A marker gene Pt8G06080 and protein for the degree of dormancy release of Pinus tabulaeformis seeds and its application in determining the dormancy degree of Pinus tabulaeformis

Abstract

The invention belongs to the field of genetic engineering and agricultural biotechnology, and specifically relates to a marker gene for the degree of dormancy release of Pinus tabulaeformis seeds. Pt8G06080 The invention discloses a protein and its application in determining the dormancy degree of Pinus tabulaeformis seeds. Pt8G06080 The present invention also provides a primer pair for specifically amplifying the above gene, and the primer pair can be used to specifically amplify the above gene. Pt8G06080 Genes are detected by comparing the test sample with the reference sample. Pt8G06080 The expression level of the gene can accurately determine the dormancy release status of the sample to be tested, with higher precision, stronger scientificity and good repeatability.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and agricultural biotechnology, and specifically relates to a marker gene for the degree of dormancy release of Pinus tabulaeformis seeds. Pt8G06080 and protein and its application in determining the dormancy degree of Pinus tabulaeformis. Background Art

[0002] Pinus tabulaeformis Pinus tabuliformis Carrière) is one of the important afforestation tree species in northern my country. It is mainly distributed in Shaanxi, Gansu, Ningxia, Shanxi, Hebei, Inner Mongolia and other provinces. It grows in mountainous and hilly areas, has strong adaptability, and is not demanding on soil. Pinus tabulaeformis has extremely high economic and ecological value. Pinus tabulaeformis wood is rich in rosin, from which turpentine can be extracted. In addition, Pinus tabulaeformis has good corrosion resistance, the wood is light and soft, with clear texture. It is often used as raw material for construction, shipbuilding, furniture, etc., and can also be used in papermaking. In addition, Pinus tabulaeformis plays an important role in ecological environmental restoration. Its well-developed root system has a good effect on maintaining water and soil. It is one of the important vegetation for preventing and controlling soil erosion. At the same time, Pinus tabulaeformis has a wide crown, a majestic tree shape, and needles that do not fall in all seasons, which can also play a role in preventing wind and sand.

[0003] As a high-quality tree species with extremely high value, the dormancy release and germination of Pinus tabulaeformis seeds are affected by many internal factors, including seed maturity, seed dormancy state, seed nutrient reserves, seed moisture content, etc. How to obtain high-quality seeds and how to quickly determine the dormancy release state of a batch of Pinus tabulaeformis seeds during the planting process determine the quality of subsequent Pinus tabulaeformis seedling cultivation. Otherwise, under traditional conditions, after sowing Pinus tabulaeformis seeds that have not yet been released from dormancy and are still in a dormant state, it is found that they cannot germinate normally after a long period of cultivation, which is time-consuming and labor-intensive, and is not conducive to the development of afforestation.

[0004] ac 4 C (N4-acetylcytidine) is a type of acetylation modification that can occur on eukaryotic mRNA. It can regulate RNA stability and promote RNA translation. 4 The C-modified gene is more stable, has a longer gene expression period and is easier to detect than the unmodified gene, making it more suitable as a marker gene to determine the degree of dormancy release in seeds. 4 There are no reports on genes modified by C acetylation and their related research on Pinus tabulaeformis dormancy. Summary of the invention

[0005] The purpose of the present invention is to provide a marker gene for the degree of dormancy release of Pinus tabulaeformis seeds Pt8G06080 and protein and its application in determining the dormancy degree of Pinus tabulaeformis, the marker gene for the dormancy release degree of Pinus tabulaeformis seeds Pt8G06080Can accurately and efficiently determine the dormancy degree of Pinus tabulaeformis seeds ， Provide basic support for selecting high-quality Pinus tabulaeformis seeds that can germinate normally.

[0006] The present invention provides a method based on ac 4 C acetylation-modified marker gene for dormancy release in Pinus tabulaeformis seeds Pt8G06080 , the dormancy release marker gene of Pinus tabulaeformis seeds Pt8G06080 The encoded amino acid sequence is shown in SEQ ID NO: 2.

[0007] Preferably, the dormancy release marker gene of Pinus tabulaeformis seeds Pt8G06080 The nucleotide sequence is shown in SEQ ID NO: 1.

[0008] The present invention also provides a marker gene for the degree of dormancy release of Pinus tabulaeformis seeds described in the above technical solution. Pt8G06080 Encoded by Pt8G06080 protein.

[0009] The present invention also provides a method for amplifying the marker gene for the degree of dormancy release of Pinus tabulaeformis seeds described in the above technical solution. Pt8G06080 primer pairs.

[0010] Preferably, the primer pair comprises an upstream primer and a downstream primer, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0011] The present invention also provides a kit, which comprises the primer pair and PCR amplification reagent described in the above technical solution.

[0012] Preferably, the kit further comprises one or more of an internal reference gene primer, an RNA extraction reagent and a reverse transcription reagent.

[0013] The present invention also provides a marker gene for the degree of dormancy release of Pinus tabulaeformis seeds described in the above technical solution. Pt8G06080 Or the use of the Pt8G06080 protein described in the above technical solution, or the primer pair described in the above technical solution, or the kit described in the above technical solution in determining the dormancy degree of Pinus tabulaeformis seeds.

[0014] The present invention also provides a method for determining the dormancy degree of Pinus tabulaeformis seeds, comprising the following steps:

[0015] Using the cDNA of the sample to be tested and the cDNA of the reference sample as templates, respectively use the primer pairs described in the above technical solution to perform fluorescence quantitative PCR amplification, and respectively count the cDNA of the sample to be tested and the reference sample. Pt8G06080 The expression level of the gene;

[0016] When the sample to be tested Pt8G06080 The expression of the gene is similar to that in the reference sample. Pt8G06080 When there is no significant difference in the expression levels of the genes, it is determined that the dormancy release degree of the test sample is the same as that of the reference sample;

[0017] When the sample to be tested Pt8G06080 The expression of the gene was significantly higher than that in the reference sample. Pt8G06080 When the expression level of the gene is greater than that of the reference sample, it is determined that the dormancy release degree of the sample to be tested is higher than that of the reference sample;

[0018] When the sample to be tested Pt8G06080 The expression of the gene was significantly lower than that in the reference sample. Pt8G06080 When the expression level of the gene is greater than that of the reference sample, it is determined that the dormancy release degree of the sample to be tested is lower than that of the reference sample.

[0019] Preferably, the significant differences include P Significant difference at <0.05.

[0020] Beneficial effects:

[0021] The present invention provides a method based on ac 4 C acetylation-modified marker gene for dormancy release in Pinus tabulaeformis seeds Pt8G06080 , the dormancy release marker gene of Pinus tabulaeformis seeds Pt8G06080 The amino acid sequence encoded is shown in SEQ ID NO: 2. The present invention uses Pinus tabulaeformis seeds to be sown as sample materials, and obtains the ac-based 4 C acetylation-modified marker gene for dormancy release in Pinus tabulaeformis seeds Pt8G06080 The expression level in the seeds of Pinus tabulaeformis in the dormancy release state is significantly different from that in the seeds in the dormancy state. Pt8G06080 The expression level of the gene is relatively high and more stable, and the Pt8G06080 protein encoded by the gene is further provided. Pt8G06080 Both the gene and the Pt8G06080 protein can be used to determine the dormancy release degree of Pinus tabulaeformis seeds. Through experiments, it was found that compared with the traditional judgment method that relies on observing the seed germination rate and physiological indicators, the technical solution provided by the present invention can accurately reflect the dormancy release degree of Pinus tabulaeformis seeds, with higher accuracy, stronger scientificity and good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1To prepare the same batch of seeds in the same treatment conditions shown in the example, which were photographed at the same time after 14 days in a deep dormancy state and could not germinate normally, and seeds that germinated normally after dormancy was released;

[0024] Figure 2 For Example 1 Pt8G06080 Acetylation modification peak diagram on genes;

[0025] Figure 3 The pine seeds in the dormancy release state after creating the dormancy release conditions in Example 2 Pt8G06080 Genes and other 4 C Comparative bar graph of the expression levels of acetylation-modified genes;

[0026] Figure 4 The same batch of seeds were grown under the conditions of dormancy, dormancy release and germination in Example 3. 4 C acetylated Pt8G06080 Gene expression comparison bar graph. DETAILED DESCRIPTION

[0027] The present invention provides a method based on ac 4 C acetylation-modified marker gene for dormancy release in Pinus tabulaeformis seeds Pt8G06080 , the dormancy release marker gene of Pinus tabulaeformis seeds Pt8G06080 The encoded amino acid sequence is shown in SEQ ID NO: 2.

[0028] In the present invention, the amino acid sequence shown in SEQ ID NO: 2 is as follows: MAEYDLTSKIAPHLDRHLVFPLLEFLQEKQLYPDEQILKSKIDLLSKTNMVDYAMDIHKSLYRTEDVPQEMTDRRIEVVARLKSLEEMAAPLISFLQNPSLVQELRSDKQYNLQMLNDRYQIGPNQIEALYQYAKFQFECGNYSGAADFLYQYRTFCTNSERSLSALWGKLAAEILMQNWDIALEELNRLKEIIDSKNFASPLNQLQNRTWLMH WSLFIFFNHENGRNGIIDLFFQERYLNAIQTNAHHLLRYLATAVVVNKRRRNMLKELIKVIQQEQYTYKDPITEFLECLYVNYDFEGAQQKLVECEKVILNDPFLGKQVQEGNF VTVPLRDEFLENARLFIFETYCRIHQCIDIRMLAEKLSMSYDEAERWIVNLVKNAKLDAKIDSTSGTVVMGTNHTNVYEQIIENTKQLSMRTYVLANNVASIAVPVTQVPTIR*.

[0029] As an embodiment, the pine seed dormancy release degree marker gene Pt8G06080

[0030] The present invention also provides a marker gene for the degree of dormancy release of Pinus tabulaeformis seeds described in the above technical solution. Pt8G06080 The amino acid sequence of the Pt8G06080 protein of the present invention is shown in SEQ ID NO:2.

[0031] In the present invention, the dormancy release degree marker gene of Pinus tabulaeformis seeds Pt8G06080 The Pt8G06080 protein encoded by it can be used to determine the degree of dormancy release of Pinus tabulaeformis seeds.

[0032] The present invention also provides a method for amplifying the dormancy release marker gene of Pinus tabulaeformis seeds described in the above technical solution. Pt8G06080 As an embodiment, the primer pair includes an upstream primer and a downstream primer, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively. The upstream primer and the downstream primer shown in SEQ ID NO:3 and SEQ ID NO:4 of the present invention can specifically amplify the dormancy release degree marker gene of Pinus tabulaeformis seeds. Pt8G06080 .

[0033] The present invention also provides a kit, which includes the primer pair and PCR amplification reagent described in the above technical solution. As an embodiment, the PCR amplification reagent can be a fluorescent quantitative PCR amplification reagent; as another embodiment, the fluorescent quantitative PCR amplification reagent can include SYBR Green DNA polymerase, dNTPs and PCR buffer. As an embodiment, the kit also includes one or more of internal reference gene primers, RNA extraction reagents and reverse transcription reagents, that is, the kit of the present invention contains primer pairs and PCR amplification reagents, and the kit containing any one or more of internal reference gene primers, RNA extraction reagents and reverse transcription reagents belongs to the protection scope of the kit of the present invention. As an embodiment, the internal reference gene can be Actin gene; as another embodiment, the Actin The nucleotide sequences of the upstream primer and the downstream primer of the gene primer are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The present invention has no special restrictions on the source and amount of the PCR amplification reagent, RNA extraction reagent and reverse transcription reagent, which can be conventionally set and selected according to needs.

[0034] Based on the dormancy release degree marker gene of Pinus tabulaeformis seeds Pt8G06080The Pt8G06080 protein can accurately determine the degree of dormancy release of Pinus tabulaeformis seeds, and the effect is higher than the traditional judgment method that relies on observing seed germination rate and physiological indicators.

[0035] Based on the above advantages, the present invention also provides a marker gene for the degree of dormancy release of Pinus tabulaeformis seeds described in the above technical solution. Pt8G06080 Or the use of the Pt8G06080 protein described in the above technical solution, or the primer pair described in the above technical solution, or the kit described in the above technical solution in determining the dormancy degree of Pinus tabulaeformis seeds.

[0036] Specifically, the present invention also provides a method for determining the dormancy degree of Pinus tabulaeformis seeds, comprising the following steps:

[0037] Using the cDNA of the sample to be tested and the cDNA of the reference sample as templates, respectively use the primer pairs described in the above technical solution to perform fluorescence quantitative PCR amplification, and respectively count the cDNA of the sample to be tested and the reference sample. Pt8G06080 The expression level of the gene;

[0038] When the sample to be tested Pt8G06080 The expression of the gene is similar to that in the reference sample. Pt8G06080 When there is no significant difference in the expression levels of the genes, it is determined that the dormancy release degree of the test sample is the same as that of the reference sample;

[0039] When the sample to be tested Pt8G06080 The expression of the gene was significantly higher than that in the reference sample. Pt8G06080 When the expression level of the gene is greater than that of the reference sample, it is determined that the dormancy release degree of the sample to be tested is higher than that of the reference sample;

[0040] When the sample to be tested Pt8G06080 The expression of the gene was significantly lower than that in the reference sample. Pt8G06080 When the expression level of the gene is greater than that of the reference sample, it is determined that the dormancy release degree of the sample to be tested is lower than that of the reference sample.

[0041] The present invention uses the cDNA of the sample to be tested and the cDNA of the reference sample as templates, respectively uses the primer pairs described in the above technical solution to perform fluorescent quantitative PCR amplification, and respectively counts the cDNA of the sample to be tested and the cDNA of the reference sample. Pt8G06080The expression level of the gene. The present invention does not specifically limit the preparation method of the cDNA, and the conventional cDNA preparation method in the art can be used. As an embodiment, the test sample and the reference sample of the present invention are Pinus tabulaeformis seeds of the same variety. As an embodiment, the reference sample is a Pinus tabulaeformis seed that is in a dormancy-released state and can germinate normally; as another embodiment, the reference sample can be a seed that has been treated with cold stratification. As another embodiment, the temperature of the cold stratification treatment is 4°C and the time is 7 to 10 days; as another embodiment, the time of the cold stratification treatment is 7 days. The Pinus tabulaeformis seeds treated with cold stratification of the present invention have a germination rate of >70%, are in a dormancy-released state, and have normal germination ability.

[0042] As an embodiment, the reaction system of the fluorescent quantitative PCR amplification is 20 μL, including: 2 × SuperReal PreMix Plus (SYBR Green) 10 μL, cDNA template 1 μL, 100 μM upstream primer 0.8 μL, 100 μM downstream primer 0.8 μL, and RNase-free water is added to 20 μL. As an embodiment, the reaction procedure of the fluorescent quantitative PCR amplification is: 95°C pre-denaturation for 2 minutes, 94°C denaturation for 5 seconds, 60°C annealing for 30 seconds, and 39 cycles. In this way, the specific amplification of the cDNA of the sample to be tested and the cDNA of the reference sample can be achieved respectively.

[0043] The sample to be tested and the reference sample are obtained Pt8G06080 After the expression level of the gene is determined, the present invention is based on the expression level of the gene in the sample to be tested and the reference sample. Pt8G06080 The expression level of the gene is used to determine the result: when the sample to be tested contains Pt8G06080 The expression of the gene is similar to that in the reference sample. Pt8G06080 If there is no significant difference in the expression of the gene, it is determined that the dormancy release degree of the test sample is the same as that of the reference sample; Pt8G06080 The expression of the gene was significantly higher than that in the reference sample. Pt8G06080 When the expression level of the gene in the test sample is greater than that of the reference sample, the dormancy release degree of the test sample is determined to be higher than that of the reference sample; Pt8G06080 The expression of the gene was significantly lower than that in the reference sample. Pt8G06080 When the expression level of the gene is greater than that of the reference sample, it is determined that the dormancy release degree of the sample to be tested is lower than that of the reference sample. As an embodiment, the significant difference includes P<0.05. The present invention has no special requirements for the equipment of the fluorescent quantitative PCR amplification and the analysis method of the gene expression, and the technology well known in the art can be used. In a specific embodiment of the present invention, the equipment model used for fluorescent quantitative PCR amplification is: CFX ConnectTM Optics Module, and the specific number of the equipment is: 788BR04747; the software used for gene expression data analysis is: Bio-Rad CFX Manager.

[0044] Experiments have shown that compared with the traditional identification method that relies on observing seed germination rate and physiological indicators, the technical solution provided by the present invention can accurately reflect the degree of dormancy release of Pinus tabulaeformis seeds, with higher accuracy, stronger scientificity and good repeatability.

[0045] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] Preparation Example

[0047] Pinus tabulaeformis seeds: purchased from Zhongtiaoshan State Forest Farm in Yuanqu County, Yuncheng City, Shanxi Province; Yuanqu Zhongtiaoshan State Forest Farm is located in a fault basin with an average annual temperature of 22°C, a maximum temperature of 40°C, and a minimum temperature of -11°C; the merchant collected mature Pinus tabulaeformis cones from well-growing mature Pinus tabulaeformis mother plants, dried them at room temperature, collected the seeds, and mixed them evenly;

[0048] The Pinus tabulaeformis seeds purchased from different batches were transported back to the laboratory of Beijing Forestry University. The quality of Pinus tabulaeformis seeds obtained from different batches was different. Germination conditions were created to count the germination rates of seeds from different batches. The specific process was as follows: Sterilize gauze, pure water, etc. in advance, and lay 6 layers of gauze moistened with sterile water in the culture dish to ensure that the seeds are provided with sufficient water during the subsequent culture process. After washing the seeds with water, disinfect the seeds with 3% hydrogen peroxide solution for 15 minutes, and then wash them repeatedly with sterile water at least 3 times; evenly place 45 seeds on the moist gauze laid flat in the culture dish. Seeds purchased from the same batch were treated in one culture dish, and a total of 3 batches of seeds were treated.

[0049] The culture dish containing the processed seeds was sealed and placed flat in an incubator at 22°C, 16h light and 8h dark for incubation. The number of germinated seeds was observed and recorded at the same time every day. The criterion for seed germination was the cracking of the outer seed coat and the exposure of the radicle. The seeds that never germinated were broken with tweezers to test whether the seeds were viable (vitality judgment criteria: if the embryo of the seed was white, plump and solid, the seed was judged to be viable; if the embryo was moldy, dark brown or wrinkled, and broke when touched with tweezers, the seed was judged to be inactive). The results are shown in Table 1.

[0050] Table 1 Germination rate of different batches of Pinus tabulaeformis seeds

[0051]

[0052] As can be seen from Table 1, there are obvious differences in the germination rates of different batches of Pinus tabulaeformis seeds. The germination rate of seeds that have been released from dormancy is high, exceeding 70%, while the germination rate of seeds in a dormant state is low, with a germination rate of about 20% for seeds in mild dormancy and less than 10% for seeds in deep dormancy. The phenotypes of seeds that cannot germinate normally in a deep dormancy state and those that germinate normally after dormancy is released are as follows: Figure 1 As shown. Therefore, it is necessary to create dormancy release conditions. Sterilize gauze, pure water, etc. in advance, and lay 6 layers of gauze moistened with sterile water in the culture dish to ensure that the seeds are provided with sufficient water during the subsequent culture process. After washing the seeds with water, disinfect the seeds with 3% hydrogen peroxide solution for 15 minutes, and then wash them repeatedly with sterile water at least 3 times; 45 seeds are evenly placed on the moist gauze laid flat in the culture dish. The culture dish is sealed and completely wrapped with tin foil and placed in a refrigerator at 4°C for 7 days of cold stratification. After 7 days, transfer and culture in an incubator at 22°C, 16h light, and 8h dark for cultivation. The germination rate of the seeds after 14 days is re-counted to obtain the results. The germination rate of Pinus tabulaeformis seeds in different batches is >70%.

[0053] Example 1

[0054] Based on ac 4 C acetylation-modified dormancy-release marker genes Pt8G06080 Filter by:

[0055] 1) Sampling of dormancy-released pine seeds

[0056] Pinus tabulaeformis seeds treated with cold stratification for 7 days before the experiment were taken as samples, with a total of 3 biological replicates, 30 seeds in each replicate. The above materials were immediately frozen with liquid nitrogen after sampling and stored in a -80℃ ultra-low temperature refrigerator to protect biological molecules such as DNA, RNA and protein in the samples from degradation and ensure sample quality.

[0057] 2) RNA quality control, library construction, ac 4 CRIP sequencing

[0058] The total RNA of the reference samples was extracted by Trizol method, and the RNA concentration of each sample was measured by NanoDrop ND-1000 (ThermoFisher Scientific, Waltham, MA, USA). The OD260 / OD280 value was used as the RNA purity index to judge the RNA quality and complete RNA quality control. RNA acetylation sequencing service was provided by Shanghai Yunxu Biotechnology Co., Ltd. The RNA acetylation kit of GenSeq was used for ac 4 C-IP reaction. Using GenSeq ® The RNA sequencing library was constructed using the Low Input WholeRNA Library Prep Kit (GenSeq, Inc.). The constructed library was quality controlled using an Agilent 2100 bioanalyzer and then subjected to high-throughput sequencing on an Illumina NovaSeq sequencer.

[0059] 3) Data quality control and analysis

[0060] After sequencing, image analysis, base recognition and quality control by Illumina NovaSeq 6000 sequencer, raw reads (Raw Data) were generated. First, Q30 was used for quality control. Q30>80% indicated good sequencing quality. Then, cutadapt software (v1.9.3) was used to remove adapters and low-quality reads to obtain high-quality clean reads. BWA software was used to match the clean reads of the input library to the reference genome. Hisat2 software (v2.0.4) was used to match the clean reads of all samples to the reference genome. MACS software was then used to identify acetylation peaks in each sample. Differential acetylation peaks were identified using diffReps software. Peaks located on mRNA exons were screened using a proprietary program and corresponding annotations were made. GO and Pathway analysis was performed on differentially acetylated coding genes.

[0061] 4) Gene expression analysis

[0062] The clean reads of each sample were mapped to the reference genome of Chinese pine ( Pinus tabuliformis) genome (https: / / www.ncbi.nlm.nih.gov / bioproject / PRJNA784915) to obtain the expression level of mRNA, and 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 genes.

[0063] 5) Weighted gene co-expression network analysis

[0064] 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 analyzed 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 modules, and PC1 was used to represent the module's indicator, namely the module eigenvector (MEs), and the modules were screened based on the change trend of MEs values. The top 1% of genes in the two modules were retained for hub gene network construction. 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 combined with the intersection of 12 algorithms such as MMC to obtain the ac-based 4 C acetylation-modified dormancy-release marker genes Pt8G06080 , Pt8G06080 The CDS sequence of the gene is shown in SEQ ID NO: 1. Pt8G06080 The acetylation peak of the gene is Figure 2 As shown, DR rep1, rep2 and rep3 represent three groups of biological replicates (biological replicates are parallel measurements of different biological samples) for the location of acetylation peaks in dormancy-released Pinus tabulaeformis seeds. Pt8G06080 The gene transcript structure is shown in ac 4 Above the C peak graph, the blue lines on the transcripts are intron regions, the blue rectangles are exon regions, and the arrows on the transcripts pointing to the left indicate that the gene is on the negative strand. The ac 4 C modification sites are marked in dark blue.

[0065] Example 2

[0066] Based on ac 4 C acetylation-modified dormancy-release marker genes Pt8G06080 Verification

[0067] 1) Primer design

[0068] Based on ac 4 C acetylation-modified dormancy-release marker genes Pt8G06080 And other devices with ac in the sleep state 4 C acetylation modified genes Pt2G02970 , Pt9G14330 The CDS sequences were used to design primers using the NCBI online primer design website Primer-BLAST. Pt2G02970

[0069] Pt9G14330

[0070] Pt8G06080 The primer sequences for the genes are:

[0071] Upstream primer: 5′-ATGACAGACAGGCGCATTGA-3′ (SEQ ID NO: 3);

[0072] Downstream primer: 5'-CCCCACAAGGCACTCAAACT-3' (SEQ ID NO: 4).

[0073] Pt2G02970 The primer sequences for the genes are:

[0074] Upstream primer: 5′-ATGCAGAGGCATCAGGCAAA-3′ (SEQ ID NO: 9);

[0075] Downstream primer: 5′-AAGCGCCCATTTGGTGAAAG-3′ (SEQ ID NO: 10);

[0076] Pt9G14330 The primer sequences for the genes are:

[0077] Upstream primer: 5′-ATTTTATGCCACCGCCGAGA-3′ (SEQ ID NO: 11);

[0078] Downstream primer: 5'-GCATCTGGCTCCTGCTCTAC-3' (SEQ ID NO: 12).

[0079] 2) Sampling of dormancy-released pine seeds

[0080] The same batch of Pinus tabulaeformis seeds (named DR) that were in a dormancy-released state after treatment were selected. The above materials were immediately quick-frozen with liquid nitrogen after collection and stored in an ultra-low temperature refrigerator at -80°C.

[0081] 3) Extraction of total RNA from samples

[0082] 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.

[0083] 4) Reverse transcription of total RNA from samples

[0084] In an RNase free PCR tube, complete the reverse transcription reaction of the total RNA of the sample in step 3) and remove the genomic DNA. Prepare the reverse transcription system according to Table 2 below:

[0085] Table 2 Reverse transcription system

[0086]

[0087] The above systems were gently mixed, incubated at 42°C for 15 min in a PCR instrument, and then heated at 85°C for 5 s to completely inactivate RT / RI Enzyme and gDNA Remover, and the product cDNAs were stored at -20°C.

[0088] 5) In the same batch of dormant seeds Pt8G06080 Gene and Pt2G02970、Pt9G14330 Comparison of real-time fluorescence quantitative PCR:

[0089] The cDNA generated by reverse transcription in step 4) was appropriately diluted and used as a template, and qRT-PCR was performed according to the qRT-PCR reaction system and reaction conditions in Table 3 below:

[0090] Table 3 qRT-PCR reaction system

[0091]

[0092] Note: The equipment model used for fluorescence quantitative PCR verification is: CFX ConnectTM Optics Module, the specific equipment number is: 788BR04747; the software used for gene expression data analysis is: Bio-Rad CFX Manager.

[0093] The SYBR Green chimeric fluorescence method was used for the reaction. The pre-denaturation reaction program was set as: 95°C pre-denaturation for 2 min, the PCR reaction program was set as: 94°C denaturation for 5 s, 60°C annealing for 30 s, 39 cycles; the melting curve analysis reaction program was set as 65-95°C with an increase of 0.5°C every 5 s;

[0094] Each sample to be tested was repeated 3 times technically and 3 times biologically, and an independent internal reference gene was added to each plate sample. Actin . Reference gene Actin The primer sequences are shown in SEQ ID NO:5 to SEQ ID NO:6:

[0095] Upstream primer: 5′- GGCATACCGGCAGCTCTTC-3′ (SEQ ID NO: 5);

[0096] Downstream primer: 5'- AAGTTGTTGGCGGCGTCTT-3' (SEQ ID NO: 6).

[0097] The expression of pine seeds in the dormancy release state was counted in the expression instrument and software Bio-Rad CFX Manager. Pt8G06080 Genes and Pt2G02970、Pt9G14330 The expression of genes is shown in Figure 3 and Table 4, in Figure 3 ** and *** indicate significant differences P <0.05 and P <0.001.

[0098] Table 4 Seeds that have been released from dormancy Pt8G06080 , Pt2G02970、Pt9G14330 Gene expression

[0099]

[0100] From Table 4 and Figure 3 It can be seen that in the dormancy-released state of Pinus tabulaeformis seeds, based on ac 4 C acetylation-modified dormancy-release marker genes Pt8G06080 The expression level is higher than that of ac 4 C acetylated Pt2G02970、Pt9G14330 The expression level of the gene indicates that the ac 4 C acetylation-modified dormancy-release marker genes Pt8G06080 Relative to Pt2G02970 and Pt9G14330 Genes are more easily detected, so it can be seen that based on ac 4 C acetylation-modified dormancy-release marker genes Pt8G06080 It can better reflect the degree of seed dormancy release and is more suitable as a marker for detecting whether Pinus tabulaeformis seeds have released dormancy.

[0101] Example 3

[0102] Based on ac 4 C acetylation-modified dormancy-release marker genes Pt8G06080 Method for judging the degree of dormancy release of the sample to be tested

[0103] 1) Primer design

[0104] Based on ac 4 C acetylation-modified dormancy-release marker genes Pt8G06080 The CDS sequence of the primers was designed using the NCBI online primer design website Primer-BLAST. Pt8G06080 The primer sequences for the genes are:

[0105] Upstream primer: 5′-ATGACAGACAGGCGCATTGA-3′ (SEQ ID NO: 3);

[0106] Downstream primer: 5'-CCCCACAAGGCACTCAAACT-3' (SEQ ID NO: 4).

[0107] 2) Sampling of Pinus tabulaeformis seeds with different treatments

[0108] The same batch of Pinus tabulaeformis seeds that were dormant after 15 days of heat stress at 37°C (named DS), dormancy released after 7 days of cold stratification at 4°C (named DR), and germinated and whitened (named GS) were selected. The above materials were collected at the same time and immediately quick-frozen with liquid nitrogen and stored in an ultra-low temperature refrigerator at -80°C.

[0109] 3) Extraction of total RNA from samples

[0110] The total RNA of the three 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 by Nano micro-spectrophotometer and 1% agarose gel electrophoresis, respectively.

[0111] 4) Reverse transcription of total RNA from samples

[0112] In an RNase free PCR tube, complete the reverse transcription reaction of the total RNA of the three samples in step 3) and remove the genomic DNA. Prepare the reverse transcription system according to Table 5 below:

[0113] Table 5 Reverse transcription system

[0114]

[0115] The above systems were gently mixed, incubated at 42°C for 15 min in a PCR instrument, and then heated at 85°C for 5 s to completely inactivate RT / RI Enzyme and gDNA Remover. The three product cDNAs were stored at -20°C respectively.

[0116] 5) Seeds with different degrees of dormancy release Pt8G06080 Real-time fluorescence quantitative PCR verification of genes

[0117] The cDNA generated by reverse transcription in step 4) was appropriately diluted and used as a template, and qRT-PCR was performed according to the qRT-PCR reaction system and reaction conditions in Table 6 below:

[0118] Table 6 qRT-PCR reaction system

[0119]

[0120] Note: The equipment model used for fluorescence quantitative PCR verification is: CFX ConnectTM Optics Module, the specific equipment number is: 788BR04747; the software used for gene expression data analysis is: Bio-Rad CFX Manager.

[0121] The SYBR Green chimeric fluorescence method was used for the reaction. The pre-denaturation reaction program was set as: 95°C pre-denaturation for 2 min, the PCR reaction program was set as: 94°C denaturation for 5 s, 60°C annealing for 30 s, 39 cycles; the melting curve analysis reaction program was set as 65-95°C with an increase of 0.5°C every 5 s;

[0122] Each sample to be tested was repeated 3 times technically and 3 times biologically, and an independent internal reference gene was added to each plate sample. Actin . Reference gene Actin The primer sequences are shown in SEQ ID NO:5 to SEQ ID NO:6:

[0123] Upstream primer: 5′-GGCATACCGGCAGCTCTTC-3′ (SEQ ID NO: 5);

[0124] Downstream primer: 5'-AAGTTGTTGGCGGCGTCTT-3' (SEQ ID NO: 6).

[0125] The expression of different dormancy release degrees in Pinus tabulaeformis seeds was counted in the expression instrument and software Bio-Rad CFX Manager. Pt8G06080 The expression of genes is shown in Figure 4 and Table 7, in Figure 4 DS refers to the content of pine seeds after 15 days of heat stress at 37℃. Pt8G06080 The expression level of the gene, DR, represents the expression level of the gene in Pinus tabulaeformis seeds after 7 days of cold stratification at 4℃. Pt8G06080 The expression level of the gene, GS, represents the expression level of the gene in Pinus tabulaeformis seeds after germination and whitening. Pt8G06080 The expression level of the gene Figure 3 ** and **** indicate significant differences P <0.05 and P <0.0001.

[0126] Table 7 Seeds in different treatments Pt8G06080 Gene expression

[0127]

[0128] From Table 7 and Figure 4 It can be seen that as the dormancy of seeds is released, Pt8G06080 The expression of the gene is increasing, and when the seeds germinate normally, Pt8G06080 The expression level of the gene is also higher than that in the dormant state; Pt8G06080 The expression level of the gene was positively correlated with the degree of dormancy release.

[0129] In summary, the technical solution provided by the present invention can accurately determine the degree of dormancy release of the sample to be tested, and in particular can determine the degree of dormancy release by directly testing the seeds of Pinus tabulaeformis. Compared with the traditional discrimination method that relies on observing the germination rate of seeds and physiological indicators (i.e., the method in the preparation example of the present invention), the technical solution provided by the present invention can reduce randomness and uncertainty, accurately reflect the degree of dormancy release of Pinus tabulaeformis seeds, and has higher accuracy, stronger scientificity, and good repeatability, avoiding the loss of time and financial resources caused by finding that the seeds cannot germinate normally after planting.

[0130] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method based on AC 4 C acetylation-modified marker gene for dormancy release in Pinus tabulaeformis seeds Pt8G06080 , the dormancy release marker gene of Pinus tabulaeformis seeds Pt8G06080 The encoded amino acid sequence is shown in SEQ ID NO:

2.

2. The dormancy release marker gene for Pinus tabulaeformis seeds according to claim 1 Pt8G06080 , characterized in that, The dormancy release degree marker gene of Pinus tabulaeformis seeds Pt8G06080 The nucleotide sequence is shown in SEQ ID NO:

1.

3. The dormancy release marker gene for Pinus tabulaeformis seeds according to claim 1 or 2 Pt8G06080 Encoded by Pt8G06080 protein.

4. A marker gene for detecting the degree of dormancy release of Pinus tabulaeformis seeds according to claim 1 or 2 Pt8G06080 A primer pair, characterized in that The primer pair includes an upstream primer and a downstream primer, and the nucleotide sequences of the upstream primer and the downstream primer are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

5. A kit, characterized in that: The kit comprises the primer pair according to claim 4 and a PCR amplification reagent.

6. The kit according to claim 5, characterized in that The kit also includes one or more of an internal reference gene primer, an RNA extraction reagent and a reverse transcription reagent.

7. Use of the primer pair according to claim 4 or the kit according to claim 5 or 6 in determining the dormancy degree of Pinus tabulaeformis seeds.

8. A method for determining the dormancy degree of Pinus tabulaeformis seeds, characterized in that: The steps include: Using the cDNA of the sample to be tested and the cDNA of the reference sample as templates, respectively using the primer pair described in claim 4 to perform fluorescent quantitative PCR amplification, and respectively counting the cDNA of the sample to be tested and the reference sample. Pt8G06080 The expression level of the gene; When the sample to be tested Pt8G06080 The expression of the gene is similar to that in the reference sample. Pt8G06080 When there is no significant difference in the expression levels of the genes, it is determined that the dormancy release degree of the test sample is the same as that of the reference sample; When the sample to be tested Pt8G06080 The expression level of the gene was significantly higher than that in the reference sample Pt8G06080 When the expression level of the gene is greater than that of the reference sample, it is determined that the dormancy release degree of the sample to be tested is higher than that of the reference sample; When the sample to be tested Pt8G06080 The expression of the gene was significantly lower than that in the reference sample. Pt8G06080 When the expression level of the gene is greater than that of the reference sample, it is determined that the dormancy release degree of the sample to be tested is lower than that of the reference sample; Said Pt8G06080 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

9. The method according to claim 8, characterized in that The significant differences include P Significant difference when <0.05.

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