Application of PtXG24780 gene and its protein in determining drought stress response of pinus tabulaeformis seedlings

Abstract

The application belongs to the technical field of biology, and particularly relates to PtXG24780 application of a gene and a protein thereof in determining drought stress response of Pinus tabulaeformis seedlings PtXG24780 The amino acid sequence coded by the gene is shown as SEQ ID NO. 2, and the gene and the protein thereof can be used in determining the drought stress response of Pinus tabulaeformis seedlings. PtXG24780 In addition, the application further provides a primer pair for specifically amplifying the gene, and the primer pair can be used for specifically amplifying the gene. PtXG24780 By comparing the expression amount of the gene in the to-be-tested sample and the reference sample, the drought stress response of the to-be-tested sample can be accurately determined. Experiments prove that the technical scheme provided by the application can accurately reflect the damage degree of Pinus tabulaeformis seedlings in the drought stress response, and has higher precision, stronger scientificity and better repeatability.

Description

Technical Field

[0001] This invention belongs to the field of gene and agricultural biotechnology, specifically involving PtXG24780 Application of genes and their proteins in determining the degree of drought stress response of Pinus tabuliformis seedlings. Background Technology

[0002] Pinus tabuliformis ( Pinus tabuliformis Pinus tabuliformis (Carrière) is an evergreen coniferous tree belonging to the genus Pinus in the family Pinaceae of the phylum Gymnosperm. It exhibits strong tolerance to extreme environments and plays a crucial role in soil and water conservation and ecological restoration. The root system of Pinus tabuliformis forms a three-dimensional soil-stabilizing network, effectively enhancing the erosion resistance of slope soils and significantly reducing soil loss. Simultaneously, the canopy intercepts rainfall, and the fallen needles and other debris improve soil structure and increase organic matter content, thereby enhancing the soil's water-holding capacity. Pinus tabuliformis also has extremely high economic value; its timber is a traditional high-quality material, commonly used in the furniture manufacturing industry. The seeds of Pinus tabuliformis have a high oil content, rich in unsaturated fatty acids, and have edible value. Aromatic essential oils can also be extracted from pine needles for use in the development of fragrances and daily chemical products.

[0003] As a highly valuable tree species with strong tolerance to extreme drought environments, the seedling stage of Pinus tabuliformis is the most sensitive period to drought stress. How to cultivate superior seedlings and quickly assess the drought stress status of a batch of seedlings during planting determines the subsequent growth quality of the seedlings. Otherwise, under traditional conditions, it is impossible to promptly and intuitively judge the degree of drought stress from appearance. Cultivation may reveal slow growth and inability to develop normally, leading to a significant increase in labor and time costs, severely restricting the sustainable development of the Pinus tabuliformis industry.

[0004] Plant adaptation to drought stress largely depends on cellular and tissue structural remodeling, with the cortical microtubule network being a key regulator of this process. Microtubule-associated proteins play a crucial regulatory role, embodying high dynamic plasticity in the microtubule network by altering its stability, tortuosity, and cross-linking degree, enabling it to respond rapidly to water deficit signals. However, the existence of response gene markers for drought stress in the seedling stage of *Pinus tabuliformis* cytoskeleton remains inconclusive.

[0005] Therefore, exploring marker genes for 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] The purpose of this invention is to provide a marker gene for determining the degree of drought stress response in Pinus tabuliformis seedlings. PtXG24780 And proteins and their application in determining the drought stress response marker genes of Pinus tabuliformis seedlings under drought stress conditions, said Pinus tabuliformis seedling drought stress response marker genes PtXG24780 It can accurately and efficiently determine the degree of drought stress in Pinus tabuliformis seedlings.， This provides fundamental support for selecting high-quality Pinus tabuliformis seedlings.

[0007] A method for determining the degree of drought stress response in Pinus tabuliformis seedlings PtXG24780 Genes, the ones mentioned PtXG24780 The amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0008] Preferably, the PtXG24780 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0009] Another object of the present invention is to provide a PtXG24780 protein for determining the degree of drought stress response of Pinus tabuliformis seedlings, wherein the amino acid sequence of the PtXG24780 protein is shown in SEQ ID NO.2.

[0010] Another object of the present invention is to provide the above-mentioned PtXG24780 The detection primers for the gene are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0011] Another object of the present invention is to provide an amplification of the above. PtXG24780 A gene assay kit, the kit comprising the detection primers described above.

[0012] Preferably, the kit further includes PCR amplification reagents, which include SYBR Green DNA polymerase, dNTPs, PCR buffer, and internal reference gene primers.

[0013] More preferably, the nucleotide sequences of the internal reference gene primers are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0014] Another object of the present invention is to provide the above-mentioned PtXG24780 Application of the gene, the PtXG24780 protein, the detection primers, or the kit in determining the degree of drought stress on Pinus tabuliformis seedlings.

[0015] Another objective of this invention is to provide a method for determining the degree of drought stress experienced by Pinus tabuliformis seedlings, comprising the following steps: using cDNA from the test sample and cDNA from the reference sample as templates, performing quantitative real-time PCR amplification using the aforementioned detection primers, and statistically analyzing the cDNA levels in the test sample and the reference sample. PtXG24780 Gene expression levels;

[0016] When the sample to be tested contains PtXG24780 The gene expression level was significantly lower than that in the reference sample. PtXG24780When 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... PtXG24780 The gene expression level was significantly higher than that in the reference sample. PtXG24780 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 PtXG24780 The amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0017] Preferably, the reference sample is a sample of Pinus tabuliformis seedlings treated with the same degree of drought stress but different durations of drought stress.

[0018] Beneficial effects: This invention provides a marker gene for determining the degree of drought stress response in Pinus tabuliformis seedlings. PtXG24780 The drought stress response marker gene of Pinus tabuliformis seedlings PtXG24780 The encoded amino acid sequence is shown in SEQ ID NO.2. This invention used 43-day, 47-day, and 54-day-old *Pinus tabuliformis* seedlings as sample materials, and obtained marker genes based on the degree of drought stress response during the seedling stage of *Pinus tabuliformis* through high-throughput sequencing and analysis screening. PtXG24780 The expression level of this gene in drought-stricken Pinus tabuliformis seedlings differed significantly from that in normal seedlings, enabling accurate determination of the drought stress level in the test samples and further providing the protein encoded by this gene, PtXG24780. PtXG24780 Both the gene and the PtXG24780 protein can be used to determine the degree of drought stress response in Pinus tabuliformis seedlings. Experiments have shown that, compared with traditional methods that rely on observing the state of Pinus tabuliformis needles and physiological indicators, the technical solution provided by this invention can accurately reflect the degree of drought stress on Pinus tabuliformis seedlings, with higher precision, stronger scientific basis, and better reproducibility. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly introduced below.

[0020] Figure 1 The results of agarose gel electrophoresis in Example 2 of this invention; Figure 2 The images shown in Example 2 depict the seedlings of Pinus tabuliformis under the same treatment conditions, in a non-drought-stress state (i.e., 0 days) and under drought-stress conditions for 3 days, 7 days, and 14 days. Figure 3 The needles of Pinus tabuliformis seedlings in Example 2 under both non-drought and drought conditions after drought conditions were created. PtXG24780 Bar chart comparing gene expression levels; Figure 4 Example 3 illustrates the determination of drought severity in Pinus tabuliformis, specifically within the sample to be tested. PtXG24780 Gene expression levels were compared with those within 0d, 3d, 7d, and 14d, respectively. PtXG24780 Analysis of significant differences in gene expression levels, among which represent P<0.0001 . Detailed Implementation

[0021] This invention provides a marker gene for determining the degree of drought stress response in Pinus tabuliformis seedlings. PtXG24780 Its nucleotide sequence is shown in SEQ ID NO.1, which is a marker gene for the degree of drought stress response in the Pinus tabuliformis seedlings. PtXG24780 The encoded amino acid sequence is shown in SEQ ID NO.2.

[0022] In this invention, the marker gene for the drought stress response of the Pinus tabuliformis seedlings is... PtXG24780 Both the PtXG24780 protein encoded by it can be used to determine the degree of drought stress in Pinus tabuliformis seedlings.

[0023] This invention also provides amplification of marker genes for the drought stress response of the above-mentioned Pinus tabuliformis seedlings. PtXG24780 The primer pair comprises an upstream primer and a downstream primer, the nucleotide sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0024] This invention also provides a kit comprising the aforementioned primer pairs and PCR amplification reagents. As one embodiment, the PCR amplification reagent can be a real-time quantitative PCR amplification reagent; as another embodiment, the real-time quantitative PCR amplification reagent can include SYBR Green DNA polymerase, dNTPs, and PCR buffer. As one embodiment, the kit further includes one or more of an internal reference gene primer, RNA extraction reagent, and reverse transcription reagent. That is, kits containing primer pairs and PCR amplification reagents, with the addition of any one or more of an internal reference gene primer, RNA extraction reagent, and reverse transcription reagent, are all within the protection scope of this invention. As one embodiment, the internal reference gene can be... Actin Gene; as another implementation, the ActinThe nucleotide sequences of the upstream and downstream primers for the gene are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively. This invention does not impose any special limitations on the source and amount of the PCR amplification reagents, RNA extraction reagents, and reverse transcription reagents; they can be conventionally set and selected according to needs.

[0025] Based on the marker genes of drought stress response of the Pinus tabuliformis seedlings PtXG24780 The PtXG24780 protein can accurately determine the degree of drought stress response in Pinus tabuliformis seedlings, and the effect is more accurate than the traditional method that relies on observing the state of Pinus tabuliformis needles.

[0026] Based on the above advantages, the present invention also provides the drought stress response marker gene for Pinus tabuliformis seedlings described in the above technical solution. PtXG24780 The application of the PtXG24780 protein, primer pairs, or kits described in the above technical solutions in determining the degree of drought stress in Pinus tabuliformis seedlings.

[0027] Specifically, the present invention also provides a method for determining the degree of drought stress in 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 primer pairs described in the above technical solution. The levels of cDNA in the test sample and the reference sample were then statistically analyzed. PtXG24780 Gene expression levels; When the sample to be tested contains PtXG24780 Gene expression levels compared to the reference sample PtXG24780 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 PtXG24780 The gene expression level was significantly lower than that in the reference sample. PtXG24780 When the gene expression level is considered, the drought stress response of the test sample is determined to be higher than that of the reference sample. When the sample to be tested contains PtXG24780 The gene expression level was significantly higher than that in the reference sample. PtXG24780 When the gene expression level is measured, the drought stress level of the test sample is determined to be lower than that of the reference sample.

[0028] This invention uses cDNA from the test sample and cDNA from the reference sample as templates, and performs real-time quantitative PCR amplification using the primer pairs described in the above-described technical solution, respectively, and statistically analyzes the cDNA content in the test sample and the reference sample. PtXG24780Gene expression levels. This invention does not specifically limit the preparation method of the cDNA; conventional cDNA preparation methods in the art can be used. In one embodiment, the test sample and reference sample are Pinus tabuliformis seedlings of the same variety. In another embodiment, the reference sample is Pinus tabuliformis seedlings subjected to the same degree of drought stress but different drought stress durations; in yet another embodiment, the reference sample is 40-day-old Pinus tabuliformis seedlings with uniform growth.

[0029] In one implementation method, the 20 μL reaction system for quantitative real-time PCR amplification includes: 10 μL of 2 × SuperReal PreMix Plus (SYBR Green), 1 μL of cDNA template, 0.4 μL of 100 μM upstream primer, 0.4 μL of 100 μM downstream primer, and RNase-free water to bring the total to 20 μL. The preferred reaction program for quantitative 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 allows for the specific amplification of cDNA from the test sample and the reference sample, respectively.

[0030] This invention does not impose special requirements on the equipment used for quantitative real-time PCR amplification or the method for analyzing gene expression levels; techniques well-known in the art can be used. In a specific embodiment of this invention, the equipment used for quantitative real-time PCR amplification is the CFX Connect™ Optics Module, specifically numbered 788BR04747; the software used for gene expression data analysis is Bio-Rad CFX Manager.

[0031] Experiments have shown that, compared with traditional methods that rely on observing the phenotypic state and physiological indicators of Pinus tabuliformis, the technical solution provided by this invention can accurately reflect the drought response of Pinus tabuliformis seedlings, with higher precision, stronger scientific basis, and better repeatability.

[0032] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0033] Preparation before the experiment: 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 for germination on sphagnum moss. The specific process was as follows: First, the pine seeds were placed in a 4℃ refrigerator for cold stratification for 7 days to ensure normal germination. Then, the seeds were disinfected with 0.5% sodium hypochlorite for 20 minutes, followed by thorough rinsing with sterile water to remove any sodium hypochlorite residue and prevent it from affecting germination. Sterile sphagnum moss moistened with sterile water was placed in a culture box, and the seeds were evenly placed on the surface. Finally, sterile water was sprayed on top and the box was covered. The culture boxes containing the seeds were placed in an incubator at 22℃ with a light condition of 16 hours of light and 8 hours of darkness. The number of germinating seeds was recorded daily, and sterile distilled water was added to the culture dishes as needed. After 30 days of germination, seedlings with uniform growth were transferred to four 24-well hydroponic boxes without drought treatment for 10 days to allow them to recover. Once stabilized, drought treatment was then carried out.

[0034] Example 1 marker genes for drought stress response PtXG24780 Filtering: 1. Sampling of Pinus tabuliformis seedlings under different drought stress treatments Based on the pre-experimental preparations, 40-day-old (10 days after acclimatization) Pinus tabuliformis seedlings were transferred to a hydroponic system simulating drought treatment with 300 mM mannitol. The water was changed every 3 days to replenish dissolved oxygen and maintain normal root physiological function and growth environment. Samples of Pinus tabuliformis were taken on days 3, 7, and 14 (43, 47, and 54).

[0035] Three biological replicates were used, with 10 seedlings per replicate, to ensure the accuracy of subsequent experimental results. After sampling, the above materials were immediately flash-frozen with liquid nitrogen and stored in an ultra-low temperature freezer at -80°C to protect the biomolecules in the samples from degradation, thereby maintaining the integrity and quality of the samples.

[0036] 2. RNA extraction, library construction, and transcriptome sequencing According to the extraction requirements, appropriate samples (non-drought-stressed (i.e., 0 days) and drought-stressed pine seedlings at 3, 7, and 14 days) were collected and sent to Annoroad Gene Technology (Beijing) Co., Ltd. Total RNA was extracted using a column chromatography method. RNA purity was detected using a NanoDrop 2000 / 8000 micro-spectrophotometer, and RNA concentration and integrity were detected using a 5400 detection system. 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-tailed sequencing adapter addition, and fragments of approximately 350 bp were selected for PCR enrichment 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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. PtXG24780 , PtXG24780 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.

[0041]

[0042] MQMNSARRLLSATTTPGGSVDPFRVELDRLQNQLKDKDRELGDAQAEIKALKLTERLKEKAVEELTNTLGKLEEKLKTNEALLESKNLEIKKLNDEKKAALATQFAVESALRRVYAAQKDEDMIPLEALLAPFEAEEIKATRHHI ILLQEDYRALERLTKSKEAALLEAEKSVQVAQAKADMVDDLQNKNQELTRQLEICQEENKIIDRMHRVKVSEVERLSQTICELEEAILNGGAAINALHDYERKVYELTEEKRTLERELTRAKITENRVATAVVGHEWRDGNDKVI PLKRWLEERRFIQGEMQQLREKVALAERTAKAEEQLKDKFKLRLKVLEEAVRGVGAFRSAQQNEYQRRASRTNGSSRCGQLVCGDDSARTLQSNGIGNSLETVLTWKHSVSSNNNTQKETPQLGVVVDGTINAASDLINSLSNEH VELNNGDNGLSTNTDNSHSENNKICKEIVECNAGSDDTVSGVFYDILQREVIALRKACHHKDLNVKHKEDVIKVLSKKVDTLKRAMDVEAKKMRREVAAREKEVASMRVEQDNRGESRRSNITSKGPTNTNTSPRVPGRISTNSQ , SEQ ID NO.2.

[0043] Example 2 marker genes for drought stress response PtXG24780 Verification 1. Primer design marker genes based on drought stress response PtXG24780 Primers were designed using Primer-BLAST, an online primer design website developed by NCBI. PtXG24780 The primer sequences for the gene are: Upstream primer: 5'-CAAGCAGAACGAATGGCTCG-3' (SEQ ID NO.3); Downstream primer: 5'-TCCCATCAACAACCACACCA-3' (SEQ ID NO.4).

[0044] Upstream primer for internal control: 5'-GGCTGACACCATCACCAGAATC-3' (SEQ ID NO.5); Downstream primer for internal control: 5'-GTTGGTCGCCCTCGTCATACT-3' (SEQ ID NO.6).

[0045] 2. Sampling of Pinus tabuliformis under different drought stress days Ten *Pinus tabuliformis* seedlings from the same batch that underwent mannitol drought treatment for 3, 7, and 14 days, as well as ten seedlings that were not drought treated (i.e., 0 days), were collected from each batch for phenotypic observation (results are attached). Figure 2 (As shown in Table 1), all samples were immediately flash-frozen with liquid nitrogen and stored in an ultra-low temperature freezer at -80°C.

[0046] Table 1. Phenotypic observation of Pinus tabuliformis seedlings under drought stress

[0047] 3. Extraction of total RNA from samples Total RNA was extracted from the 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 measured using a Nano micro spectrophotometer and a 1% agarose gel electrophoresis apparatus, respectively. Swimming assay. Results are attached. Figure 1 As shown in Table 2: Table 2. Results of RNA concentration and purity

[0048] 4. Reverse transcription of total RNA from the sample In an RNase-free PCR tube, perform reverse transcription of the total RNA from the sample in step 3, and remove genomic DNA. Prepare the reverse transcription system according to Table 3 below: Table 3 Reverse transcription system

[0049] The above systems were gently mixed and incubated in a PCR instrument at 42°C for 15 min. Then, they were heated at 85°C for 5 s to completely inactivate the RT / RIEnzyme and gDNA Remover. The cDNA products were stored at -20°C.

[0050] 5. Among the same batch of Pinus tabuliformis seedlings treated with drought stress and those treated without drought stress... PtXG24780 Comparison of genes by real-time quantitative PCR: After appropriately diluting the cDNA generated by reverse transcription in step 4, use it as a template (with Actin as the internal reference gene) and perform qRT-PCR according to the qRT-PCR reaction system and reaction conditions in Table 4 below: Table 4 qRT-PCR reaction system

[0051] Note: The device used for quantitative PCR validation is the CFX Connect™ Optics Module, with the device serial number 788BR04747; the software used for gene expression data analysis is Bio-Rad CFX Manager.

[0052] The reaction was performed using the SYBR Green chimeric fluorescence assay. The pre-denaturation reaction program was set as follows: 95℃ for 2 min. The PCR reaction program was set as follows: 94℃ for 5 s denaturation, 60℃ for 30 s annealing, 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 9 technical replicates and 3 biological replicates.

[0053] Expression levels in the needles of Pinus tabuliformis seedlings were analyzed using the Bio-Rad CFX Manager instrument and software after 0, 3, 7, and 14 days of drought treatment. PtXG24780 Gene expression levels, results are attached. Figure 3 And Table 5, in the appendix Figure 3 middle Indicates significance of difference P<0.0001 .

[0054] Table 5. Pine seedlings under drought and non-drought conditions PtXG24780 Gene expression level

[0055] From Table 5 and Appendix Figure 3 It can be seen that as the degree of drought stress experienced by Pinus tabuliformis seedlings increases, PtXG24780 Gene expression levels are decreasing; in Pinus tabuliformis seedlings PtXG24780 Gene expression levels were negatively correlated with the degree of drought stress, gradually decreasing as the degree of drought stress on Pinus tabuliformis seedlings increased.

[0056] Example 3 To further explain and verify the above results, this embodiment is an example of determining the degree of drought of the sample to be tested.

[0057] The PCR method described in Example 2 was used to test the sample. PtXG24780 Genes were amplified, and their expression levels are shown in Table 6. Analyses of significant differences are provided in the appendix. Figure 4 .

[0058] Table 6. Samples to be tested PtXG24780 Gene expression level

[0059] Based on the sample to be tested and the attached Figure 4 Real-time quantitative PCR results, PtXG24780 The expression level of the sample determines the degree of drought stress experienced by the sample. PtXG24780 The expression level was significantly lower than that of the reference sample on day 0, indicating that the drought stress experienced by this sample was greater than that experienced by the reference sample on day 0; in the test sample PtXG24780 The gene expression level was significantly higher than that of the test sample 3 days ago. PtXG24780 The expression level of genes can determine that the drought stress level of the test sample is lower than that of the test sample 3 days ago, proving that the drought level of the test sample is between 0 days and 3 days ago.

[0060] 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 condition and physiological indicators of Pinus tabuliformis seedlings, 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 precision, stronger scientific basis, and better repeatability. It avoids the time and financial losses caused by finding that the seedlings grow slowly or cannot grow normally after planting.

[0061] 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 PtXG24780 gene for determining the degree of drought stress response in Pinus tabuliformis seedlings, characterized in that, The amino acid sequence encoded by the PtXG24780 gene is shown in SEQ ID NO.2, and the nucleotide sequence of the PtXG24780 gene is shown in SEQ ID NO.

1.

2. The detection primers for the PtXG24780 gene as described in claim 1, characterized in that, The nucleotide sequences of the detection primers are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

3. A kit for amplifying the PtXG24780 gene as described in claim 1, characterized in that, The kit includes the detection primers as described in claim 2.

4. The reagent kit according to claim 3, characterized in that, The kit also includes PCR amplification reagents, which include SYBR Green DNA polymerase, dNTPs, PCR buffer, and internal reference gene primers.

5. The reagent kit according to claim 4, 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.

6. The application of the PtXG24780 gene of claim 1, the detection primer of claim 2, or the kit of any one of claims 3-5 in determining the degree of drought stress suffered by Pinus tabuliformis seedlings.

7. A method for determining the degree of drought stress suffered by Pinus tabuliformis seedlings, characterized in that, The procedure includes the following steps: using the cDNA of the sample to be tested and the cDNA of the reference sample as templates, performing real-time PCR amplification using the detection primers described in claim 2, and counting the expression levels of the PtXG24780 gene in the sample to be tested and the reference sample, respectively. When the expression level of the PtXG24780 gene in the test sample is significantly lower than that in the reference sample, the drought stress level of the test sample is determined to be higher than that of the reference sample; when the expression level of the PtXG24780 gene in the test sample is significantly higher than that in the reference sample, the drought stress level of the test sample is determined to be lower than that of the reference sample. The amino acid sequence encoded by the PtXG24780 gene is shown in SEQ ID NO.2, and the nucleotide sequence of the PtXG24780 gene is shown in SEQ ID NO.

1.

8. The method according to claim 7, characterized in that, The reference sample is a sample of Pinus tabuliformis seedlings treated with the same degree of drought stress but different durations of drought stress.

Images