Application of Pt6G00980 gene and protein thereof in determining germination activity of Chinese pine seeds

Abstract

The invention discloses an application of a Pt6G00980 gene and a protein thereof in determining the germination activity of Chinese pine seeds, and relates to the technical field of biology. The expression quantity of the gene is in negative correlation with the germination activity and is gradually reduced along with the germination of Chinese pine seeds. The invention also provides a primer group for detecting the gene, the cDNA of a sample to be detected and the cDNA of a reference sample are subjected to quantitative PCR amplification by using the primer group provided by the invention, and the expression quantity of the Pt6G00980 gene in the sample to be detected and the expression quantity of the Pt6G00980 gene in the reference sample are compared, so that the germination activity of the sample to be detected can be accurately judged. Compared with a traditional distinguishing method depending on observation of the seed germination rate and physiological indexes, the technical scheme provided by the invention can accurately reflect the germination activity of the Chinese pine seeds, and is higher in precision, stronger in scientificity and good in repeatability.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and more specifically to the application of a Pt6G00980 gene and a protein thereof in determining the germination vitality of Pinus tabulaeformis seeds. Background Art

[0002] Pinus tabulaeformis Carr., an evergreen coniferous tree of the genus Pinus in the Pinaceae family, is a species unique to China and is widely distributed in the Northeast, Central Plains, Northwest and Southwest regions. Pinus tabulaeformis not only has excellent soil and water conservation and water conservation functions, but also plays an important role in improving soil and preventing wind and sand. It is an indispensable key tree species in barren hill afforestation, afforestation and urban greening. In addition, the wood of Pinus tabulaeformis is hard and durable, and is widely used in construction, bridges, railways and other industries. The branches, leaves, root bark, resin and other parts of Pinus tabulaeformis also have high medicinal value and are often used in the extraction of Chinese medicinal materials. Therefore, its comprehensive economic value is very considerable and has important ecological and economic significance.

[0003] However, due to the large differences in the germination of Pinus tabulaeformis seeds and the influence of the dormancy level of the seeds themselves, uneven germination of seeds or failure of some seeds to germinate successfully often occurs. Therefore, the traditional method of determining the germination of Pinus tabulaeformis seeds relies on the observation of seed germination rate or physiological indicators, but this method has shortcomings in practical applications. Usually, the germination of seeds can only be observed with the naked eye, and it is greatly affected by external factors such as the environment and climate, resulting in poor accuracy and repeatability of the evaluation results. Therefore, the traditional method is not only time-consuming and labor-intensive, but also unable to efficiently and accurately evaluate the germination of Pinus tabulaeformis seeds, and cannot meet the efficient needs of modern forestry production and ecological restoration.

[0004] Therefore, developing a high-efficiency and high-accuracy method for determining the germination of Pinus tabulaeformis seeds is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides an application of a Pt6G00980 gene and a protein thereof in determining the germination vitality of Pinus tabulaeformis seeds.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The Pt6G00980 gene, the amino acid sequence encoded by the Pt6G00980 gene is shown in SEQ ID NO.2.

[0008] Preferably, the nucleotide sequence of the Pt6G00980 gene is as shown in SEQ ID NO.1.

[0009] Another object of the present invention is to provide a Pt6G00980 protein, the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] Another object of the present invention is to provide a primer set for amplifying the above-mentioned Pt6G00980 gene, the nucleotide sequences of the primer set are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0011] Preferably, the nucleotide sequence of the primer set also includes internal reference primers as shown in SEQ ID NO.5 and SEQ ID NO.6.

[0012] Another object of the present invention is to provide a kit for amplifying the above-mentioned Pt6G00980 gene, wherein the kit comprises the above-mentioned primer set.

[0013] Preferably, the kit further comprises reagents for extracting total RNA, and / or reagents for reverse transcribing total RNA as a template into cDNA, and / or reagents for quantitative PCR of cDNA.

[0014] Another object of the present invention is to provide the use of the above-mentioned Pt6G00980 gene, or the above-mentioned Pt6G00980 protein, or the above-mentioned primer set, or the above-mentioned kit in detecting the germination vitality of Pinus tabulaeformis seeds.

[0015] Another object of the present invention is to provide a method for determining the germination vitality of Pinus tabulaeformis seeds, comprising the following steps:

[0016] S1: using the cDNA of the sample to be tested and the cDNA of the reference sample as templates, using the above primer set or the above kit to perform quantitative PCR amplification on the templates, and respectively counting the expression levels of the Pt6G00980 gene in the sample to be tested and the reference sample;

[0017] S2: When there is no significant difference between the expression amount of the Pt6G00980 gene in the test sample and the expression amount of the Pt6G00980 gene in the reference sample, it is determined that the germination activity of the test sample is the same as the germination activity of the reference sample;

[0018] When the expression level of the Pt6G00980 gene in the test sample is significantly lower than the expression level of the Pt6G00980 gene in the reference sample, it is determined that the germination activity of the test sample is higher than that of the reference sample;

[0019] When the expression level of the Pt6G00980 gene in the test sample is significantly higher than the expression level of the Pt6G00980 gene in the reference sample, it is determined that the germination activity of the test sample is lower than that of the reference sample;

[0020] The reference samples are Pinus tabulaeformis seeds subjected to heat stress at 37°C for 15 days (named H15d), Pinus tabulaeformis seeds subjected to cold stratification at 4°C for 7 days (named C7d), and Pinus tabulaeformis seeds in the germination and whitening period (named GW).

[0021] Preferably, the quantitative PCR amplification reaction system is 20 μL, including: 10 μL of 2× SuperRealPreMix 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 is added to 20 μL;

[0022] The reaction procedure of the quantitative PCR amplification was: pre-denaturation at 95°C for 2 min, denaturation at 94°C for 5 s, annealing at 60°C for 30 s, and 39 cycles.

[0023] Beneficial effects:

[0024] The present invention provides a Pt6G00980 gene and a Pt6G00980 protein encoded by the gene. The expression level of the gene is negatively correlated with the germination vigor, and gradually decreases with the germination of Pinus tabulaeformis seeds. Based on the above technical advantages, the present invention provides a method for determining the germination vigor of Pinus tabulaeformis seeds. By using the primer set provided by the present invention to perform quantitative PCR amplification on the cDNA of the test sample and the cDNA of the reference sample, and comparing the expression level of the Pt6G00980 gene in the test sample and the reference sample, the germination vigor of the test sample can be accurately determined. Compared with the traditional discrimination method that relies on observing the seed germination rate and physiological indicators, the technical solution provided by the present invention can accurately reflect the germination vigor of Pinus tabulaeformis seeds, and has higher accuracy, stronger scientificity and good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0026] Figure 1 The attached figure shows the expression results of Pt6G00980 gene in samples of different germination degrees of Pinus tabulaeformis seeds detected by real-time fluorescence quantitative PCR.

[0027] Figure 2 The attached figure shows the expression results of the Pt6G00980 gene in different test samples detected by real-time fluorescence quantitative PCR in Example 4. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The embodiments of the present invention disclose the application of the Pt6G00980 gene in determining the germination vitality of Pinus tabulaeformis seeds. The materials and methods involved are conventional materials and methods unless otherwise mentioned, and will not be described in detail here.

[0030] Example 1

[0031] Pinus tabulaeformis seeds: purchased from Zhongtiaoshan State Forest Farm in Yuanqu County, Yuncheng City, Shanxi Province; Yuanqu Zhongtiaoshan Forest Farm belongs to a fault basin with an average annual temperature of 22°C, a maximum temperature of 40°C, and a minimum temperature of -11°C; mature Pinus tabulaeformis cones were collected from well-grown mature Pinus tabulaeformis mother plants, the seeds were collected after drying at room temperature, and mixed evenly.

[0032] The above seeds were tested for vitality and germination in distilled water. The specific process was as follows: 6 layers of sterilized gauze were laid in a petri dish and soaked with sterile water to keep the gauze moist. After washing the seeds with water, the seeds were disinfected with 0.5% sodium hypochlorite for 20 minutes and then fully washed with sterile water; 25 seeds were placed in the petri dish and the seeds were evenly placed in the petri dish so that each seed was in contact with the moist gauze. One petri dish was one treatment, and 4 parallel replicates were set for each treatment.

[0033] Place the culture dish containing the seeds in an incubator at 22°C, with 8 hours of light and 16 hours of darkness, and add sterile distilled water to the culture dish as needed. The germination criterion is that the radicle protrudes the seed coat. Use tweezers to break the ungerminated seeds to test whether the seeds are viable (vitality judgment standard: if the embryo of the seed is white and solid, the seed is considered viable; if the embryo is moldy or breaks when touched with tweezers, the seed is considered inactive)

[0034] The seeds were treated by the method described in the existing patent (CN 117887734A) to prepare seeds with different treatments: Whitening seeds (GW): Seeds with radicles breaking through the seed coat were considered to have germinated, i.e. whitening seeds. Seeds with radicles extending out of the seed coat <3 mm were taken out of the culture dish, quickly frozen in liquid nitrogen, and stored in a -80°C refrigerator. Non-dormant seeds (C7d): Vigorous Pinus tabulaeformis seeds were evenly placed in a culture dish padded with 6 layers of gauze (the gauze was sterilized), and sterilized distilled water was added to the culture dish to make the gauze saturated and moist. The culture dish was wrapped with tin foil and then placed in a black bag. The culture dish containing the seeds was placed in a refrigerator at 4°C for 7 days of cold stratification, after which the seeds were taken out and quickly frozen in liquid nitrogen and stored in a -80°C refrigerator. Induce seed dormancy (H15d): Place the non-dormant seeds obtained by cold stratification treatment in a petri dish with 6 layers of moist gauze, wrap the petri dish with tin foil, and place it in a completely dark incubator at 37°C (i.e. heat stress treatment). After 15 days of heat stress, take out the seeds and quickly freeze them in liquid nitrogen and store them in a -80°C refrigerator.

[0035] The treated seeds were subjected to germination test in distilled water (the specific process of germination test is as described above), the number of germinated seeds was recorded, and the vitality of ungerminated seeds was tested. During the germination test, one culture dish was one treatment, each treatment was repeated 4 times in parallel, and each culture dish contained 25 seeds. The results are shown in Table 1.

[0036] Table 1 Germination rate or germination rate of Pinus tabulaeformis seeds under different treatments

[0037] Example 2

[0038] Screening of germination vigor marker gene Pt6G00980

[0039] 1) Sampling of Pinus tabulaeformis seeds with different dormancy degrees Seeds after 15 days of heat stress, seeds after 7 days of cold stratification, and white seeds prepared before the experiment were taken as samples. Each group of samples had 3 biological replicates, and each replicate had 25 seeds. The above materials were immediately quick-frozen with liquid nitrogen after sampling and stored in a -80℃ ultra-low temperature refrigerator to protect the biological molecules (such as DNA, RNA and protein) in the samples from being degraded, thereby maintaining the integrity and quality of the samples.

[0040] 2) RNA extraction, library construction, and transcriptome sequencing

[0041] The total RNA of the reference samples (seeds after 15 days of heat stress, seeds after 7 days of cold stratification, and seeds with whitening) was extracted by the Trizol method. After quality inspection, the transcriptome library was constructed using the NEB #7530 kit (#E7530 purchased from New England Biolabs). After the library passed the quality inspection, it was sent to Shanghai Yunxu Biotechnology Co., Ltd. for high-throughput sequencing on the Illumina nova-6000 platform.

[0042] 3) Data quality control and reference genome comparison

[0043] Perform data quality control on raw reads, filter reads with base N ratio greater than 10%, filter reads with quality value Q≤20 and base number accounting for more than 50% of the whole read, truncate adaPtor and the part after it, filter reads shorter than 50bp after truncation of adaPtor, and obtain high quality clean reads;

[0044] The high-quality Chinese pine genome (https: / / www.ncbi.nlm.nih.gov / bioproject / PRJNA784915) was used as a reference genome, and the quality control data were compared with it to obtain the matching data (mapped reads) for subsequent transcript assembly and expression analysis.

[0045] 4) Gene expression analysis

[0046] The clean reads of each sample were mapped to the reference genome of Pinus tabulaeformis to obtain the expression level of mRNA. The RNA-Seq data quantification software Kallisto was used to calculate the expression abundance of each uigene, and the TPM (TranscriPt per million) normalization method was selected to characterize the expression of the gene.

[0047] 5) Weighted gene co-expression network analysis

[0048] Genes with expression levels of TPM < 1 in all samples were filtered out, and the filtered gene set was soft-thresholded with "power=15" to construct a gene network. The parameters for network construction and module merging were "deepSplit =2; minModuleSize=100; mergeCutHeight = 0.20". The obtained modules were analyzed for correlation between modules and between samples and modules. The former was clustered by module gene expression, and the latter was calculated 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 the module, and PC1 was used to represent the module index, namely the module eigenvector (MEs), and the module was screened based on the change trend of MEs values. The top 1% genes of the two modules were retained to construct the hub gene network. The network was visualized and hub genes were screened using Cytoscape software. The subnetwork was extracted using the MCODE plug-in, and then the CytoHubba plug-in was used to combine the intersection of 12 algorithms such as MMC to obtain the germination vitality marker gene Pt6G00980. The CDS sequence of the Pt6G00980 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.

[0049]

[0050] Amino acid sequence of Pt6G00980 protein: MAKVQAWFAILTVIITGNILRSKSGEPLVPALFIFGDSLADAGNNNNLTTLAKANFLPYGRAFPDSVPTGRFSNGYNSMDFLCFKLGLPIIPIYADPNTKGGNLLRGVNYASGASGIENYSGRSFGELIPLEDQVNNFMNTKQEIIDVLGEEGASQLISKAVFSIITGNNDWLNT YLFPFSPLRVLYTVDQFRDNLINKLALQIQKLYSNGARNFVVAGLSALGCLPSQLNRYNSNGSCIDFLNDMAQNFNEALRLRVQDLNSQLPNSTFLFNNLYTPLYEAFHNPAAYGFIYVNEACCGIGKFGGFLICVPEFPVCSNEEDYLFWDAYHTTDKMFKQMVDLMWENGPPYSYPISGKEAVYRTM, SEQ ID NO.2.

[0051] Example 3

[0052] Verification of germination vigor marker genes

[0053] 1) Primer design

[0054] According to the CDs sequence of the germination vigor marker gene Pt6G00980, primers were designed using the NCBI online primer design website Primer BLAST. The primer sequence of the Pt6G00980 gene is:

[0055] Upstream primer: 5′-CCCTTCTCACCCCTTCGAGT-3′ (SEQ ID NO. 3);

[0056] Downstream primer: 5'-TTTGGGCCATGTCGTTCAGA-3' (SEQ ID NO. 4).

[0057] 2) Sampling of Pinus tabulaeformis seeds at different germination stages

[0058] Pinus tabulaeformis seeds subjected to heat stress at 37°C for 15 days (named H15d), Pinus tabulaeformis seeds subjected to cold stratification at 4°C for 7 days (named C7d), and Pinus tabulaeformis seeds at the germination and whitening stage (named GW) were selected. All of the above materials were immediately quick-frozen with liquid nitrogen after treatment and stored in an ultra-low temperature refrigerator at -80°C.

[0059] 3) Extraction of total RNA from samples

[0060] The total RNA of the samples collected in step 2) was extracted using RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441) from Tiangen Biochemical Technology (Beijing) Co., Ltd. The RNA concentration and integrity were determined using a Nano micro-spectrophotometer and 1% agarose gel electrophoresis, respectively.

[0061] 4) Reverse transcription of total RNA from samples

[0062] In an RNase free PCR tube, complete the reverse transcription reaction of the total RNA obtained in step 3) and remove the genomic DNA. Prepare the reverse transcription system according to Table 2 below:

[0063] Table 2 Reverse transcription system

[0064] The above system was gently mixed, incubated in a PCR instrument at 42 °C for 15 min, and then heated at 85 °C for 5 s to completely inactivate RT / RI Enzyme and gDNA Remover. The product was stored at -20 °C.

[0065] 5) Real-time fluorescence quantitative PCR verification of Pt6G00980 gene

[0066] The cDNA generated by reverse transcription in step 4) was diluted 5 times and used as a template, and qRT-PCR was performed according to the qRT-PCR reaction system and reaction conditions in Table 3 below:

[0067] Table 3 qRT-PCR reaction system

[0068] The SYBR Green chimeric fluorescence method was used for the reaction. The pre-denaturation reaction program was set at 95°C for 2 min, the PCR reaction program was set at 94°C for 5 s, 60°C for 30 s, and 39 cycles. The melting curve analysis reaction program was set at 65-95°C with an increase of 0.5°C every 5 s. Each sample was repeated 3 times technically and 3 times biologically, and an independent internal reference Actin was added to each plate sample.

[0069] Internal reference forward primer: 5′-GGCATACCGGCAGCTCTTC-3′ (SEQ ID NO. 5);

[0070] Internal reference reverse primer: 5′-AAGTTGTTGGCGGCGTCTT-3′ (SEQ ID NO. 6);

[0071] The results are shown in Table 4 and Appendix Figure 1As shown, with the increase of seed germination vitality and the establishment of seedlings, the expression level of Pt6G00980 gene decreased, indicating that the expression level of Pt6G00980 gene in Pinus tabulaeformis seeds was negatively correlated with germination vitality and gradually decreased with the germination of Pinus tabulaeformis seeds.

[0072] Table 4 qPCR test results

[0073] Example 4

[0074] Method for judging germination vitality of samples based on germination vitality marker gene Pt6G00980 of Pinus tabulaeformis seeds

[0075] 1. Extraction of total RNA from Pinus tabulaeformis seeds

[0076] (1) Viable Pinus tabulaeformis seeds are the sample materials to be tested.

[0077] (2) Reference sample settings

[0078] Pinus tabulaeformis seeds subjected to heat stress at 37°C for 15 days (named H15d), Pinus tabulaeformis seeds subjected to cold stratification at 4°C for 7 days (named C7d), and Pinus tabulaeformis seeds at the germination and whitening stage (named GW) were selected as reference samples, and the expression level of the Pt6G00980 gene in these samples was used as the standard for indicating low germination activity of Pinus tabulaeformis seeds.

[0079] Use Adela EASYspin Plus Plant RNA Kit for extraction, and follow the instructions for specific steps.

[0080] The RNA concentration and integrity were determined by Nano micro-spectrophotometer and 1% agarose gel electrophoresis, respectively.

[0081] 2. Synthesis of the first-strand cDNA

[0082] Prepare the reverse transcription reaction system according to the reagent addition amounts in Table 5.

[0083] Table 5 Reverse transcription reaction system

[0084] The above system was gently mixed, incubated in a PCR instrument at 42 °C for 15 min, and then heated at 85 °C for 5 s to completely inactivate RT / RI Enzyme and gDNA Remover. The product was stored at -20 °C.

[0085] 3. Real-time fluorescence quantitative PCR

[0086] The real-time fluorescence quantitative PCR reaction system was prepared according to the reagent addition amounts in Table 6.

[0087] Table 6 Reaction system of real-time fluorescence quantitative PCR

[0088] The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3; the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4. The nucleotide sequence of the internal reference forward primer is shown in SEQ ID NO.5; and the nucleotide sequence of the internal reference negative primer is shown in SEQ ID NO.6.

[0089] The SYBR Green chimeric fluorescence method was used for the reaction. The pre-denaturation reaction program was set at 95°C for 2 min, the PCR reaction program was set at 94°C for 5 s, 60°C for 30 s, and 39 cycles; the melting curve analysis reaction program was set at 65-95°C with an increase of 0.5°C every 5 s. Each sample was repeated 3 times in technical replicates and 3 times in biological replicates, and an independent internal reference Actin was added to each plate sample. The results are shown in Table 7 and Appendix. Figure 2 shown.

[0090] Table 7 qPCR test results

[0091] The germination activity of the tested samples was determined based on the expression of Pt6G00980 in the real-time fluorescence quantitative PCR results of the tested samples and the reference samples. There was no significant difference in the expression of Pt6G00980 between the tested sample 1 and the reference sample H15d, and its germination activity was similar to that of the reference sample H15d.

[0092] There was no significant difference in the expression level of Pt6G00980 between the test sample 2 and the reference sample C7d, and its germination activity was similar to that of the reference sample C7d; there was no significant difference in the expression level of Pt6G00980 between the test sample 3 and the reference sample GW, and its germination activity was similar to that of the reference sample GW.

[0093] Therefore, the technical solution provided by the present invention can accurately reflect the germination vitality of Pinus tabulaeformis seeds with higher precision, stronger scientificity and good repeatability.

[0094] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0095] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Pt6G00980 gene, characterized in that The amino acid sequence encoded by the Pt6G00980 gene is shown in SEQ ID NO.

2.

2. The Pt6G00980 gene according to claim 1, characterized in that The nucleotide sequence of the Pt6G00980 gene is shown in SEQ ID NO.

1.

3. Pt6G00980 protein, characterized in that The amino acid sequence of the Pt6G00980 protein is shown in SEQ ID NO.

2.

4. A primer set for amplifying the Pt6G00980 gene according to claim 1 or 2, characterized in that: The nucleotide sequences of the primer set are shown in SEQ ID NO.3 and SEQ ID NO.

4.

5. The primer set according to claim 4, characterized in that: The nucleotide sequence of the primer set also includes internal reference primers as shown in SEQ ID NO.5 and SEQ ID NO.

6.

6. A kit for amplifying the Pt6G00980 gene according to claim 1 or 2, characterized in that: The kit comprises the primer set according to claim 4 or 5.

7. The kit according to claim 6, characterized in that The kit also includes reagents for extracting total RNA, and / or reagents for reverse transcribing total RNA as a template into cDNA, and / or reagents for quantitative PCR of cDNA.

8. Use of the Pt6G00980 gene according to claim 1 or 2, or the Pt6G00980 protein according to claim 3, or the primer set according to claim 4 or 5, or the kit according to claim 6 or 7 in detecting the germination vigor of Pinus tabulaeformis seeds.

9. A method for determining the germination vitality of Pinus tabulaeformis seeds, characterized in that: The steps include: S1: using the cDNA of the sample to be tested and the cDNA of the reference sample as templates, using the primer set described in claim 4 or 5 or the kit described in claim 6 or 7 to perform quantitative PCR amplification on the templates, and respectively counting the expression levels of the Pt6G00980 gene in the sample to be tested and the reference sample; S2: When there is no significant difference between the expression amount of the Pt6G00980 gene in the test sample and the expression amount of the Pt6G00980 gene in the reference sample, it is determined that the germination activity of the test sample is the same as the germination activity of the reference sample; When the expression level of the Pt6G00980 gene in the test sample is significantly lower than the expression level of the Pt6G00980 gene in the reference sample, it is determined that the germination activity of the test sample is higher than that of the reference sample; When the expression level of the Pt6G00980 gene in the test sample is significantly higher than the expression level of the Pt6G00980 gene in the reference sample, it is determined that the germination activity of the test sample is lower than that of the reference sample; The reference samples are Pinus tabulaeformis seeds subjected to heat stress at 37°C for 15 days, Pinus tabulaeformis seeds subjected to cold stratification at 4°C for 7 days, and Pinus tabulaeformis seeds in the germination and whitening period.

10. The determination method according to claim 9, wherein the reaction system of the quantitative PCR amplification is 20 μL and comprises: 10 μL of 2× SuperReal PreMix Plus, 1 μL of cDNA template, 0.8 μL of 100 μM upstream primer, 0.8 μL of 100 μM downstream primer, and add RNase-free water to 20 μL; The reaction procedure of the quantitative PCR amplification was: pre-denaturation at 95°C for 2 min, denaturation at 94°C for 5 s, annealing at 60°C for 30 s, and 39 cycles.

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