Application of Pt7G25060 gene and protein thereof in judging drought stress degree of Chinese pine seedlings

Abstract

The invention discloses application of a Pt7G25060 gene and a protein thereof in judging drought stress degree of Chinese pine seedlings, and belongs to the technical field of biology. The expression quantity of the gene is in negative correlation with the drought stress degree, and obvious expression difference is shown in the early stage of drought stress of Chinese pine seedlings. Based on the technical advantages, the invention provides the method for judging the drought stress degree of the Chinese pine seedlings. The cDNA of a sample to be detected and the cDNA of a reference sample are subjected to fluorescent quantitative PCR amplification by using the detection primer provided by the invention, and the expression quantity of the Pt7G25060 gene in the sample to be detected and the expression quantity of the Pt7G25060 gene in the reference sample are compared, so that the drought stress degree of the sample to be detected can be accurately judged. Compared with a traditional distinguishing method depending on observation of seedling needle leaf sallow degree and physiological indexes, the method provided by the invention can sensitively and accurately reflect the drought stress degree of the Chinese pine seedlings, and is higher in precision, stronger in scientificity and good in repeatability.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a... Pt7G25060 Application of genes and their proteins in determining the degree of drought stress in Pinus tabuliformis seedlings. Background Technology

[0002] *Pinus tabuliformis* Carriere, a coniferous evergreen tree belonging to the genus *Pinus* in the family Pinaceae, possesses strong resistance to cold, drought, and poor soil conditions. It plays a significant role in soil and water conservation, effectively preventing soil erosion, improving soil structure, and enhancing soil fertility. It also plays a crucial role in windbreak and sand fixation, climate regulation, and vegetation restoration, making it an important tree species for afforestation of barren mountains, soil and water conservation, and urban greening. Furthermore, *Pinus tabuliformis* wood is hard, corrosion-resistant, and has a beautiful grain, making it widely used in construction, bridges, railway sleepers, mine props, and furniture manufacturing. The resin, needles, knots, and bark of *Pinus tabuliformis* contain abundant medicinal components, commonly used to extract turpentine and rosin, which have effects such as dispelling wind and dampness, promoting blood circulation and relieving pain, and antibacterial and anti-inflammatory properties, making them highly valuable in traditional Chinese medicine and chemical products.

[0003] However, with the intensification of global warming, droughts are becoming more frequent in many regions, severely impacting the normal cultivation of Pinus tabuliformis seedlings. The economic losses of drought stress to Pinus tabuliformis plantations cannot be ignored: in arid and semi-arid regions, drought-induced slow seedling growth and reduced survival rates directly increase seedling costs and replanting expenses. Furthermore, drought-induced seedling mortality and stand degradation further exacerbate the risk of soil erosion, weakening the crucial role of Pinus tabuliformis in soil and water conservation and ecological restoration, resulting in significant economic losses to forestry production and ecological environment construction.

[0004] Pinus tabuliformis possesses a certain degree of drought resistance, making it sometimes difficult to determine from phenotype whether a plant is under drought stress. The cell wall is the first interface between plant cells and the external environment, making it an ideal location for sensing environmental changes (including water deficit). When drought occurs, it often leads to changes in cell wall tension, pressure, or structure, thereby activating receptor kinases on the cell membrane and initiating intracellular signal transduction. Whether there are response gene markers to drought stress in the cell wall of Pinus tabuliformis seedlings remains inconclusive.

[0005] Therefore, exploring biomarkers of drought stress response in Pinus tabuliformis seedlings is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] This invention discloses a Pt7G25060Application of genes and their proteins in determining the degree of drought stress in Pinus tabuliformis seedlings. The method for determining the degree of drought stress in Pinus tabuliformis seedlings presented in this invention is an earlier and more accurate method for drought diagnosis. It has significant theoretical and practical implications for guiding scientific water management in Pinus tabuliformis seedling cultivation, improving seedling survival rates, reducing forestry economic losses, and ensuring the effectiveness of ecological restoration projects.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A sort of Pt7G25060 Genes, the ones mentioned Pt7G25060 The amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0009] As a preferred technical solution, the Pt7G25060 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0010] The present invention also provides a Pt7G25060 protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0011] The present invention also provides the above. Pt7G25060 The detection primers for the gene are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0012] The present invention also provides an amplification of the above. Pt7G25060 A gene assay kit, the kit comprising the detection primers described above.

[0013] As a preferred technical solution, the kit further includes PCR amplification reagents, which include SYBR Green DNA polymerase, dNTPs, PCR buffer, and internal reference gene primers.

[0014] As a more preferred technical solution, the nucleotide sequences of the internal reference gene primers are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0015] The present invention also provides the above. Pt7G25060 Application of the gene, the Pt7G25060 protein, the detection primers, or the kit in determining the degree of drought stress on Pinus tabuliformis seedlings.

[0016] The present invention also provides a method for determining the degree of drought stress suffered by Pinus tabuliformis seedlings, comprising the following steps: Using cDNA from the test sample and cDNA from the reference sample as templates, quantitative real-time PCR amplification was performed using the aforementioned detection primers. The levels of cDNA in the test sample and reference sample were then statistically analyzed. Pt7G25060Gene expression levels; When the sample to be tested contains Pt7G25060 Gene expression levels compared to reference samples Pt7G25060 If there is no significant difference in gene expression levels, the drought stress level of the test sample is determined to be the same as that of the reference sample. When the sample to be tested contains Pt7G25060 The gene expression level was significantly lower than that in the reference sample. Pt7G25060 When the gene expression level is measured, it is determined that the drought stress level of the test sample is higher than that of the reference sample. When the sample to be tested contains Pt7G25060 The gene expression level was significantly higher than that in the reference sample. Pt7G25060 When the gene expression level is measured, it is determined that the drought stress level of the test sample is lower than that of the reference sample. The Pt7G25060 The amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0017] As a preferred technical solution, the reference sample is a sample of Pinus tabuliformis seeds treated with the same degree of drought stress but different durations of drought stress.

[0018] As a preferred technical solution, the significant difference includes significant differences when P < 0.05.

[0019] As a preferred technical solution, the reaction system for the real-time PCR amplification is 20 μL, comprising: 10 μL of 2× SuperReal PreMix Plus (SYBR Green), 1 μL of cDNA template, 0.8 μL of 100 μM upstream primer, 0.8 μL of 100 μM downstream primer, and RNase-Free Water to make up to 20 μL.

[0020] As a preferred technical solution, the reaction program for the fluorescence quantitative PCR amplification is as follows: pre-denaturation at 95℃ for 2 min, denaturation at 94℃ for 5 s, annealing at 60℃ for 30 s, and running for 39 cycles.

[0021] Beneficial effects: This invention provides a cell wall-related biomarker for drought stress response in Pinus tabuliformis seedlings. Pt7G25060The gene, whose nucleotide sequence is shown in SEQ ID NO.1, is described. The expression level of this gene is negatively correlated with the degree of drought stress, gradually decreasing as the drought stress on Pinus tabuliformis seedlings increases. Furthermore, the gene shows significant expression differences in the early stages of drought stress on Pinus tabuliformis seedlings. Based on the above technical advantages, this invention provides a method for determining the degree of drought stress on Pinus tabuliformis seedlings. The cDNA of the test sample and the cDNA of the reference sample are amplified by real-time quantitative PCR using the detection primers provided in this invention, and the expression levels in the test sample and the reference sample are compared. Pt7G25060 Gene expression levels can accurately determine the degree of drought stress experienced by the test sample. Experiments have shown that, compared with traditional methods that rely on observing the degree of yellowing of seedling needles and physiological indicators, the technical solution provided by this invention can sensitively and accurately reflect the degree of drought stress in Pinus tabuliformis seedlings, with higher precision, stronger scientific basis, and better reproducibility. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 The needle phenotypes of Pinus tabuliformis seedlings subjected to different degrees of drought stress are shown; from left to right, the mannitol concentrations are 0 mM, 100 mM, 200 mM, 300 mM, and 400 mM, respectively. Figure 2 For the *Pinus tabuliformis* seedlings after different drought stress treatments in Example 2 Pt7G25060 Gene expression level; D0d represents the expression level of *Pinus tabuliformis* seedlings under drought stress for 0 days. Pt7G25060 Gene expression levels, D3d represents the expression level of *Pinus tabuliformis* seedlings subjected to drought stress for 3 days. Pt7G25060 Gene expression levels, D7d represents the expression level of *Pinus tabuliformis* seedlings under drought stress for 7 days. Pt7G25060 Gene expression levels.

[0024] Figure 3 For the sample to be tested in Example 4 Pt7G25060 Gene expression levels were compared with those in the reference sample at D0d, D3d, and D7d, respectively. Pt7G25060 Analysis of significant differences in gene expression levels, among which represent P< 0.05. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0027] This invention provides a cell wall-related drought stress marker gene for Pinus tabuliformis seedlings. Pt7G25060, The drought stress marker gene of Pinus tabuliformis seedlings Pt7G25060 The nucleotide sequence is shown in SEQ ID NO.1. This gene can be used to determine the degree of drought stress suffered by Pinus tabuliformis seedlings.

[0028] This invention also provides the above-mentioned drought stress marker gene for Pinus tabuliformis seedlings. Pt7G25060 The encoded amino acid sequence, as shown in SEQ ID NO.2, indicates that this protein can also be used to determine the degree of drought stress experienced by Pinus tabuliformis seedlings.

[0029] This invention also provides amplification of the drought stress marker gene in the above-mentioned Pinus tabuliformis seedlings. Pt7G25060 The detection primer pair consists of an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, specifically: 5'-ACACCATTGGATCCGGTCAC-3'; the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4, specifically: 5'-ACCCGGTGAGAGGACTGATG-3'. This primer pair can specifically amplify the drought stress marker gene of the *Pinus tabuliformis* seedlings. Pt7G25060 .

[0030] This invention also provides a kit comprising the above-described detection primer pair, and further comprising PCR amplification reagents. As one embodiment, the PCR amplification reagent can be a real-time PCR amplification reagent, comprising SYBR Green DNA polymerase, dNTPs, PCR buffer, and an internal reference gene primer; as another embodiment, the internal reference gene primer pair consists of an upstream primer and a downstream primer, the nucleotide sequences of which are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively. The source and amount of the SYBR Green DNA polymerase, dNTPs, PCR buffer, and internal reference gene primers are not particularly limited, and techniques well known in the art can be used.

[0031] The present invention also provides the technical solutions described above. Pt7G25060 The application of the gene, the aforementioned Pt7G25060 protein, the aforementioned detection primer pairs, or the aforementioned detection kit in the sensitive and accurate determination of the degree of drought stress suffered by Pinus tabuliformis seedlings. Furthermore, the method of determining whether Pinus tabuliformis seedlings are under drought stress is more timely and sensitive than the traditional method that relies on observing the phenotype and physiological indicators of seedling needles.

[0032] Specifically, the present invention also provides a method for determining the degree of drought stress suffered by Pinus tabuliformis seedlings, comprising the following steps: Using cDNA from the test sample and cDNA from the reference sample as templates, quantitative real-time PCR amplification was performed using the aforementioned detection primer pairs. The levels of cDNA in the test sample and reference sample were then statistically analyzed. Pt7G25060 Gene expression levels; When the sample to be tested contains Pt7G25060 Gene expression levels compared to reference samples Pt7G25060 If there is no significant difference in gene expression levels, the drought stress level of the test sample is determined to be the same as that of the reference sample. When the sample to be tested contains Pt7G25060 The gene expression level was significantly lower than that in the reference sample. Pt7G25060 When the gene expression level is measured, it is determined that the drought stress level of the test sample is higher than that of the reference sample. When the sample to be tested contains Pt7G25060 The gene expression level was significantly higher than that in the reference sample. Pt7G25060 When the gene expression level is measured, it is determined that the drought stress level of the test sample is lower than that of the reference sample.

[0033] This invention uses cDNA from the test sample and cDNA from the reference sample as templates, performs quantitative real-time PCR amplification using the aforementioned detection primer pairs, and statistically analyzes the cDNA content in the test sample and the reference sample. Pt7G25060Gene expression levels. This invention does not specifically limit the preparation method of the cDNA; conventional cDNA preparation methods in the art are acceptable. The reference samples preferably include Pinus tabuliformis seedlings treated with drought for 0 days; Pinus tabuliformis seedlings treated with 300mM mannitol for 3 days; and Pinus tabuliformis seedlings treated with 300mM mannitol for 7 days.

[0034] After obtaining the cDNA of the test sample and the cDNA of the reference sample, this invention uses the cDNA of the test sample and the cDNA of the reference sample as templates to perform real-time PCR amplification using the aforementioned detection primer pairs. In this invention, the reaction system for real-time PCR amplification is 20 μL, preferably comprising: 10 μL of 2× SuperReal PreMix Plus (SYBR Green), 1 μL of cDNA template, 0.8 μL of 100 μM upstream primer, 0.8 μL of 100 μM downstream primer, and RNase-free water to bring the total to 20 μL. The preferred reaction program for real-time PCR amplification is: 95℃ pre-denaturation for 2 min, 94℃ denaturation for 5 s, 60℃ annealing for 30 s, for 39 cycles. This enables specific amplification of the cDNA of the test sample and the cDNA of the reference sample, respectively.

[0035] After the quantitative PCR amplification, the present invention statistically analyzes the levels of the test sample and the reference sample. Pt7G25060 The gene expression level is determined and assessed. When the sample to be tested contains... Pt7G25060 Gene expression levels compared to reference samples Pt7G25060 If there is no significant difference in gene expression levels, the drought stress level of the test sample is determined to be the same as that of the reference sample. When the sample to be tested contains Pt7G25060 The gene expression level was significantly lower than that in the reference sample. Pt7G25060 When the gene expression level is measured, it is determined that the drought stress level of the test sample is higher than that of the reference sample; when the test sample contains... Pt7G25060 The gene expression level was significantly higher than that in the reference sample. Pt7G25060 When analyzing gene expression levels, the drought stress level of the test sample is determined to be lower than that of the reference sample. As one implementation method, the significant difference includes a significant difference when P < 0.05. This invention does not have special requirements for the equipment used in the quantitative PCR amplification or the gene expression analysis method; techniques well-known in the art can be used. In a specific embodiment of this invention, the equipment used for quantitative PCR amplification is the CFX Connect™ Optics Module, specifically numbered 788BR04747; the software used for gene expression data analysis is Bio-Rad CFX Manager.

[0036] Experiments have shown that, compared with traditional methods that rely on observing the degree of yellowing of the needles and physiological indicators of Pinus tabuliformis seedlings, the technical solution provided by this invention can more sensitively and accurately reflect the degree of drought stress suffered by Pinus tabuliformis seedlings, and has higher precision, stronger scientific basis, and better repeatability.

[0037] To further illustrate the present invention, the following description, in conjunction with the accompanying drawings and embodiments, provides a method for... Pt7G25060 The application of genes and their proteins in determining the degree of drought stress in Pinus tabuliformis seedlings is described in detail, but they should not be construed as limiting the scope of protection of this invention.

[0038] Example 1: Determination of Drought Stress Conditions Pinus tabuliformis seeds: purchased from Zhongtiao Mountain State-owned Forest Farm, Yuanqu County, Yuncheng City, Shanxi Province; Zhongtiao Mountain Forest Farm in Yuanqu is a fault basin with an average annual temperature of 22℃, a maximum temperature of 40℃, and a minimum temperature of -11℃; the seller collects mature Pinus tabuliformis cones from healthy, mature Pinus tabuliformis mother trees, dries them at room temperature, collects the seeds, and mixes them evenly. The purchased pine seeds were transported back to the laboratory of Beijing Forestry University. The seeds were then placed in sterile distilled water for viability screening, removing empty seeds that floated to the surface. The specific disinfection and germination process was as follows: The seeds were disinfected with a 0.5% sodium hypochlorite solution for 20 minutes, with slow stirring to ensure uniform disinfection. Afterward, they were rinsed repeatedly five times with sterile distilled water to thoroughly remove any residual disinfectant. Sterile sphagnum moss was placed in a petri dish and moistened with sterile water to ensure sufficient moisture for the seeds during subsequent cultivation. Several seeds were placed in the petri dish, ensuring each seed was in contact with the moistened sphagnum moss.

[0039] Place the seed-containing petri dishes in an artificial climate incubator at 22℃, 70% relative humidity, and a light / dark cycle of 16 hours light / 8 hours darkness for germination culture. Observe the seed germination status daily and add sterile distilled water to the petri dishes as needed. The criterion for seed germination is the cracking of the outer seed coat and the emergence of the radicle. After 20 days of germination, transfer the seedlings to a hydroponic incubator for two weeks, after which different degrees of drought treatment can be applied.

[0040] Drought treatment design: Mannitol concentrations of 0 mM, 100 mM, 200 mM, 300 mM, and 400 mM were set to simulate drought stress treatments. Thirty-six seedlings of uniform growth were selected from each treatment group and transferred to hydroponic incubators with different mannitol concentrations. After 14 days of cultivation, the phenotype of the Pinus tabuliformis seedlings in each treatment group was observed. (See attached...) Figure 1It can be seen that after 14 days of treatment with 300mM mannitol, the seedlings of Pinus tabuliformis showed obvious yellowing of needles, but the vitality of the seedlings was not seriously damaged. Therefore, 300mM mannitol can be used as a reasonable drought stress condition for Pinus tabuliformis in subsequent experiments.

[0041] After determining the mannitol concentration for simulating drought stress, prepare the pine seedlings to be drought treated according to the method described in the pre-experiment preparation example. Place the pine seedlings in a hydroponic box with a concentration of 300mM mannitol for drought treatment. Take samples on day 0, day 3 and day 7 of drought treatment. Quickly freeze the pine seedlings under different drought treatments in liquid nitrogen and store them in a freezer at -80℃.

[0042] Example 2 Screening of drought stress marker genes 1. Sampling of Pinus tabuliformis seedlings under different drought stress treatments Pinus tabuliformis seedlings from Example 1, after being subjected to drought treatment simulating 300 mM mannitol for 0 days, 3 days, and 7 days, were taken as samples, with a total of 3 biological replicates, each with 14 seedlings. All materials were immediately flash-frozen with liquid nitrogen after sampling and stored in an ultra-low temperature freezer at -80°C. This helps to protect the biomolecules (such as DNA, RNA, and proteins) in the samples from degradation, thereby maintaining the integrity and quality of the samples.

[0043] 2. RNA extraction, library construction, and transcriptome sequencing According to the extraction requirements, appropriate amounts of reference samples (Pinus tabuliformis seedlings after 0, 3, and 7 days of drought stress) were collected and sent to Annoroad Gene Technology (Beijing) Co., Ltd. The company used a column chromatography method to extract total RNA, and used a NanoDrop 2000 / 8000 micro-spectrophotometer to detect RNA purity and a 5400 detection system to detect RNA concentration and integrity. Library construction involved enriching eukaryotic mRNA with Oligo(dT) magnetic beads, followed by fragmentation to synthesize double-stranded cDNA. The purified double-stranded DNA underwent end repair, A-tailing, and sequencing adapter addition. Fragments of approximately 350 bp were selected and enriched by PCR to obtain the cDNA library. After passing quality control using Qubit 3.0 and Agilent 2100, the library was sequenced using the MGI high-throughput sequencing platform with a PE150 sequencing strategy.

[0044] 3. Data quality control and reference genome alignment The raw reads from the sequencing machine were quality controlled and filtered as follows: reads containing adapter contamination (more than 5 bp of adapter contamination bases) were removed; low-quality reads (more than 50% of the total bases had a quality value of Q ≤ 19) were removed; and reads containing more than 5% N were removed. The filtered results yielded high-quality sequences (Clean Reads) for subsequent analysis.

[0045] Using the high-quality Chinese pine genome (https: / / www.ncbi.nlm.nih.gov / bioproject / PRJNA784915) as a reference genome, clean reads were aligned with the reference genome using HISAT2 software to obtain mapped reads, which were then used for subsequent transcript assembly and expression analysis. The alignment rate for all samples in this project was above 99%, indicating a good match between the sequencing data and the reference genome.

[0046] 4. Gene expression level analysis Clean reads from each sample were mapped onto the Pinus tabuliformis reference genome to obtain mRNA expression levels. The expression abundance of each uigene was calculated using the RNA-Seq data quantification software Kallisto, and the gene expression level was characterized by the TPM (TranscriPt permillion) normalization method.

[0047] 5. Weighted gene co-expression network analysis Genes with expression levels TPM < 1 in each sample were filtered out, retaining the high-expression gene set for weighted gene co-expression network analysis. A gene co-expression network was constructed with a soft threshold power=15, and the network construction and module merging parameters were set to "deepSplit=2; minModuleSize=100; mergeCutHeight=0.20". Correlation analyses were performed on the obtained modules, including inter-module correlation analysis and inter-sample correlation analysis. The former was performed by clustering module gene expression levels, and the latter by Pearson correlation analysis to calculate the correlation between module eigenvalues ​​and sample expression matrices. Finally, PCA analysis was performed on the gene expression levels in each module, using PC1 to represent the module's eigenvector (MEs), and module selection was based on the trend of MEs values. The top 1% of genes from two modules were retained for hub gene network construction. Cytoscape software was used for network visualization and hub gene selection. Sub-networks were extracted using the MCODE plugin, and then drought stress marker genes were obtained by combining the intersection of 12 algorithms, including MMC, using the CytoHubba plugin. Pt7G25060 , Pt7G25060 The CDS sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it expresses is shown in SEQ ID NO.2.

[0048] MARMKHIPGLTLQFQSLLITGAALFLWIQTSDAQDCNGLSHHFYQKSCPNAQAIIKSVVSDAVKKEARMAASLLRLHFHDCFVQGCDASILLDDTASFTGEKTANPNRNSVRGFEVVDKIKSKLEEACPGVVSCADILAVAARDSVGFSVGPHWEVLLGRRDSKTASKSG ANNDIPAPNSTHQTLETKFKLQGLNVLDLVALSGSHTIGLARCSSFKARLYNQTVNGKPDPTLDKSYLKKLRAVCPQTGTDDNRTTPLDPVTPIKFDINYYRNLVEGTGLLSSDEILYSTKGSRTASLVRFYSTNTPAFFKQFAASMIKMGNISPLTGSNGEIRKNCRKRN , SEQ ID NO.2.

[0049] Example 3: Validation of a cell wall-related marker gene for drought stress in Pinus tabuliformis seedlings 1. Primer design Based on drought stress marker genes Pt7G25060 The CDS sequence was used to design primers using NCBI's online primer design website, Primer-BLAST. Pt7G25060 The primer sequences for the gene are: Upstream primer: 5'-ACACCATTGGATCCGGTCAC-3' (SEQ ID NO.3); Downstream primer: 5'-ACCCGGTGAGAGGACTGATG-3' (SEQ ID NO.4).

[0050] 2. Sampling of Pinus tabuliformis seedlings under different drought stress levels Pinus tabuliformis seedlings subjected to drought stress for 0 days (named D0d), 3 days (named D3d), and 7 days (named D7d) were selected as reference samples. All samples were immediately flash-frozen with liquid nitrogen after collection and stored in an ultra-low temperature freezer at -80℃.

[0051] 3. Extraction of total RNA from reference samples Total RNA was extracted from the three samples collected in step 2 using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441) from Tiangen Biotech (Beijing) Co., Ltd. RNA concentration and integrity were determined using a Nano micro spectrophotometer and 1% agarose gel electrophoresis, respectively.

[0052] 4. Reverse transcription of total RNA from reference sample In RNase-free PCR tubes, reverse transcription of total RNA from the three samples in step 3 was performed, and genomic DNA was removed. The reverse transcription system was prepared according to Table 1 below: Table 1 Reverse transcription system

[0053] After gently mixing the reaction system in Table 1, incubate it in a PCR instrument at 42°C for 30 min; then heat it at 85°C for 5 s to completely inactivate TransScript RT / RI and gDNA Remover, and store the cDNA at -20°C.

[0054] 5. Reference samples of Pinus tabuliformis seedlings under different drought stresses Pt7G25060 Real-time quantitative PCR detection of genes Using the cDNA generated from reverse transcription in step 4 as a template (with Actin as the internal reference gene, ensuring the Ct value of the internal reference gene is between 18 and 22), qRT-PCR was performed according to the qRT-PCR reaction system and reaction conditions in Table 2 below: Table 2 qRT-PCR reaction system

[0055] Note: The device used for quantitative PCR validation is the CFX Connect™ Optics Module, with the specific serial number 788BR04747; the software used for gene expression data analysis is Bio-Rad CFX Manager.

[0056] The reaction was carried out using the SYBR Green intercalation fluorescence method. The pre-denaturation reaction program was set as follows: 95℃ pre-denaturation for 2 min, 94℃ denaturation for 5 s, 60℃ annealing for 30 s, for 39 cycles. The melting curve analysis reaction program was set as follows: 65~95℃, increasing by 0.5℃ every 5 s. Each sample to be tested was subjected to three technical replicates and three biological replicates, and an independent internal control gene, Actin, was added to each plate of samples. The primer sequences for the internal control gene Actin are shown in SEQ ID NO.5~SEQ ID NO.6: Upstream primer: 5'-GGCTGACACCATCACCAGAATC-3' (SEQ ID NO.5); Downstream primer: 5'-GTTGGTCGCCCTCGTCATACT-3' (SEQ ID NO.6).

[0057] Expression levels in *Pinus tabuliformis* seedlings under different drought stress conditions were statistically analyzed using the expression measurement instrument and software Bio-Rad CFX Manager. Pt7G25060 Gene expression levels, results are attached. Figure 2 And Table 3.

[0058] Table 3 Seedlings under different drought treatments Pt7G25060 Gene expression level

[0059] From Table 3 and Appendix Figure 2 It can be seen that as the degree of drought stress experienced by Pinus tabuliformis seedlings increases, Pt7G25060 Gene expression levels are decreasing; in Pinus tabuliformis seedlings Pt7G25060 Gene expression levels were negatively correlated with the degree of drought stress, gradually decreasing as the degree of drought stress in Pinus tabuliformis seedlings increased.

[0060] Example 4: Marker genes for drought stress in Pinus tabuliformis seedlings Pt7G25060 Methods for determining the degree of drought stress in the sample to be tested The samples of Pinus tabuliformis seedlings to be tested were subjected to total RNA extraction, reverse transcription, and real-time quantitative PCR detection according to the method in Example 3. The obtained samples were then analyzed. Pt7G25060 Gene expression levels and reference samples Pt7G25060 The expression levels of genes were analyzed for significant differences to determine the degree of drought stress in the samples being tested.

[0061] In the sample to be tested Pt7G25060 Gene expression levels are shown in Table 4, and the analysis of significant differences is shown in the appendix. Figure 3 .

[0062] Table 4. Samples to be tested Pt7G25060 Gene expression level

[0063] Based on the real-time quantitative PCR results of the test sample and the reference sample Pt7G25060 The expression level is used to determine the degree of drought stress experienced by the test sample. The test sample and the reference sample D3d are compared. Pt7G25060 There was no significant difference in expression levels, and the degree of drought stress experienced was similar to that of the reference sample D3d; in the test sample Pt7G25060 The gene expression level was significantly lower than that in the reference sample D0d. Pt7G25060 Gene expression levels can determine whether the drought stress experienced by the test sample is higher than that experienced by the reference sample D0d; test sample Pt7G25060 The expression level was significantly higher than that of the reference sample D7d. Pt7G25060 The expression level can determine that the drought stress level of the sample under test is lower than that of the reference sample D7d.

[0064] In summary, the technical solution provided by this invention can accurately determine the degree of drought stress in the tested samples, especially by directly testing Pinus tabuliformis seedlings. Compared with traditional methods that rely on observing the degree of yellowing of seedling needles and physiological indicators (i.e., the method in the preparation example of this invention), the technical solution provided by this invention can reduce randomness and uncertainty, accurately reflect the degree of drought stress in Pinus tabuliformis seedlings, and has higher sensitivity, stronger scientific basis, and better repeatability. It avoids the increase in seedling costs and replanting expenses caused by slow growth and reduced survival rate of Pinus tabuliformis seedlings due to drought.

[0065] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A kind Pt7G25060 Genes, characterized by, The Pt7G25060 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

2. As described in claim 1 Pt7G25060 Genes, characterized by, The Pt7G25060 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. A Pt7G25060 protein, characterized in that, The amino acid sequence of the Pt7G25060 protein is shown in SEQ ID NO.

2.

4. The claim 1 Pt7G25060 Gene detection primers, characterized in that, The nucleotide sequences of the detection primers are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

5. An amplification method according to claim 1 Pt7G25060 Gene kit, characterized in that, The kit includes the detection primers as described in claim 4.

6. The reagent kit according to claim 5, characterized in that, The kit also includes PCR amplification reagents, which include SYBR Green DNA polymerase, dNTPs, PCR buffer, and internal reference gene primers.

7. The reagent kit according to claim 6, characterized in that, The nucleotide sequences of the internal reference gene primers are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

8. The claim 1 or 2 Pt7G25060 The application of the gene, the Pt7G25060 protein of claim 3, the detection primer of claim 4, or the kit of any one of claims 5-7 in determining the degree of drought stress suffered by Pinus tabuliformis seedlings.

9. A method for determining the degree of drought stress suffered by Pinus tabuliformis seedlings, characterized in that, Includes the following steps: Using the cDNA of the test sample and the cDNA of the reference sample as templates, real-time quantitative PCR amplification was performed using the detection primers described in claim 4, and the concentrations of cDNA in the test sample and the reference sample were statistically analyzed. Pt7G25060 Gene expression levels; When the sample to be tested contains Pt7G25060 Gene expression levels compared to reference samples Pt7G25060 If there is no significant difference in gene expression levels, the drought stress level of the test sample is determined to be the same as that of the reference sample. When the sample to be tested contains Pt7G25060 The gene expression level was significantly lower than that in the reference sample. Pt7G25060 When the gene expression level is measured, it is determined that the drought stress level of the test sample is higher than that of the reference sample. When the sample to be tested contains Pt7G25060 The gene expression level was significantly higher than that in the reference sample. Pt7G25060 When the gene expression level is measured, it is determined that the drought stress level of the test sample is lower than that of the reference sample. The Pt7G25060 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

10. The method for determining the degree of drought stress suffered by Pinus tabuliformis seedlings according to claim 9, characterized in that, The reference sample is a sample of Pinus tabuliformis seeds treated with the same degree of drought stress but different durations of drought stress.

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