according to the methods of Example 1 and Example 2. The expression level of HY5 at each time point was detected, and the evaluation indicators of four dimensions were calculated.

[0060] The results showed that the HY5 expression levels of germplasm A at 6 h and 12 h (5.60 and 7.04, respectively) were significantly higher than those of germplasm B (3.20 and 4.50, respectively, P<0.05); the HY5 / CBF1 ratio of germplasm A at 6 h was 1.75, significantly higher than that of germplasm B (1.35); the AUC value of germplasm A was 101.085, significantly higher than that of germplasm B (65.320); after 24 h of recovery culture, the HY5 expression level of germplasm A had dropped to 1.20, while that of germplasm B remained at 2.80.

[0061] Based on the comprehensive evaluation results across four dimensions, germplasm A exhibited a faster initiation of its low-temperature response, a higher peak value, higher transcriptional activation efficiency, greater cumulative response intensity, and stronger recovery ability, indicating that germplasm A possesses a stronger low-temperature response capacity and better low-temperature tolerance potential than germplasm B. After simultaneously subjecting both germplasms to 4°C low-temperature stress for 72 hours followed by restoration to room temperature culture, the recovery growth rate of germplasm A (85%) was significantly higher than that of germplasm B (52%), validating the accuracy of the above evaluation results.

[0062] This embodiment demonstrates that the comprehensive evaluation method provided by the present invention can rapidly evaluate the low-temperature response capability of tomato germplasm within 24 hours of low-temperature treatment. Compared with the traditional phenotypic screening method that requires several days to several weeks, it significantly shortens the screening cycle and improves breeding efficiency. Example 4

[0063] The application of the evaluation method described in this invention in low-temperature early warning for tomato greenhouse cultivation is as follows: In winter and spring greenhouse tomato production, when encountering a sudden drop in temperature, follow these procedures: (1) After the temperature drop, plant leaf samples were collected at 1h, 3h, 6h, 12h and 24h respectively; (2) The expression level of HY5 gene at each time point was detected according to the method in Example 1, and dynamic curves were plotted to determine the response initiation point and peak point; (3) Detect indicators in four dimensions during the critical period: If the HY5 expression level is ≥4.0 at 6h (with 1h as the baseline value of 1), it indicates that the plant has initiated a low-temperature defense response.

[0064] If the HY5 expression level is ≥6.0 at 12h, it indicates that the plant is in the peak period of low temperature response.

[0065] If the HY5 / CBF1 ratio is ≥1.5 at 6h, it indicates that the transcriptional activation efficiency is high.

[0066] Calculate the AUC value to assess the overall response strength.

[0067] (4) Determine the plant’s low-temperature response status based on the comprehensive evaluation results of the four dimensions.

[0068] If all four indicators reach the above thresholds, it indicates that the plant has initiated an efficient low-temperature defense response. It is recommended to continue monitoring and take preventive insulation measures.

[0069] If the dynamic characteristic indicators do not meet the standard (6h expression level <4.0), it indicates that the plant has not yet initiated a significant response and close observation is required.

[0070] If the dynamic characteristics meet the standards but the ratio or integral characteristics are low, it indicates that although the response has been initiated, the efficiency is not high. It is recommended to take remedial measures such as heating, covering and insulating, or spraying exogenous conditioning substances in a timely manner.

[0071] Compared with traditional methods that rely on visual observation of chilling injury phenotypes (which usually appear after 24 hours), this method can complete a multi-dimensional comprehensive assessment within 24 hours after the temperature drop occurs, and can know the plant's low-temperature response status at least 12-24 hours before the appearance of chilling injury phenotypes, thus gaining valuable time for timely remedial measures.

[0072] Other aspects of this invention that are not detailed herein are all conventional techniques known to those skilled in the art.

[0073] It should be noted that the terms “comprising,” “including,” or any other variations are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0074] The scope of protection of this invention is not limited to the technical solutions disclosed in the specific embodiments. Any modifications, equivalent substitutions, improvements, etc., made to the above embodiments based on the technical essence of this invention shall fall within the scope of protection of this invention.

Claims

1. A comprehensive evaluation method for the low-temperature response status of the tomato HY5 gene based on time series analysis, characterized in that, Includes the following steps: (1) Low temperature stress treatment was applied to tomato seedlings; (2) Tomato leaf samples were collected at multiple consecutive time points after low-temperature treatment; (3) Detect the expression level of HY5 gene in samples at each time point, plot the dynamic curve of the expression level of HY5 gene with the low temperature treatment time based on the expression level of HY5 gene at each time point, determine the response initiation point and peak point of HY5 gene to initiate low temperature response based on the dynamic curve, and determine the continuous interval from the response initiation point to the peak point as the key period for HY5 gene to initiate low temperature response. (4) During the aforementioned critical period, the following four dimensions of indicators will be monitored: a) Dynamic characteristics: Expression levels of the HY5 gene at the response initiation and peak points; b) Ratio characteristics: The expression ratio of the HY5 gene to its downstream target genes; c) Recovery characteristics: The rate of decline in HY5 expression levels during the recovery process after the low-temperature treatment and subsequent return to room temperature culture; d) Integral characteristics: time-integrated expression level of the HY5 gene during low-temperature treatment; (5) Compare the four indicators in step (4) with the reference benchmark respectively, and comprehensively determine the low temperature response status of the tomato plant based on the number of indicators that meet the standards in each dimension.

2. The method according to claim 1, characterized in that: The temperature of the low-temperature stress treatment is 2-6℃.

3. The method according to claim 1, characterized in that: The multiple consecutive time points include at least 1 hour, 3 hours, 6 hours, 12 hours and 24 hours after the low temperature treatment.

4. The method according to claim 1, characterized in that: In step (3), the expression level of the HY5 gene is detected by real-time PCR.

5. The method according to claim 1, characterized in that: In step (3), the expression level at the early time point of low temperature treatment is used as the baseline value.

6. The method according to claim 1, characterized in that: In step (3), the significant increase is defined as a statistical significance test showing a probability P-value of less than 0.

05.

7. The method according to claim 1, characterized in that: The downstream target gene is at least one of SlCBF1, SlNCED6, or SlMIPS3.

8. The method according to claim 1, characterized in that: The time integral expression is calculated using the following formula: AUC = Σ[(Ei + Ei+1) / 2 × Δti] Where Ei is the relative expression level at the i-th time point, and Δti is the time interval between two adjacent time points.

9. The application of the method according to any one of claims 1 to 8 in the screening of low-temperature tolerant tomato germplasm.

10. The application of the method according to any one of claims 1 to 8 in low-temperature early warning for tomato greenhouse cultivation.

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