Application of Pt9G10460 gene and its encoded protein in judging whether Pinus tabulaeformis seedlings have entered the dark morphogenesis stage

Abstract

The present invention discloses the application of the Pt9G10460 gene and its encoding protein in judging whether the Chinese pine seedlings have entered the dark morphology construction period. It belongs to the field of agricultural biotechnology. The present invention provides a marker gene Pt9G10460 and its encoding protein, and the nucleotide sequence of the gene is shown in SEQ ID NO.1. The gene and its encoding protein can be used to judge whether the Chinese pine seedlings have entered the dark morphology construction period. The present invention also provides a primer pair for specifically amplifying the above-mentioned gene, and the Pt9G10460 gene can be specifically amplified by the primer pair. The expression amount of the Pt9G10460 gene in different state tissues of Chinese pine is analyzed by qRT-PCR technology, so as to efficiently and accurately judge whether the sample to be tested is in the dark morphology construction period. Experiments have shown that the technical solution provided by the present invention can accurately judge whether the Chinese pine seedlings have entered the dark morphology construction period, and has higher accuracy, stronger scientificity and good repeatability.

Description

Technical Field

[0001] The invention relates to the field of agricultural biotechnology, and more specifically to the application of a Pt9G10460 gene and a protein encoded therein in determining whether a Chinese pine seedling has entered a dark morphological construction period. Background Art

[0002] Pinus tabuliformis is a gymnosperm and tree of the Pinaceae family. It is mainly distributed in Jilin, Henan, Hebei, Shandong, Shanxi and other places. It has strong adaptability and likes dry and cold climates. It mainly reproduces by sowing. All parts of Pinus tabuliformis have medicinal value, and its wood can be used in construction and furniture manufacturing, and is also suitable for gardening. It has a wide range of uses.

[0003] During the germination process of plant seeds, they may be temporarily unable to receive sunlight due to cover materials such as soil, fallen leaves, and weeds. In dark conditions, seeds use their own nutrients to germinate and grow. This process is called dark morphogenesis, which is characterized by elongation of the hypocotyl, failure to unfold the apical hook, and yellowing of the seedling, which helps the seedlings to emerge from the soil as soon as possible and receive sunlight.

[0004] Traditionally, the determination of dark morphogenesis in Pinus tabulaeformis seedlings mainly relies on phenotypic observation and empirical inference, but this method is easily affected by environmental and subjective factors, and different researchers may have different judgments on the characteristics, so the accuracy is limited. For many years, experts and scholars generally believe that in addition to the common phenotypic changes, the dark morphogenesis process is often accompanied by changes in physiological metabolic levels and changes in gene expression levels.

[0005] In recent years, with the development of molecular biology, regulatory factors related to light and hormone signaling pathways have been discovered in a variety of angiosperms, and these factors are related to the dark morphogenesis of seedlings. Due to differences in genomes, long evolutionary distances, different biological functions, characteristics of adaptive evolution, and the limitations of the depth and breadth of current research, marker genes found in angiosperms cannot be directly applied to gymnosperms. These factors have led to the fact that some marker genes unique to angiosperms may not have homologous sequences or have changed functions in gymnosperms, so the marker genes of angiosperms cannot be simply applied to the study of gymnosperms. At present, in gymnosperms such as Pinus tabulaeformis, marker genes that can clearly determine the dark morphogenesis period have not been identified. Determining whether Pinus tabulaeformis seedlings are in the dark morphogenesis period has always been a difficult problem in research.

[0006] In summary, how to provide a marker gene for determining whether Pinus tabulaeformis seedlings have entered the dark morphogenesis period is an urgent problem that technicians in this field need to solve. Summary of the invention

[0007] In view of this, the present invention provides the use of the Pt9G10460 gene and its encoded protein in determining whether a Chinese pine seedling has entered the dark morphogenesis period. The gene and its encoded protein can efficiently and accurately determine whether a Chinese pine seedling has entered the dark morphogenesis period, providing a scientific reference for rapid detection of the growth status of Chinese pine seedlings.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] The present invention provides a Pt9G10460 gene for judging whether a Pinus tabulaeformis seedling has entered a dark morphogenesis period. The nucleotide sequence of the Pt9G10460 gene is shown in SEQ ID NO.1.

[0010] The present invention provides a Pt9G10460 protein encoded by the Pt9G10460 gene in the above technical solution, and the amino acid sequence of the Pt9G10460 protein is shown in SEQ ID NO.2.

[0011] A primer pair for amplifying the Pt9G10460 gene described in the above technical solution, wherein the primer pair comprises an upstream primer and a downstream primer;

[0012] The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3;

[0013] The nucleotide sequence of the downstream primer is shown in SEQ ID NO.4.

[0014] The invention provides a kit for judging whether a Chinese pine seedling has entered a dark morphological construction period. The kit comprises a primer pair and a PCR amplification reagent according to the technical solution.

[0015] Furthermore, the PCR amplification reagent includes SYBR Green DNA polymerase, dNTPs, PCR buffer and internal reference gene primers.

[0016] The present invention provides the use of the Pt9G10460 gene, the Pt9G10460 protein, the primer pair or the kit described in the above technical solution in determining whether the Pinus tabulaeformis seedlings have entered the dark morphogenesis period.

[0017] The present invention provides a method for judging whether a Chinese pine seedling has entered a dark morphological construction period, comprising the following steps:

[0018] The pine seedlings growing normally under light conditions were set as reference samples, and the cDNA of the sample to be tested and the cDNA of the reference sample were used as templates. The above primer pairs were used to perform fluorescence quantitative PCR amplification, and the expression levels of the Pt9G10460 gene in the sample to be tested and the reference sample were counted respectively;

[0019] When there is no significant difference between the expression level of Pt9G10460 gene in the test sample and the expression level of Pt9G10460 gene in the reference sample, it is determined that Pinus tabulaeformis has not entered the dark morphological construction period;

[0020] When the expression level of the Pt9G10460 gene in the test sample is significantly lower than that in the reference sample, it is determined that the Pinus tabulaeformis seedlings have entered the dark morphogenesis stage;

[0021] The nucleotide sequence of the Pt9G10460 gene is shown in SEQ ID NO.1.

[0022] Furthermore, the significant difference includes the significant difference when P<0.05.

[0023] Furthermore, the reaction system of the fluorescent quantitative PCR amplification is 20 μL, including: 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 RNase-free water is added to 20 μL.

[0024] Furthermore, the reaction procedure of the fluorescent 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 running for 39 cycles.

[0025] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0026] The present invention provides a Pt9G10460 gene, the nucleotide sequence of which is shown in SEQ NO. 1. On this basis, the present invention further provides a Pt9G10460 protein encoded by the gene. Both the Pt9G10460 gene and the Pt9G10460 protein can be used to determine whether a Chinese pine seedling has entered a dark morphogenesis period.

[0027] Based on the above technical advantages, the present invention provides a method for judging whether the Chinese pine seedlings have entered the dark morphogenesis period. By using the primers provided by the present invention to perform fluorescent quantitative PCR amplification on the cDNA of the test sample and the cDNA of the reference sample, and comparing the expression levels of the Pt9G10460 gene of the two samples, it is possible to accurately judge the state of the Chinese pine seedlings. The experimental results show that compared with the traditional method that relies on the dark morphogenesis phenotype and empirical judgment, the technical scheme of the present invention can more accurately reflect whether the Chinese pine seedlings are in the dark morphogenesis period, and has higher accuracy, stronger scientificity and better repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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.

[0029] Figure 1 The results of agarose gel electrophoresis in Example 2 of the present invention are as follows;

[0030] Figure 2 is the expression level of the Pt9G10460 gene in the transcriptome sequencing results of Pinus tabulaeformis seedlings grown under light and dark conditions in Example 1 of the present invention;

[0031] Figure 3 The expression level of the Pt9G10460 gene in the Pinus tabulaeformis seedling sample under light conditions (reference sample) and the Pinus tabulaeformis seedling sample under dark conditions (test sample) is detected by real-time fluorescence quantitative PCR in Example 2 of the present invention;

[0032] Figure 4 The phenotypes of the reference sample and the test sample in different states in Example 3 of the present invention. DETAILED DESCRIPTION

[0033] 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.

[0034] The present invention provides a Pt9G10460 gene, the nucleotide sequence of which is shown in SEQ ID NO.1, specifically:

[0035]

[0036] This gene can be used to determine whether Pinus tabulaeformis seedlings have entered the dark morphogenesis period.

[0037] In the present invention, unless otherwise specified, all reagents and materials used are purchased conventionally.

[0038] The present invention also provides a protein encoded by the Pt9G10460 gene described in the above technical solution, and its amino acid sequence is shown in SEQ ID NO.2, specifically:

[0039] MAQERIPQAMPNDFYGQEKPCAANMMSNQEKASNATMIVVEQKCCVYPARPSDQKYCDLTTFDLSFVTFYYNQKIILYPSPAQGFASAVESLKKSLSEALVHFYPLAGRLCLDDDGI LKVHCDNAGVDFIEASSDVVGVATLMDCDSSSEVMQQLVPYVDNLNVDGFFLPLVGVQVTKLRDGVAVGIAVNHLIVDGYSTWHFIRSWADLCRGSSVIYLPPSHDRAMARNTKVKL NLNPPNGDSNAKGNKTEKHPLKGKTFHFSKEMMEEIKSRANKNREGKPLSSFQSLGAHLWQAVTKARKLAPEDIALFAVLMDCRAKVSPPLPKSYFGNAIQSIKVVTSVGHLLSNDL SFAANMLQQVIDSHGANAIKQKNEEWERDPKLYGFSDAARNCVVVGSSPRFEENDFGWGKPVRVKSGCNNKFDGMVYLYPGQAGRGSVDVEITLLPTTMDLLESDPQFRLTN*, SEQ ID NO.2.

[0040] This protein can also be used to determine whether Pinus tabulaeformis seedlings have entered the dark morphogenesis stage.

[0041] The present invention provides a primer pair for amplifying the Pt9G10460 gene described in the above technical solution, wherein the primer pair comprises an upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, specifically: 5'-GCTTTATGGATTCAGCGATGC-3', SEQ ID NO.3; the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4, specifically: 5'-GCGGAATTGAGGGTCAGACTC-3', SEQ ID NO.4. The primer pair can be used to specifically amplify the Pt9G10460 gene.

[0042] The present invention provides a detection kit, which includes the primer pair and PCR amplification reagent described in the above technical solution. In the present invention, the PCR amplification reagent preferably includes SYBR Green DNA polymerase, dNTPs, PCR buffer and internal reference gene primers; the sources and amounts of the SYBR Green DNA polymerase, dNTPs, PCR buffer and internal reference gene primers are not particularly limited, and can be used by techniques well known in the art.

[0043] The present invention provides the use of the Pt9G10460 gene, the Pt9G10460 protein, the primer pair or the detection kit described in the above technical solution in determining whether a Chinese pine seedling has entered a dark morphogenesis period. The method can quickly and accurately determine the state of the Chinese pine seedling.

[0044] The present invention provides a method for judging whether a Chinese pine seedling has entered a dark morphology construction period. The method comprises the following steps: setting a Chinese pine seedling grown under light conditions as a reference sample, using the reference sample cDNA and the sample to be tested cDNA as templates, respectively using the primer pairs described in the above technical solution to perform fluorescent quantitative PCR amplification, and respectively counting the expression levels of the Pt9G10460 gene in the reference sample and the sample to be tested; when there is no significant difference between the expression level of the Pt9G10460 gene in the sample to be tested and the expression level of the Pt9G10460 gene in the reference sample, the sample to be tested has not entered a dark morphology construction period; when the expression level of the Pt9G10460 gene in the sample to be tested is significantly lower than the expression level of the Pt9G10460 gene in the reference sample, it is determined that the sample to be tested has entered a dark morphology construction period. The significant difference preferably includes a significant difference when P<0.05.

[0045] The present invention preferably prepares cDNA of reference samples and samples to be tested respectively. In the present invention, the reference sample and the sample to be tested are preferably of the same species; the sample to be tested preferably includes pine seedlings after shading treatment; the pine seedlings under dark conditions should be in a normal state, without wilting, drying up, etc., and it is appropriate to grow 7 to 9 days after germination of pine seeds; the reference sample preferably includes pine seedlings growing normally under long-day conditions (16h light, 8h dark); there is no special requirement for the culture method. There are no special requirements for the method and reagents for preparing cDNA in the present invention, and the technology well known in the art can be used.

[0046] After obtaining the cDNA of the reference sample and the sample to be tested, the present invention uses the two cDNAs as templates and respectively uses the primer pairs described in the above technical scheme to perform fluorescent quantitative PCR amplification. In the present invention, the reaction system of the fluorescent quantitative PCR amplification is based on 20 μL, preferably including: 10 μL of 2×SuperReal PreMix Plus (SYBR Green), 1 μL of cDNA template, 0.8 μL of 100 μM upstream primer, 0.8 μL of 100 μM downstream primer, and RNase-free water is supplemented to 20 μL; the reaction procedure of the fluorescent quantitative PCR amplification is preferably: 95°C pre-denaturation for 2 minutes, 94°C denaturation for 5 seconds, 60°C annealing for 30 seconds, and running 39 cycles. In this way, the specific amplification of the Pt9G10460 gene in the reference sample and the sample to be tested can be achieved respectively.

[0047] After the fluorescent quantitative PCR amplification, the present invention counts the relative expression levels of the Pt9G10460 gene in the reference sample and the sample to be tested, and makes a judgment.

[0048] The present invention has no special requirements for the equipment for fluorescence quantitative PCR amplification and the analysis method of gene expression, and the technology well known in the art can be used. In a specific embodiment of the present invention, the equipment model for fluorescence quantitative PCR amplification is: CFX Connect TM -Optics Module, the specific number of the device is: 788BR04747; the software used for gene expression data analysis is: Bio-Rad CFX Manager.

[0049] Experiments have shown that compared with the traditional reliance on dark morphogenesis phenotypes and experience to judge whether the Pinus tabulaeformis seedlings have entered the dark morphogenesis period, the technical solution provided by the present invention can accurately reflect the status of the Pinus tabulaeformis seedlings with higher accuracy, stronger scientificity and good repeatability.

[0050] To further illustrate the present invention, the application of a Pt9G10460 gene and its protein provided by the present invention in determining whether Chinese pine seedlings have entered the dark morphological construction period is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0051] Experimental Materials:

[0052] Pinus tabulaeformis seeds: purchased from Zhongtiaoshan State Forest Farm in Yanqu County, Yuncheng City, Shanxi Province; Yanqu Zhongtiaoshan Forest Farm is located in a fault basin with an average annual temperature of 22℃, a maximum temperature of 40℃, and a minimum temperature of -11℃; the merchant collected mature Pinus tabulaeformis cones from well-grown mature Pinus tabulaeformis mother plants, dried them at room temperature, collected the seeds, and mixed them evenly.

[0053] The above-mentioned Chinese pine seeds were transported to the laboratory, and cultured under different conditions in a seedling culture box with wild sphagnum moss as the medium. The specific process is as follows: after the wild sphagnum moss is soaked, it is spread flat in the seedling culture box; the Chinese pine seeds are washed with clean water for 3 times, and then soaked in sodium hypochlorite disinfectant with a mass fraction of 0.5% for 20 minutes, and washed with clean water again for 3 times; the clean Chinese pine seeds are evenly sown on the soaked wild sphagnum moss medium, sprayed with an appropriate amount of clean water and covered with a lid; the seedling culture boxes with Chinese pine seeds are placed in light culture boxes respectively, one of which is germinated and grown under normal plant growth conditions, and the other is shaded with a black plastic bag; during the germination and growth process, clean water is sprayed appropriately to ensure the growth of the seedlings; after 7 to 9 days after germination, Chinese pine seedlings under dark conditions and Chinese pine seedlings under light conditions are obtained. By observing the phenotype, it was found that the pine seedlings under dark conditions had obvious hypocotyl elongation, overall yellowing of the seedlings, and apical hooks, indicating that the pine seedlings under dark conditions have entered the dark morphogenesis period. The seedlings were removed and cleaned, then quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator.

[0054] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0055] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0056] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0057] Example 1

[0058] Screening of the Pt9G10460 gene that can determine the dark morphogenesis period of Pinus tabulaeformis seedlings

[0059] 1) Sampling of Pinus tabulaeformis seedlings under different treatment conditions

[0060] The samples frozen in the dark and light conditions at -80°C were taken, and each group of samples had 3 biological replicates, with each replicate weighing 3 g.

[0061] 2) RNA extraction, library construction, and transcriptome sequencing

[0062] The total RNA of the reference samples was extracted by the Trizol method, and after quality inspection, the transcriptome library was constructed using the NEB #7530 kit (#E7530, New England Biolabs). After the library passed the quality inspection, it was sent to Annoroad Gene Technology (Beijing) Co., Ltd. for high-throughput sequencing on the Illuminanova-6000 platform.

[0063] 3) Data quality control and reference genome comparison

[0064] The raw data (rawreads) were subjected to data quality control, and reads with a base N ratio greater than 10% were filtered out, reads with a quality value Q≤20 accounting for more than 50% of the entire read were filtered out, the adapter and the subsequent part were truncated, and reads shorter than 50bp after truncating the adapter were filtered out to obtain high quality cleanreads.

[0065] The high-quality Chinese pine genome (https: / / www.ncbi.nlm.nih.gov / bioproject / PRJNA784915) was used as the 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.

[0066] 4) Gene expression analysis

[0067] 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.

[0068] 5) Weighted gene co-expression network analysis

[0069] 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 levels, 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. The CytoHubba plug-in was then used to combine the intersection of 12 algorithms such as MMC to obtain the Pt9G10460 gene. The nucleotide sequence of the Pt9G10460 gene is shown in SEQ ID NO.1.

[0070] Example 2

[0071] Verification of marker gene Pt9G10460

[0072] 1) Primer design

[0073] According to the CDS sequence of the Pt9G10460 gene, primers were designed using the NCBI online primer design website Primer-BLAST. The primer sequence of the Pt9G10460 gene is:

[0074] Upstream primer: 5'-GCTTTATGGATTCAGCGATGC-3', SEQ ID NO. 3;

[0075] Downstream primer: 5'-GCGGAATTGAGGGTCAGACTC-3', SEQ ID NO.4.

[0076] 2) Sampling of Pinus tabulaeformis seedlings under different treatments

[0077] The above-mentioned Pinus tabulaeformis seedlings under light conditions and dark conditions were selected. The above-mentioned materials were immediately quick-frozen with liquid nitrogen after collection and stored in a -80°C ultra-low temperature refrigerator.

[0078] 3) Extraction of total RNA from samples

[0079] 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. The results are shown in Figure 1 , as shown in Table 1:

[0080] Table 1 RNA concentration and purity results

[0081] sample Concentration (ng / μL) Volume (μL) OD260 / 280 OD260 / 230 Samples under lighting conditions 591 25 2.1 2.0 Samples in dark conditions 579 25 2.1 2.1

[0082] 4) Reverse transcription of total RNA of samples

[0083] In an RNase free PCR tube, use the TransScript One-Step gDNA Removal and cDNA Synthesis Kit from Beijing Quanshijin Biotechnology Co., Ltd. to 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:

[0084] Table 2 Reverse transcription system

[0085]

[0086] 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 cDNA was stored in a -20°C refrigerator.

[0087] 5) Real-time fluorescence quantitative PCR verification of gene Pt9G10460

[0088] 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:

[0089] Table 3 qRT-PCR reaction system

[0090]

[0091]

[0092] 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 as follows: 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.

[0093] Each sample was repeated 3 times technically and 3 times biologically, and an independent internal reference gene Actin was added to each plate sample. The primer sequence of the internal reference gene Actin is as follows:

[0094] Upstream primer: 5'-GGCATACCGGCAGCTCTTC-3', SEQ ID NO.5;

[0095] Downstream primer: 5'-AAGTTGTTGGCGGCGTCTT-3', SEQ ID NO.6.

[0096] The results are as follows Figure 3 As shown. After dark treatment, the expression level of Pt9G10460 gene was significantly downregulated. This result is consistent with the transcriptome sequencing results ( Figure 2 , measured in Example 1). The gene expression level of the sample under dark conditions (test sample) was significantly lower than that under light conditions (reference sample) ( Figure 3 ), indicating that the Pinus tabulaeformis seedlings have entered the dark morphogenesis stage.

[0097] Example 3

[0098] Application Examples

[0099] Different batches of Pinus tabulaeformis seedlings were selected to carry out the above steps of sampling, RNA extraction, reverse transcription and real-time fluorescence quantitative PCR, and the gene determination results were compared with those of traditional methods to further verify the technical effect, as follows:

[0100] 1) Randomly select 10 Pinus tabulaeformis seedlings grown under light conditions (reference samples) and 10 Pinus tabulaeformis seedlings grown under dark conditions (test samples). Observe the phenotype ( Figure 4 ) and used for RNA extraction.

[0101] 2) Total RNA of the samples collected in step 1) 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 Nano micro-spectrophotometer and 1% agarose gel electrophoresis, respectively.

[0102] 3) Reverse transcription of total RNA of samples

[0103] In an RNase free PCR tube, the total RNA of the sample obtained in step 2) was reverse transcribed using the TransScript One-Step gDNA Removal and cDNA Synthesis Kit from Beijing Quanshijin Biotechnology Co., Ltd., and genomic DNA was removed. The reverse transcription system was the same as in Table 2.

[0104] 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 cDNA was stored in a -20°C refrigerator.

[0105] 4) Real-time fluorescence quantitative PCR of gene Pt9G10460

[0106] The cDNA generated by reverse transcription in step 3) was diluted 5 times and used as a template, and qRT-PCR was performed according to the qRT-PCR reaction system in Table 3 and the following reaction conditions.

[0107] 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. Each sample was repeated 3 times technically and 3 times biologically, and an independent internal reference gene Actin was added to each plate sample.

[0108] The results are as follows:

[0109] By observing the phenotypes of the reference sample and the sample to be tested, it was found that the sample to be tested had obvious phenotypes such as hypocotyl elongation, overall yellowing of the seedling, and apical hooking ( Figure 4 ), indicating that the sample to be tested has entered the dark form formation period.

[0110] The expression levels of the Pt9G10460 gene in the reference samples and the test samples and the result determination are shown in Table 4.

[0111] Table 4 Judgment results of the status of different batches of Pinus tabulaeformis seedlings

[0112]

[0113] Compared with the reference sample, the expression level of the Pt9G10460 gene in the test sample was significantly downregulated after dark treatment, indicating that the Pinus tabulaeformis seedlings (test sample) have entered the dark morphogenesis stage. The gene determination result is consistent with the traditional method.

[0114] 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.

[0115] 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. The Pt9G10460 gene for determining whether the Pinus tabulaeformis seedlings have entered the dark morphogenesis period is characterized by: The nucleotide sequence of the Pt9G10460 gene is shown in SEQ ID NO.

1.

2. The Pt9G10460 protein encoded by the Pt9G10460 gene according to claim 1, characterized in that: The amino acid sequence of the Pt9G10460 protein is shown in SEQ ID NO.

2.

3. A primer pair for amplifying the Pt9G10460 gene according to claim 1, characterized in that: The primer pair includes an upstream primer and a downstream primer; 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.

4. A kit for determining whether a Pinus tabulaeformis seedling has entered a dark morphogenesis period, characterized in that: The kit comprises the primer pair according to claim 3 and a PCR amplification reagent.

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

6. Use of the Pt9G10460 gene according to claim 1, the Pt9G10460 protein according to claim 2, the primer pair according to claim 3, or the kit according to claim 4 or 5 in determining whether a Pinus tabulaeformis seedling has entered the dark morphogenesis period.

7. A method for determining whether a Pinus tabulaeformis seedling has entered a dark morphogenesis period, characterized in that: The following steps are involved: A pine seedling grown normally under light conditions is set as a reference sample, and the cDNA of the sample to be tested and the cDNA of the reference sample are used as templates, and the primer pairs described in claim 3 are used to perform fluorescent quantitative PCR amplification, and the expression levels of the Pt9G10460 gene in the sample to be tested and the reference sample are counted respectively; When there is no significant difference between the expression level of Pt9G10460 gene in the test sample and the expression level of Pt9G10460 gene in the reference sample, it is determined that Pinus tabulaeformis has not entered the dark morphological construction period; When the expression level of the Pt9G10460 gene in the test sample is significantly lower than that in the reference sample, it is determined that the Pinus tabulaeformis seedlings have entered the dark morphogenesis stage; The nucleotide sequence of the Pt9G10460 gene is shown in SEQ ID NO.

1.

8. The method according to claim 7, characterized in that The significant differences include those when P<0.

05.

9. The method according to claim 7, characterized in that: The reaction system of the fluorescent quantitative PCR amplification is 20 μL, including: 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 RNase-free water is added to 20 μL.

10. The method according to claim 7, characterized in that The reaction procedure of the fluorescent 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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