Method for analyzing jujube ZjGGP gene to respond to chilling stress to promote ascorbic acid accumulation

Abstract

The invention provides a method for analyzing jujube ZjGGP gene response cold stress to promote ascorbic acid accumulation, and relates to the technical field of plant molecular biology, and the method comprises the following five steps: sample preparation and treatment, ascorbic acid content determination, gene expression profile analysis and key gene screening, key gene expression verification and causal relationship determination. According to the method disclosed by the invention, short-term cold treatment is an optimization process window for inducing the ascorbic acid of the jujube fruits to be accumulated to a peak value, accurate temperature and time parameters are provided for postharvest cold-chain logistics, and the core nutritional value of the jujube fruits can be actively maintained and improved through a physical regulation and control means on the premise of not depending on a chemical preservative; the shelf life and the commodity quality are obviously prolonged. Meanwhile, the determination of the ZjGGP gene provides valuable molecular targets and selection markers for molecular breeding of jujube trees.

Description

Technical Field

[0001] This invention relates to the field of plant molecular biology, and in particular to a method for analyzing the jujube ZjGGP gene's response to cold stress in promoting ascorbic acid accumulation. Background Technology

[0002] Jujubes are highly nutritious, especially rich in ascorbic acid (AsA, also known as vitamin C), making them an important fruit with both medicinal and edible properties. Low-temperature storage after harvest is a key technique for maintaining their quality and extending their shelf life. Previous studies have shown that low-temperature storage can effectively delay the ripening and senescence of many fruits (such as kiwifruit and apples) and inhibit the degradation of nutrients.

[0003] However, current research on the changes in ascorbic acid content, a core nutrient in jujube fruit, and its intrinsic regulatory mechanisms during postharvest cold storage is insufficient, and there are even gaps in our understanding. Current postharvest preservation techniques for jujube fruit largely rely on macroscopic control of external parameters such as temperature and humidity, lacking precise regulatory strategies based on the fruit's internal physiological and metabolic responses. This makes it difficult to maintain or even enhance the important nutritional value of ascorbic acid in postharvest jujube fruit through active regulation in actual production, thus hindering further improvement in the storage quality of jujube fruit.

[0004] On the other hand, cold stress is a key environmental factor affecting plant growth, development, and postharvest quality. When plants respond to low-temperature stress, they activate a series of complex molecular response mechanisms. Among these, ascorbic acid, as a key antioxidant, plays a central role in scavenging reactive oxygen species and mitigating oxidative damage. Previous studies have found that cold stress can induce the expression of key genes for ascorbic acid synthesis in crops such as kiwifruit. However, in jujube trees, the key genes that respond to cold stress and directly regulate ascorbic acid accumulation, and their mechanisms of action, remain unclear. At the genetic breeding level, although significant differences in ascorbic acid content are known among jujube varieties, traditional breeding methods mainly rely on phenotypic selection, which is time-consuming and inefficient. The lack of molecular markers or key target genes closely associated with the trait of high ascorbic acid accumulation severely limits the breeding efficiency of new jujube varieties with high nutritional quality.

[0005] Therefore, existing technologies lack a systematic understanding of the dynamic changes in ascorbic acid metabolism in post-harvest jujubes under cold storage conditions, hindering the development of precise quality control techniques. Furthermore, the molecular mechanisms by which jujube trees respond to cold stress and regulate ascorbic acid accumulation, particularly the identification and functional analysis of key genes, remain unexplored. In breeding practice, there is a lack of molecular tools and theoretical guidance for efficiently breeding jujube varieties with high ascorbic acid content. Therefore, this invention proposes a method for analyzing the jujube ZjGGP gene's response to cold stress in promoting ascorbic acid accumulation to address the problems existing in the prior art. Summary of the Invention

[0006] To address the aforementioned issues, the present invention aims to propose a method for analyzing the effect of the ZjGGP gene in jujube on cold stress to promote ascorbic acid accumulation. This method systematically elucidates the molecular physiological mechanism of jujube tree's response to cold stress, resulting in a series of innovative achievements directly serving industrial applications. By integrating physiological and biochemical assays, transcriptomic screening, and molecular biological verification, the core role of the ZjGGP gene in regulating ascorbic acid accumulation under cold stress has been clarified. This not only deepens the theoretical understanding of jujube tree stress resistance physiology but also transforms basic research findings into applied technologies with clear guiding significance.

[0007] To achieve the objective of this invention, the invention is implemented through the following technical solution: A method for analyzing the jujube ZjGGP gene's response to cold stress in promoting ascorbic acid accumulation, characterized by comprising the following steps:

[0008] Step 1: Sample Preparation and Processing

[0009] Prepare jujube biological samples and subject them to cold stress treatment at 2–8℃. Set up jujube biological samples without cold stress treatment as a control and take samples at different time points.

[0010] Step 2: Ascorbic acid content determination

[0011] The ascorbic acid content in the samples at each time point was determined by high performance liquid chromatography to identify the dynamic trend curve of its accumulation.

[0012] Step 3: Gene expression profiling and key gene screening

[0013] Transcriptome sequencing was performed on the samples prepared in step one, and differentially expressed genes in response to cold stress in the ascorbic acid metabolic pathway were screened by bioinformatics analysis.

[0014] Step 4: Validation of Key Gene Expression

[0015] Based on the screening results in step three, the expression level of the candidate gene ZjGGP in samples at each time point was specifically detected by real-time PCR, and the dynamic trend curve of the expression level of ZjGGP gene over time was obtained by calculation.

[0016] Step 5: Determining Causality

[0017] The dynamic trend curve of ascorbic acid content in step two and the dynamic trend curve of ZjGGP gene expression level in step four were correlated. If the two showed a significant positive correlation, that is, when the peak of ascorbic acid content appeared after cold stress, the expression level of ZjGGP gene also reached its peak, then ZjGGP gene was determined to be a key gene that promotes ascorbic acid accumulation in response to cold stress.

[0018] A further improvement is made in the following: In step one, the jujube biological sample is a post-harvest jujube fruit or a jujube seedling. When the jujube biological sample is a post-harvest jujube fruit, the cold stress treatment time is 3 to 13 days, while when the jujube biological sample is a jujube seedling, the cold stress treatment time is 6 to 48 hours.

[0019] A further improvement is that, in step three, the ascorbic acid metabolic pathway includes the L-galactose synthesis pathway and the ascorbic acid-glutathione cycle pathway.

[0020] A further improvement is made in step three, where the screening criteria for differentially expressed genes are: q-value < 0.05 and Fold Change ≥ 1.5.

[0021] A further improvement is made in step four, where the primer pair sequence used for quantitative real-time PCR detection of the ZjGGP gene expression level is:

[0022] Upstream primer: 5'-GCTCCCACCAAGAAAATAA-3';

[0023] Downstream primer: 5'-TCCACCCTCAAAGACAAGA-3'.

[0024] Further improvements include the following steps:

[0025] Step Six: Assisted Verification of Key Gene Functions

[0026] Subcellular localization analysis was performed on the ZjGGP gene obtained in step 5 to clarify the co-localization of ZjGGP protein in the cytoplasm and nucleus.

[0027] A further improvement is made in step six, where the specific method of analysis is as follows: by constructing the pC2300-35S-ZjGGP-GFP fusion expression vector and transforming it into tobacco leaves, the co-localization of ZjGGP protein in the cytoplasm and nucleus is determined by observation using a laser confocal microscope.

[0028] Further improvements include the following steps:

[0029] Step 7: Exploring the mechanisms of gene transcription regulation

[0030] Cis-acting elements were predicted from the promoter sequence upstream of the ATG start codon of the ZjGGP gene to analyze the presence of low-temperature response elements and binding sites for MYB and MYC transcription factors.

[0031] The beneficial effects of this invention are as follows: This invention reveals that short-term cold treatment is an optimized process window for inducing ascorbic acid accumulation to its peak in jujube fruit, providing precise temperature and time parameters for post-harvest cold chain logistics. It can actively maintain and enhance the core nutritional value of jujube fruit through physical regulation without relying on chemical preservatives, significantly extending its shelf life and commercial quality. Simultaneously, the identification of the ZjGGP gene provides valuable molecular targets and screening markers for molecular breeding of jujube trees. Using the specific primers validated in this study, breeders can efficiently identify superior germplasm resources with high ascorbic acid accumulation potential, thereby breaking through the bottleneck of phenotypic selection in traditional breeding and significantly accelerating the breeding process of new varieties with high nutritional quality and strong storage resistance. This invention, by integrating physiological and biochemical assays, transcriptomic screening, and molecular biological validation, clarifies the core role of the ZjGGP gene in regulating ascorbic acid accumulation under cold stress. This not only deepens the theoretical understanding of the stress resistance physiology of jujube trees but also transforms basic research findings into applied technologies with clear guiding significance. Attached Figure Description

[0032] Figure 1 This is a schematic diagram comparing the ascorbic acid content of jujube fruits after cold stress according to the present invention; A: jujube; B: sour jujube.

[0033] Figure 2 This is a schematic diagram comparing the results of GO enrichment analysis and KEGG enrichment analysis of the present invention.

[0034] Figure 3 This is a schematic diagram of the relative expression level of ZjGGP in jujube fruit after cold stress according to the present invention; A: jujube; B: sour jujube.

[0035] Figure 4 This is a schematic diagram of the subcellular localization of ZjGGP in tobacco leaves according to the present invention.

[0036] Figure 5 This is a schematic diagram of the ZjGGP gene promoter sequence analysis of the present invention.

[0037] Figure 6 This is a schematic diagram of the steps of the present invention. Detailed Implementation

[0038] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0039] Example 1

[0040] according to Figure 1 , Figure 2 , Figure 3 and Figure 6As shown in this embodiment, a method for analyzing the jujube ZjGGP gene's response to cold stress in promoting ascorbic acid accumulation is proposed, including the following steps:

[0041] Step 1: Sample Preparation and Processing

[0042] Biological samples of jujube were prepared. In this embodiment, 'Winter Jujube' fruits at the semi-ripe stage after harvest and 4-week-old tissue culture seedlings of sour jujube were used as experimental materials. Jujube fruits and seedlings were placed in an artificial climate chamber at 4°C for cold stress treatment. Fruits were sampled after 0, 3, 6, 9, and 13 days of treatment; seedlings were sampled after 0, 6, 12, 24, 36, and 48 hours of treatment. Each treatment was replicated three times. After sampling, the samples were immediately flash-frozen in liquid nitrogen and stored in an ultra-low temperature freezer at -80°C for later use.

[0043] Step 2: Ascorbic acid content determination

[0044] Ascorbic acid content was determined by high performance liquid chromatography (HPLC). Specifically, 1.0 g of frozen sample was accurately weighed, ground in liquid nitrogen, and extracted with pre-cooled 0.01 mol / L KH₂PO₄ (pH=2.08) solution. The mixture was sonicated in an ice bath for 20 minutes, centrifuged at 4°C and 6000 r / min for 20 minutes, and the supernatant was collected. The extraction was repeated twice, and the supernatants were combined and diluted to 10 mL. The solution was then filtered through a 0.22 μm microporous membrane before injection.

[0045] Furthermore, the chromatographic conditions were as follows: Agilent C18 column, column temperature 30℃, mobile phase 0.01 mol / L KH₂PO₄ solution at pH 2.08, flow rate 1.0 mL / min, detection wavelength 230 nm, and injection volume 10 μL. Quantification was performed using the external standard method. The results are as follows: Figure 1 ( Figure 1 As shown in the figure (A: jujube; B: sour jujube), cold storage treatment promoted the accumulation of ascorbic acid in jujube fruit in the short term, but long-term cold storage led to a significant decrease in its content. After 6 days and 9 days of post-harvest cold storage treatment, the ascorbic acid content in sour jujube and jujube reached the highest level, respectively.

[0046] Step 3: Gene expression profiling and key gene screening

[0047] Total RNA was extracted from jujube fruits in the control group (day 0) and the cold-treated group (day 6). After passing quality inspection, sequencing was performed using the Illumina Novaseq 6000 platform. The obtained Clean Data was aligned to the jujube reference genome using HISAT2 software, and differential expression analysis was performed using DESeq2 software (screening criteria: q-value < 0.05 and FoldChange ≥ 1.5).

[0048] A total of 5577 differentially expressed genes were identified. KEGG enrichment analysis showed that these differentially expressed genes were significantly enriched in pathways such as the ascorbic acid-glutathione cycle. Within this pathway, the GDP-L-galactose phosphorylase gene was significantly upregulated, and it was identified as a candidate key gene, named ZjGGP. Figure 2 As shown, the x-axis represents the number of genes with specific GO terms, and the y-axis represents the enriched GO terms. Green represents GO terms in biological processes, orange represents GO terms in cellular components, and blue represents GO terms in molecular functions. In KEGG enrichment analysis, the vertical columns represent the enrichment pathways, and the horizontal columns represent the enrichment scores. The red box represents the ascorbic acid-glutathione cycle pathway.

[0049] Step 4: Validation of Key Gene Expression

[0050] Based on the screening results from step three, the expression level of the candidate gene ZjGGP in samples at each time point was specifically detected using quantitative real-time PCR (qPCR), and the dynamic trend curve of ZjGGP gene expression over time was calculated. Specifically, total RNA was extracted from samples at each time point using the FOREGENE plant total RNA extraction kit and reverse transcribed into cDNA. Using the UBQ gene as an internal control, qPCR was performed using specific primers. The relative gene expression level was calculated using the 2^(-ΔΔCt) method, and the dynamic trend curve of ZjGGP gene expression over time was plotted. The primer pair sequences are as follows:

[0051] Upstream primer: 5'-GCTCCCACCAAGAAAATAA-3';

[0052] Downstream primer: 5'-TCCACCCTCAAAGACAAGA-3'.

[0053] Step 5: Determining Causality

[0054] The dynamic trend curve of ascorbic acid content obtained in step two was compared with the dynamic trend curve of ZjGGP gene expression obtained in step four. Specifically, the Pearson correlation coefficient of the two trend curves at the same time point was calculated using statistical software (such as SPSS). If the correlation coefficient R > 0.8 and the P-value < 0.05, it was considered a significant positive correlation. At the same time, it was visually observed whether the peaks of the two curves appeared at the same or similar time points, such as... Figure 1 and Figure 3 ( Figure 3As shown in the figure (A: jujube; B: sour jujube), in jujube fruit, both ascorbic acid content and ZjGGP gene expression level reached their peak on day 9 of cold treatment; in sour jujube fruit, both reached their peak on day 6. Statistical analysis showed a correlation coefficient R > 0.9 and a P-value < 0.01. Based on the above analysis results, the ZjGGP gene was identified as a key gene in response to cold stress promoting ascorbic acid accumulation.

[0055] Example 2

[0056] according to Figure 4 As shown, the difference between this embodiment and Embodiment 1 is that it further includes the following steps:

[0057] Step Six: Assisted Verification of Key Gene Functions

[0058] Subcellular localization analysis was performed on the ZjGGP gene obtained in step 5 to clarify the co-localization of ZjGGP protein in the cytoplasm and nucleus.

[0059] The specific method is as follows:

[0060] Using jujube fruit cDNA as a template, the coding region of the ZjGGP gene (excluding the stop codon) was amplified by high-fidelity enzyme PCR. The PCR product was then inserted into the multiple cloning site of the pC2300-35S-GFP vector using homologous recombination technology to construct the pC2300-35S-ZjGGP-GFP fusion expression vector. The primer sequences are as follows:

[0061] F: 5'-gagctcggtacccgggggatccATGTTGAGCATCAG-3'

[0062] R: 5'-cttgctcaccatggtgctcgacCTGCAGAACAAGGC-3'.

[0063] The successfully constructed recombinant plasmid was then transformed into Agrobacterium GV3101 competent cells. Positive single colonies were picked and cultured in LB broth until the OD600 reached approximately 0.8. The cells were collected by centrifugation, resuspended in MMA infiltration buffer containing 150 μmol / L acetylsyringone, and incubated in the dark at room temperature for 3 hours. The bacterial suspension was then injected into the lower epidermis of *Nicotiana benthamiana* leaves using a syringe percolation method.

[0064] After infection, tobacco plants were cultured in darkness for 24 hours, then transferred to a light incubator for another 48 hours. Infected leaves were cut and prepared with the underside facing upwards. GFP fluorescence signals were observed using a ZEISS LSM 880 laser confocal microscope. The excitation wavelength was 488 nm, and the detection wavelength was 500-550 nm. A plasma membrane marker protein (red fluorescence) was used as a reference.

[0065] The results are as follows Figure 4 As shown, the green fluorescence signal of the ZjGGP-GFP fusion protein is widely distributed in the cytoplasm and shows significant aggregation in the nucleus, while the fluorescence signal of the empty GFP control group is distributed throughout the entire cell. These results indicate that the ZjGGP protein is co-localized in the cytoplasm and nucleus, suggesting that it may perform ascorbic acid synthesis in the cytoplasm and may enter the nucleus to participate in gene transcription regulation.

[0066] Example 3

[0067] according to Figure 5 As shown, the difference between this embodiment and Embodiment 2 is that it further includes the following steps:

[0068] Step 7: Exploring the mechanisms of gene transcription regulation

[0069] The promoter sequence of the ZjGGP gene, 2000 bp upstream of the ATG start codon, was obtained from the ZjGGP genome database. Cis-regulatory elements were predicted using the PlantCARE online database. Results are as follows: Figure 5 As shown, in addition to the typical TATA-box and CAAT-box, the promoter region of the ZjGGP gene also contains multiple cis-regulatory elements related to abiotic stress and hormone responses, including but not limited to: low-temperature response elements, MYB transcription factor binding sites, MYC transcription factor binding sites, and abscisic acid response elements. This prediction provides a theoretical basis for the transcriptional regulatory mechanism of ZjGGP gene upregulation induced by cold stress.

[0070] Example 4

[0071] This embodiment provides a method for screening jujube germplasm with high ascorbic acid content using the above-mentioned findings. First, tissue-cultured seedlings from 10 different jujube germplasm resources were selected and cold-treated at 4°C for 12 hours, with untreated seedlings serving as a control. Then, following the method provided in Example 1, total RNA was extracted from the leaves and reverse-transcribed into cDNA. Next, using the specific primers provided in Example 1, the relative expression level of the ZjGGP gene in each germplasm was detected by qPCR. Finally, the fold change in ZjGGP gene expression level after cold treatment relative to the control was calculated for each germplasm. The two germplasms with the highest fold increase in expression level (e.g., germplasm A and germplasm B) were identified as potential germplasms with high ascorbic acid content. This allows for rapid and accurate screening of individuals with superior traits from a large number of germplasms, significantly shortening the breeding cycle and improving breeding efficiency.

[0072] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the present invention without departing from its framework and scope of application, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for analyzing the response of the jujube ZjGGP gene to cold stress in promoting ascorbic acid accumulation, characterized in that: Includes the following steps: Step 1: Sample Preparation and Processing Prepare jujube biological samples and subject them to cold stress treatment at 2–8℃. Set up jujube biological samples without cold stress treatment as a control and take samples at different time points. Step 2: Ascorbic acid content determination The ascorbic acid content in the samples at each time point was determined by high performance liquid chromatography to identify the dynamic trend curve of its accumulation. Step 3: Gene expression profiling and key gene screening Transcriptome sequencing was performed on the samples prepared in step one, and differentially expressed genes in response to cold stress in the ascorbic acid metabolic pathway were screened by bioinformatics analysis. Step 4: Validation of Key Gene Expression Based on the screening results in step three, the expression level of the candidate gene ZjGGP in samples at each time point was specifically detected by real-time PCR, and the dynamic trend curve of the expression level of ZjGGP gene over time was obtained by calculation. Step 5: Determining Causality The dynamic trend curve of ascorbic acid content in step two and the dynamic trend curve of ZjGGP gene expression level in step four were correlated. If the two showed a significant positive correlation, that is, when the peak of ascorbic acid content appeared after cold stress, the expression level of ZjGGP gene also reached its peak, then ZjGGP gene was determined to be a key gene that promotes ascorbic acid accumulation in response to cold stress.

2. The method for analyzing the jujube ZjGGP gene response to cold stress in promoting ascorbic acid accumulation according to claim 1, characterized in that: In step one, the jujube biological sample is either a harvested jujube fruit or a jujube seedling. When the jujube biological sample is a harvested jujube fruit, the cold stress treatment time is 3 to 13 days, while when the jujube biological sample is a jujube seedling, the cold stress treatment time is 6 to 48 hours.

3. The method for analyzing the jujube ZjGGP gene's response to cold stress in promoting ascorbic acid accumulation according to claim 1, characterized in that: In step three, the ascorbic acid metabolic pathway includes the L-galactose synthesis pathway and the ascorbic acid-glutathione cycle pathway.

4. The method for analyzing the jujube ZjGGP gene's response to cold stress in promoting ascorbic acid accumulation according to claim 1, characterized in that: In step three, the screening criteria for differentially expressed genes are: q-value < 0.05 and FoldChange ≥ 1.

5.

5. The method for analyzing the jujube ZjGGP gene response to cold stress in promoting ascorbic acid accumulation according to claim 1, characterized in that: In step four, the primer pair sequences used for quantitative real-time PCR detection of ZjGGP gene expression levels are as follows: Upstream primer: 5'-GCTCCCACCAAGAAAATAA-3'; Downstream primer: 5'-TCCACCCTCAAAGACAAGA-3'.

6. The method for analyzing the jujube ZjGGP gene's response to cold stress in promoting ascorbic acid accumulation according to claim 1, characterized in that: It also includes the following steps: Step Six: Assisted Verification of Key Gene Functions Subcellular localization analysis was performed on the ZjGGP gene obtained in step 5 to clarify the co-localization of ZjGGP protein in the cytoplasm and nucleus.

7. The method for analyzing the jujube ZjGGP gene response to cold stress in promoting ascorbic acid accumulation according to claim 6, characterized in that: In step six, the specific method of analysis is as follows: by constructing the pC2300-35S-ZjGGP-GFP fusion expression vector and transforming it into tobacco leaves, the co-localization of ZjGGP protein in the cytoplasm and nucleus is determined by observation using a laser confocal microscope.

8. The method for analyzing the jujube ZjGGP gene's response to cold stress in promoting ascorbic acid accumulation according to claim 1, characterized in that: It also includes the following steps: Step 7: Exploring the mechanisms of gene transcription regulation Cis-acting elements were predicted from the promoter sequence upstream of the ATG start codon of the ZjGGP gene to analyze the presence of low-temperature response elements and binding sites for MYB and MYC transcription factors.

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