Application of Pt6G00980 Gene and Its Protein in Determining Germination Vigor of Chinese Pine Seeds

Abstract

The present invention discloses the application of the Pt6G00980 gene and its protein in determining the germination vigor of Chinese pine seeds, which relates to the field of biotechnology. The expression level of this gene is negatively correlated with the germination vigor and gradually decreases with the germination of Chinese pine seeds. The present invention also provides a primer set for detecting this gene. By using the primer set provided by the present invention to perform quantitative PCR amplification on the cDNA of the sample to be tested and the cDNA of the reference sample, and comparing the expression levels of the Pt6G00980 gene in the sample to be tested and the reference sample, the accurate determination of the germination vigor of the sample to be tested can be achieved. 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 Chinese pine seeds, and has higher precision, stronger scientificity and good repeatability.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to the application of the Pt6G00980 gene and its protein in determining the germination vigor of Pinus tabulaeformis Carr. seeds. Background Art

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

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

[0004] Therefore, developing a method for determining the germination of Pinus tabulaeformis Carr. seeds with high efficiency and high accuracy 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 the application of the Pt6G00980 gene and its protein in determining the germination vigor of Pinus tabulaeformis Carr. seeds.

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

[0007] The Pt6G00980 gene, the amino acid sequence encoded by the Pt6G00980 gene is as 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 the Pt6G00980 protein being 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 being shown in SEQ ID NO.3 and SEQ ID NO.4.

[0011] Preferably, the nucleotide sequences of the primer set further include internal reference primers 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, the kit including the above-mentioned primer set.

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

[0014] Another object of the present invention is to provide the application 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 vigor of Chinese pine seeds.

[0015] Another object of the present invention is to provide a method for determining the germination vigor of Chinese pine 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, quantitatively PCR amplify the templates using the above-mentioned primer set or the above-mentioned kit, and respectively count 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 in the expression level of the Pt6G00980 gene in the sample to be tested and the expression level of the Pt6G00980 gene in the reference sample, it is determined that the germination vigor of the sample to be tested is the same as the germination vigor of the reference sample;

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

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

[0020] The reference samples are Pinus tabuliformis seeds under 37 °C heat stress for 15 days (named H15d), Pinus tabuliformis seeds under 4 °C cold stratification for 7 days (named C7d), and Pinus tabuliformis seeds at the germinated and radicle-emerged stage (named GW).

[0021] Preferably, the reaction system for quantitative PCR amplification is 20 μL in total, 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 nuclease-free water is added to make up to 20 μL.

[0022] The reaction program for quantitative PCR amplification is: 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 are run.

[0023] Beneficial effects:

[0024] The present invention provides the Pt6G00980 gene and the Pt6G00980 protein encoded thereby. The expression level of this gene is negatively correlated with the germination vigor and gradually decreases with the germination of Pinus tabuliformis seeds. Based on the above technical advantages, the present invention provides a method for determining the germination vigor of Pinus tabuliformis 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 levels of the Pt6G00980 gene in the test sample and the reference sample, the accurate determination of the germination vigor of the test sample can be achieved. 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 tabuliformis seeds, and has higher precision, stronger scientificity, and good repeatability. Description of the Drawings

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on the provided drawings.

[0026] Figure 1 The drawings are the results of the expression levels of the Pt6G00980 gene in the real-time fluorescence quantitative PCR detection samples of Pinus tabuliformis seeds at different germination degrees.

[0027] Figure 2 The drawings are the results of the expression levels of the Pt6G00980 gene in the real-time fluorescence quantitative PCR detection of different test samples in Example 4. Detailed Embodiments

[0028] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0029] The embodiments of the present invention disclose the application of the Pt6G00980 gene in determining the germination vigor of Chinese pine seeds. The materials and methods involved, unless otherwise specifically mentioned, are all conventional materials and methods, which will not be elaborated here one by one.

[0030] Example 1

[0031] Chinese pine seeds: Purchased from the Zhongtiao Mountain State-owned Forest Farm in Yuanqu County, Yuncheng City, Shanxi Province; the Yuanqu Zhongtiao Mountain Forest Farm belongs to a fault basin with an average annual temperature of 22°C, the highest temperature reaching 40°C, and the lowest temperature being -11°C; mature Chinese pine cones were collected from well-growing middle-aged Chinese pine mother plants, dried at room temperature, and the seeds were collected and mixed evenly.

[0032] The above seeds were tested for vigor and germination in distilled water. The specific process is as follows: Lay 6 layers of sterilized gauze in a petri dish and moisten it with sterile water so that the gauze is in a moist state. After washing the seeds with water, disinfect the seeds with sodium hypochlorite with a mass fraction of 0.5% for 20 minutes, and then wash them thoroughly with sterilized water; place 25 seeds in a petri dish and evenly place the seeds in the petri dish so that each seed comes into contact with the moist gauze. One petri dish is one treatment, and each treatment is set with 4 parallel repetitions.

[0033] Put the petri dish containing the seeds into an incubator at a temperature of 22°C with a light condition of 8 hours of light and 16 hours of darkness for cultivation, and add sterilized distilled water to the petri dish as needed. The judgment standard for germination is that the radicle protrudes from the seed coat. The ungerminated seeds were broken with forceps to detect whether the seeds had vigor (vigor judgment standard: if the embryo of the seed is white and firm, it is determined that the seed has vigor; if the embryo mildews or breaks easily when touched with forceps, it is determined that the seed has no vigor).

[0034] Treat the seeds using the method described in the existing patent (CN 117887734A) to prepare seeds after different treatments: Seeds with the radicle showing (GW): Seeds with the radicle breaking through the seed coat and regarded as germinated, i.e., seeds with the radicle protruding less than 3 mm from the seed coat are taken out of the petri dish, quickly frozen in liquid nitrogen, and stored in a -80°C refrigerator. Nondormant seeds (C7d): Viable Chinese pine seeds are evenly placed in a petri dish lined with 6 layers of sterilized gauze. Sterilized distilled water is added to the petri dish to make the gauze in a saturated and moist state. The petri dish is wrapped with tin foil and then placed in a black bag. The petri dish containing the seeds is placed in a 4°C refrigerator for 7 days of cold stratification treatment. Then the seeds are taken out and quickly frozen in liquid nitrogen, and stored in a -80°C refrigerator. Inducing seed dormancy (H15d): The nondormant seeds obtained by cold stratification treatment are placed in a petri dish lined with 6 layers of moist gauze. The petri dish is wrapped with tin foil and placed in a completely dark incubator at 37°C (i.e., heat stress treatment). After 15 days of heat stress, the seeds are taken out and quickly frozen in liquid nitrogen, and stored in a -80°C refrigerator.

[0035] Conduct germination tests on the above-treated seeds in distilled water respectively (the specific process of the germination test is as described above), record the number of germinated seeds, and detect the viability of the ungerminated seeds. During the germination test, one petri dish is one treatment, with 4 parallel replicates for each treatment, and 25 seeds in each petri dish. The results are shown in Table 1.

[0036] Table 1 Germination rate or sprouting rate of Chinese pine seeds with different treatments

[0037]

[0038] Example 2

[0039] Screening of the germination vigor marker gene Pt6G00980

[0040] 1) Sampling of Chinese pine seeds with different dormancy degrees Take the seeds after 15 days of heat stress, the seeds after 7 days of cold stratification, and the seeds with the radicle showing prepared before the experiment as samples. There are 3 biological replicates for each group of samples, with 25 seeds in each replicate. The above materials are immediately frozen in liquid nitrogen after sampling and stored in a -80°C ultra-low temperature refrigerator, which is beneficial to protecting biomolecules (such as DNA, RNA, and proteins) in the samples from degradation, thereby maintaining the integrity and quality of the samples.

[0041] 2) RNA extraction, library construction, and transcriptome sequencing

[0042] Total RNA was extracted from reference samples (seeds after 15 days of heat stress, seeds after 7 days of cold stratification, and germinated seeds) using the Trizol method. After quality inspection, the NEB #7530 kit (#E7530 purchased from New England Biolabs) was used to construct a transcriptome library. 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.

[0043] 3) Data quality control and alignment with the reference genome

[0044] The raw data (raw reads) was subjected to data quality control. Reads with a proportion of base N greater than 10% were filtered in sequence, reads with a proportion of bases with quality value Q ≤ 20 exceeding 50% of the entire read were filtered, the adapter and the subsequent part were truncated, and reads shorter than 50 bp after truncating the adapter were filtered to obtain high-quality quality control data (High quality clean reads);

[0045] Using the high-quality genome of Chinese pine, Chinese pine genome (https: / / www.ncbi.nlm.nih.gov / bioproject / PRJNA784915), as the reference genome, the quality control data was aligned with it to obtain the matching data (mapped reads) for subsequent transcript assembly and expression level analysis.

[0046] 4) Gene expression level analysis

[0047] The clean reads of each sample were mapped to the Chinese pine reference genome 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 normalization method of TPM (Transcript per million) was selected to characterize the gene expression level.

[0048] 5) Weighted gene co-expression network analysis

[0049] Genes with a TPM < 1 in all samples were filtered out. The filtered gene set was used to construct a gene network with a soft threshold of "power = 15". The parameters for network construction and module merging were "deepSplit = 2; minModuleSize = 100; mergeCutHeight = 0.20". Correlation analysis was performed between modules and between samples and modules. The former was done by clustering the gene expression levels in the modules, and the latter was done by Pearson correlation analysis, calculating the correlation between the module eigenvectors and the sample expression matrix. Finally, PCA analysis was performed on the gene expression levels in the modules. PC1 was used to represent the module index, i.e., the module eigenvector (MEs), and module screening was carried out based on the trend of the MEs values. The top 1% of genes in 2 modules were retained for hub gene network construction. The Cytoscape software was used to visualize the network and screen for hub genes. The sub-network was extracted using the MCODE plugin, and then the intersection of 12 algorithms such as MMC was obtained using the CytoHubba plugin to get the germination vigor 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.

[0050]

[0051] Amino acid sequence of Pt6G00980 protein: MAKVQAWFAILTVIITGNILRSKSGEPLVPALFIFGDSLADAGNNNNLTTLAKANFLPYGRAFPDSVPTGRFSNGYNSMDFLCFKLGLPIIPIYADPNTKGGNLLRGVNYASGASGIENYSGRSFGELIPLEDQVNNFMNTKQEIIDVLGEEGASQLISKAVFSIITGNNDWLNTYLFPFSPLRVLYTVDQFRDNLINKLALQIQKLYSNGARNFVVAGLSALGCLPSQLNRYNSNGSCIDFLNDMAQNFNEALRLRVQDLNSQLPNSTFLFNNLYTPLYEAFHNPAAYGFIYVNEACCGIGKFGGFLICVPEFPVCSNEEDYLFWDAYHTTDKMFKQMVDLMWENGPPYSYPISGKEAVYRTM, SEQ ID NO.2.

[0052] Example 3

[0053] Verification of germination vigor marker gene

[0054] 1) Primer design

[0055] According to the CDs sequence of the germination vigor marker gene Pt6G00980, primers were designed using the online primer design website Primer BLAST of NCBI. The primer sequences of the Pt6G00980 gene are as follows:

[0056] Forward primer: 5'-CCCTTCTCACCCCTTCGAGT-3' (SEQ ID NO.3);

[0057] Reverse primer: 5'-TTTGGGCCATGTCGTTCAGA-3' (SEQ ID NO.4).

[0058] 2) Sampling of Pinus tabuliformis seeds with different germination degrees

[0059] Pinus tabuliformis seeds stressed by heat at 37°C for 15 days (named H15d), Pinus tabuliformis seeds cold-stratified at 4°C for 7 days (named C7d), and Pinus tabuliformis seeds at the germination and radicle emergence stage (named GW) were selected. The above materials were immediately frozen with liquid nitrogen after treatment and stored in an ultra-low temperature freezer at -80°C.

[0060] 3) Extraction of total RNA from samples

[0061] Extract the total RNA of the collected samples using the RNAprep Pure Polysaccharide and Polyphenol 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.

[0062] 4) Reverse transcription of the total RNA of the samples

[0063] In an RNase free PCR tube, perform the reverse transcription reaction of the total RNA obtained in step 3) and remove genomic DNA. Prepare the reverse transcription system according to Table 2 below:

[0064] Table 2 Reverse transcription system

[0065]

[0066] Gently mix the above system, incubate at 42 °C for 15 min in a PCR instrument, and then heat at 85 °C for 5 s to completely inactivate the RT / RI Enzyme and gDNA Remover. Store the product at -20 °C.

[0067] 5) Real-time fluorescence quantitative PCR verification of the Pt6G00980 gene

[0068] Dilute the cDNA generated by reverse transcription in step 4) by 5 times as the template, and perform qRT-PCR according to the qRT-PCR reaction system and reaction conditions in Table 3 below:

[0069] Table 3 qRT-PCR reaction system

[0070]

[0071] Perform the reaction using the SYBR Green intercalating fluorescence method. The pre-denaturation reaction program is set to 95 °C for 2 min, the PCR reaction program is set to 94 °C for 5 s, 60 °C for 30 s, for 39 cycles; the melting curve analysis reaction program is set to increase by 0.5 °C every 5 s from 65 - 95 °C. Each sample is subjected to 3 technical replicates and 3 biological replicates, and an independent internal reference Actin is added to each plate of samples.

[0072] Forward primer for internal reference: 5'-GGCATACCGGCAGCTCTTC-3' (SEQ ID NO.5);

[0073] Reverse primer for internal reference: 5'-AAGTTGTTGGCGGCGTCTT-3' (SEQ ID NO.6);

[0074] The results are shown in Table 4 and Appendix Figure 1As shown in the figure, with the increase of seed germination vigor and the establishment of seedlings, the expression level of the Pt6G00980 gene decreases, indicating that the expression level of the Pt6G00980 gene in Pinus tabuliformis seeds is negatively correlated with germination vigor and gradually decreases with the germination of Pinus tabuliformis seeds.

[0075] Table 4 Results of qPCR detection

[0076]

[0077] Example 4

[0078] Method for judging the germination vigor of a test sample based on the germination vigor marker gene Pt6G00980 of Pinus tabuliformis seeds

[0079] 1. Extraction of total RNA from Pinus tabuliformis seeds

[0080] (1) Viable Pinus tabuliformis seeds are used as the test sample materials.

[0081] (2) Setting of reference samples

[0082] Pinus tabuliformis seeds heat-stressed at 37 °C for 15 days (named H15d), Pinus tabuliformis seeds cold-stratified at 4 °C for 7 days (named C7d), and Pinus tabuliformis seeds at the germinated and radicle-emerged stage (named GW) are used as the materials for taking reference samples, and the expression level of the Pt6G00980 gene in these samples is used as the standard indicating the low germination vigor of Pinus tabuliformis seeds.

[0083] Extract using Aidlab EASYspin Plus Plant RNA Kit, and complete the specific operation steps according to the instructions.

[0084] RNA concentration and integrity are measured using a Nano micro-spectrophotometer and 1% agarose gel electrophoresis, respectively.

[0085] 2. Synthesis of the first-strand cDNA

[0086] Prepare the reverse transcription reaction system according to the reagent addition amounts in Table 5.

[0087] Table 5 Reverse transcription reaction system

[0088]

[0089] Gently mix the above system, incubate at 42 °C in a PCR instrument for 15 min, and then heat at 85 °C for 5 s to completely inactivate the RT / RI Enzyme and gDNA Remover, and store the product at -20 °C.

[0090] 3. Real-time fluorescence quantitative PCR

[0091] Prepare the reaction system for real-time fluorescence quantitative PCR according to the reagent addition amounts in Table 6.

[0092] Table 6 Reaction System for Real-Time Fluorescence Quantitative PCR

[0093]

[0094] Among them, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.3; the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.4. The nucleotide sequence of the internal reference forward primer is as shown in SEQ ID NO.5; the nucleotide sequence of the internal reference reverse primer is as shown in SEQ ID NO.6.

[0095] The reaction is carried out by SYBR Green chimeric fluorescence method. The pre-denaturation reaction program is set at 95 °C for 2 min, the PCR reaction program is set at 94 °C for 5 s, 60 °C for 30 s, for 39 cycles; the melting curve analysis reaction program is set to increase by 0.5 °C every 5 s from 65 - 95 °C. Each sample is subjected to 3 technical replicates and 3 biological replicates, and an independent internal reference Actin is added to each plate of samples. The results are shown in Table 7 and the appendix Figure 2 as shown.

[0096] Table 7 qPCR Detection Results

[0097]

[0098] Judge the germination vigor of the test sample based on the expression level of Pt6G00980 in the real-time fluorescence quantitative PCR results of the test sample and the reference sample. There is no significant difference in the expression level of Pt6G00980 between the test sample 1 and the reference sample H15d, and its germination activity is similar to that of the reference sample H15d;

[0099] There is no significant difference in the expression level of Pt6G00980 between the test sample 2 and the reference sample C7d, and its germination activity is similar to that of the reference sample C7d; there is no significant difference in the expression level of Pt6G00980 between the test sample 3 and the reference sample GW, and its germination activity is similar to that of the reference sample GW.

[0100] Therefore, the technical solution provided by the present invention can accurately reflect the germination vigor of Chinese pine seeds, and has higher precision, stronger scientificity, and good repeatability.

[0101] The various embodiments in this specification are described in a progressive manner. Each embodiment focuses on the differences from other embodiments. The same or similar parts among the various embodiments can be referred to each other.

[0102] The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those 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. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded 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; the nucleotide sequence of the Pt6G00980 gene is shown in SEQ ID NO.1; 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 calculated in 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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