Application of PmACAA1 gene in determination of masson pine seed vigor

Abstract

The invention relates to the technical field of plant detection, in particular to application of a PmACAA1 gene in determination of masson pine seed vigor. The activity of the pinus massoniana seeds is judged by testing the relative expression quantity of the PmACAA1 gene, and if the expression quantity of acetyl coenzyme A acyltransferase is reduced, the activity of the pinus massoniana seeds is high. By means of the PmACAA1 gene, the activity of the pinus massoniana seeds can be efficiently and rapidly judged, and the method is not limited by the germination period of the seeds. According to the method provided by the invention, the efficiency of testing the activity of the pinus massoniana seeds is greatly improved, a good early-stage tool is provided for storage, later-stage cultivation and the like of the pinus massoniana seeds, and the economic benefit and the social benefit are good.

Description

Technical Field

[0001] This invention relates to the field of plant detection technology, and more particularly to a... PmACAA1 Application of the (acetyl-CoA acyltransferase-1) gene in determining the viability of Masson pine seeds. Background Technology

[0002] Masson pine is a deciduous tree belonging to the genus *Pinus* of the family Pinaceae. It flowers from April to May, and its cones mature from October to December of the following year. It is widely distributed in the humid subtropical eastern region of my country and is a major afforestation species in southern my country. Masson pine is suitable for afforestation of barren hills, ecological scenic forests, and can also be used for roadside and courtyard landscaping. Its wood can be used for papermaking, construction, and furniture making, possessing significant ecological and economic value.

[0003] Seeds are the core carrier for the preservation of forest germplasm resources. As a pioneer tree species for afforestation in southern my country and a strategic timber reserve, the seeds of Masson pine have a viability of over 90% under normal conditions. However, due to the obvious phenomenon of abundant and scarce years for this tree species, and the decline in viability during long-term storage or exposure to adverse conditions, problems such as low germination rate, uneven seed germination, poor seedling uniformity, weak seedling resistance, and low afforestation survival rate are caused.

[0004] Based on this, technicians often use the embryo mutation method to determine its viability by observing the color change of the embryo (yellow, green, white, hard). However, the TTC staining method relies on subjective judgment of staining depth (e.g., dark red, light red) and lacks clear quantitative standards, resulting in poor consistency in result interpretation and low detection efficiency. Summary of the Invention

[0005] In view of this, the present invention provides a PmACAA1 The application of genes in determining the vigor of Masson pine seeds, this invention... PmACAA1 Genes can efficiently and quickly determine the viability of Masson pine seeds, without being restricted by the seed germination cycle.

[0006] This invention provides PmACAA1 The application of genes in determining the vigor of Masson pine seeds, the aforementioned PmACAA1 The base sequence of the gene is shown in SEQ ID NO.1.

[0007] The present invention also provides the above-described solution. PmACAA1 Gene-encoded PmACAA1 The application of protein in determining the vigor of Masson pine seeds, the PmACAA1 The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0008] The present invention also provides an amplification method described above. PmACAA1 Primer pairs for genes.

[0009] Preferably, the primer pair includes a forward primer and a reverse primer, the nucleotide sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0010] The present invention also provides a kit comprising the primer pairs and PCR amplification reagents described in the above-described scheme.

[0011] Preferably, the kit further includes a cDNA template and RNase-free water.

[0012] Preferably, the kit further includes primers for an internal reference gene; the internal reference gene is... 18s rRNA Genes and UBC Gene.

[0013] This invention also provides the application of the primer pairs or kits described in the above scheme in determining the viability of Masson pine seeds.

[0014] This invention also provides a method for determining the viability of Masson pine seeds, comprising the following steps: using cDNA from the test sample and cDNA from a reference sample as templates, performing quantitative real-time PCR amplification using the primer pairs described in the above scheme, and statistically analyzing the cDNA levels in the test sample and the reference sample. PmACAA1 Gene expression levels; when the sample to be tested contains... PmACAA1 The gene expression level was lower than that in the reference sample. PmACAA1 When the gene expression level is measured, the viability of the test sample is determined to be higher than that of the reference sample.

[0015] Preferably, the reaction system for the real-time PCR amplification, in 10 µL increments, comprises: 5 µL of 2×SuperReal PreMix Plus, 1 µL of cDNA template, 0.5 µL of 100 µM upstream primer, 0.5 µL of 100 µM downstream primer, and 3 µL of RNase-free water.

[0016] The present invention has achieved the following beneficial effects: This invention has passed testing. PmACAA1 The relative expression levels of genes determine the viability of Masson pine seeds; downregulation of acetyl-CoA acyltransferase expression indicates high seed viability. This invention utilizes... PmACAA1 This invention enables efficient and rapid determination of the viability of Masson pine seeds, unaffected by the seed germination cycle. The method provided by this invention significantly improves the efficiency of Masson pine seed viability testing, offering an excellent preliminary tool for Masson pine cultivation, and yielding good economic and social benefits. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0018] Figure 1 The TTC staining results of Masson pine seeds stored at different temperatures for 1 year in Example 1 are shown in the figure (the deeper the red staining, the higher the seed viability); where a is room temperature, b is 4 degrees Celsius, and c is -20 degrees Celsius. Figure 2 These are photographs of 14 days after the germination of *Pinus massoniana* seeds stored at different temperatures for one year in Example 1; where a represents room temperature, b represents 4 degrees Celsius, and c represents -20 degrees Celsius. Figure 3 For example, the seeds of Masson pine stored at different temperatures for one year in Example 1 PmACAA1 Relative gene expression levels (different letters "a" and "b" represent significant differences in expression levels, p < 0.01); Figure 4 The images show the germination and staining of *Pinus massoniana* seeds aged 0-10 days in Example 2; where A is the germination image and B is the TTC staining image (the deeper the red staining, the higher the seed vigor; the higher the vigor, the more seeds germinate). Figure 5 In Example 2, the seeds of Masson pine aged for 0-10 days PmACAA1 Relative gene expression levels. Detailed Implementation

[0019] This invention provides PmACAA1 The application of genes in determining the vigor of Masson pine seeds, the aforementioned PmACAA1 The base sequence of the gene is shown in SEQ ID NO.1.

[0020] SEQ ID NO.1: ATGAAGAGGAGAATGGCAATGCAGAAAGGATTGCCCATTCTTGGAGTTTTCAGGAGTTTTGCAGCAGTTGGATGTGATCCAGCCGTAATGGGAATTGGTCCAGCTGTAGCAATACCAGCAGCAGTCAAGGCAGCTGGACTACAAATTGGTGATATTGATCTTTTCGAAATAAACGAGGCATTTGCCTCTCAGTATGTATACTGTTGTCAGAAGTT GGAGCTGGATCCAGAAAAAGTCAATGTAAATGGTGGAGCTATTGCGCTCGGTCACCCACTTGGAGCAACAGGAGCTCGCTGTGTTGGAACATTACTCCATGAAATGAAACGACGCGGTAAAGATTGTAGGTTTGGAGTAATCTCCATGTGTATAGGGACTGGAATGGGAGCAGCTGCAGTGTTTGAGCGTGGCACCAATGCCAGATCAATCTGA.

[0021] The present invention also provides the above-described solution. PmACAA1 Gene-encoded PmACAA1 The application of protein in determining the vigor of Masson pine seeds, the PmACAA1 The amino acid sequence of the protein is shown in SEQ ID NO.2.

[0022] SEQ ID NO.2: MKRRMAMQKGLPILGVFRSFAAVGCDPAVMGIGPAVAIPAAVKAAGLQIGDIDLFEINEAFASQYVYCCQKLELDPEKVNVNGGAIALGHPLGATGARCVGTLLHEMKRRGKDCRFGVISMCIGTGMGAAAVFERGTNARSI*.

[0023] The present invention also provides an amplification method described above. PmACAA1 Primer pairs for genes.

[0024] In this invention, the primer pair preferably includes a forward primer and a reverse primer; the nucleotide sequences of the forward primer and the reverse primer are preferably shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The primer pair provided by this invention can specifically amplify... PmACAA1 Gene.

[0025] SEQ ID NO.3: CTCCATGAAATGAAACGACG.

[0026] SEQ ID NO.4: TGCCACGCTCAAACACTG.

[0027] The present invention also provides a kit comprising the primer pairs and PCR amplification reagents described in the above-described scheme.

[0028] In this invention, the PCR amplification reagent is preferably a real-time PCR amplification reagent; the real-time PCR amplification reagent is preferably 2× SuperReal PreMix Plus.

[0029] In this invention, the kit preferably further includes a cDNA template and RNase-free water.

[0030] In this invention, the volume ratio of the PCR amplification reagent to the cDNA template is preferably 5:1; the volume ratio of the PCR amplification reagent to RNase-free water is preferably 5:3.

[0031] In this invention, the volume ratio of the PCR amplification reagent to the upstream primer is preferably 10:1; the concentration of the upstream primer is preferably 100µM; the volume ratio of the PCR amplification reagent to the downstream primer is preferably 10:1; and the concentration of the downstream primer is preferably 100µM.

[0032] In this invention, the kit preferably also includes primers for an internal reference gene.

[0033] In this invention, the internal reference gene is preferably... 18s rRNA Genes and UBC Gene.

[0034] In this invention, the 18s rRNA The nucleotide sequences of the upstream and downstream primers of the gene primers are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0035] SEQ ID NO.5: GCCTTTCGAAGCACTTTCAC.

[0036] SEQ ID NO.6: AACCAAACCTCCATGCTCAC.

[0037] In this invention, the UBC The nucleotide sequences of the upstream and downstream primers of the gene primers are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0038] SEQ ID NO.7: GATTTATTTCATTGGCAGGC.

[0039] SEQ ID NO.8: AGGATCAGGATTTGGGT.

[0040] This invention also provides the application of the primer pairs or kits described in the above scheme in determining the viability of Masson pine seeds.

[0041] This invention also provides a method for determining the viability of Masson pine seeds, comprising the following steps: using cDNA from the test sample and cDNA from a reference sample as templates, performing quantitative real-time PCR amplification using the primer pairs described in the above scheme, and statistically analyzing the cDNA levels in the test sample and the reference sample. PmACAA1 Gene expression levels; when the sample to be tested contains... PmACAA1 The gene expression level was lower than that in the reference sample. PmACAA1 When the gene expression level is measured, the viability of the test sample is determined to be higher than that of the reference sample.

[0042] In this invention, the test sample and the reference sample are seeds of the same variety of Masson pine; the reference sample is preferably Masson pine seeds that are sealed and stored at room temperature (26 degrees Celsius), 4 degrees Celsius or -20 degrees Celsius respectively.

[0043] In this invention, the reaction system for the real-time PCR amplification is 10 µL, preferably comprising: 5 µL of 2×SuperReal PreMix Plus, 1 µL of cDNA template, 0.5 µL of 100 µM upstream primer, 0.5 µL of 100 µM downstream primer, and 3 µL of RNase-free water.

[0044] In this invention, the preferred reaction program for the real-time PCR amplification is: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 5 s, 55℃ annealing for 30 s, for 40 cycles.

[0045] In this invention, the preferred model of the equipment used for quantitative real-time PCR amplification is BIO-RadCFX96.

[0046] In this invention, the analysis of relative gene expression levels is preferably performed using Bio RadCFXManager software.

[0047] To further illustrate the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0048] Example 1 PmACAA1 Relative expression levels of genes in Masson pine seeds at different storage temperatures: 1) This embodiment uses Masson pine seeds collected from the National Masson Pine Seed Base in Ma'anshan Forest Farm, Duyun City, Guizhou Province, and stores them in sealed containers for one year at room temperature (26 degrees Celsius), 4 degrees Celsius, or -20 degrees Celsius. The seeds used in this embodiment... PmACAA1 Primer pairs and internal reference genes for genes ( 18s rRNA and UBC The sequences of the genes are shown in Table 1: Table 1. Sequences of primer pairs and internal reference genes

[0049] 2) Amplification process: RNA extraction: Masson pine seeds were soaked in distilled water at room temperature (26°C) for 24 hours. The softened seeds were then peeled to remove the seed coat. Total RNA was collected from the samples using the RNAprepPure polysaccharide and polyphenol plant total RNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd. The purity of the extracted RNA was determined by measuring the ratio of the extracted RNA quality using 1% agarose gel electrophoresis. A ratio of approximately 2.0 indicates high RNA sample purity.

[0050] Reverse transcription of total RNA: The RNA obtained from RNA extraction was reverse transcribed using the Fastking one-step genomic cDNA first-strand synthesis kit from Tiangen Biotech (Beijing) Co., Ltd. The reverse transcription system is shown in Table 2. Table 2 Reverse Transcription System

[0051] Add the above system sequentially to RNase-free PCR tubes and mix gently. Place the tubes in a PCR instrument for reverse transcription. Set the program to incubate at 42°C for 15 min and heat at 95°C for 3 min. Store the final cDNA product in a -20°C freezer.

[0052] 3) Real-time quantitative PCR verification: The cDNA prepared in this invention was diluted to 100 ng / µL and used as a template for quantitative real-time PCR amplification using the primer pairs described above. The quantitative real-time PCR amplification reaction system, in 10 µL increments, included: 5 µL of 2×SuperRealPreMixPlus, 1 µL of cDNA template, 0.5 µL of 100 µM upstream primer, 0.5 µL of 100 µM downstream primer, and 3 µL of RNase-free water. The quantitative real-time PCR amplification reaction program was: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 5 s, 55℃ annealing for 30 s, for 40 cycles. The instrument used for quantitative real-time PCR amplification was a BIO-RadCFX96; the software used for gene expression data analysis was Bio-RadCFXManager. Results are as follows: Figure 3 As shown.

[0053] according to Figure 3 It can be seen that, PmACAA1 Gene expression differed in Masson pine seeds treated under three different temperature conditions. PmACAA1 The gene was most highly expressed in pine seeds stored at room temperature and least expressed in pine seeds stored at 4 degrees Celsius.

[0054] 4) TTC seed vigor test: Ten randomly selected *Pinus massoniana* seeds were soaked in distilled water at room temperature (26°C) for 24 hours to soften them. The seed coats were then removed from the softened seeds, and the embryo tissue was exposed by longitudinally cutting along its length. The treated seeds were then immersed in a 0.5 wt% TTC solution (tetracycline red solution), ensuring complete contact between the embryo and the TTC solution. The seeds were then incubated in a 35-37°C incubator for 1 hour in the dark. The seeds were then removed with tweezers and immediately rinsed three times with distilled water to terminate the reaction. The seeds were then observed and photographed directly under a stereomicroscope. The results are shown below. Figure 1 As shown.

[0055] according to Figure 1 It can be seen that the seeds of *Pinus massoniana* stored at room temperature showed the lightest staining, while those stored at 4°C and -20°C showed much darker staining. This indicates that the viability of *Pinus massoniana* seeds stored at room temperature was the lowest, while the viability of *Pinus massoniana* seeds stored at 4°C and -20°C was relatively high.

[0056] 5) Germinate the Masson pine seeds stored under the above conditions under the same environment, observe their growth, and the results are as follows: Figure 2 As shown. According to Figure 2 It can be seen that the seeds of Masson pine stored at room temperature basically did not germinate, while the seeds of Masson pine stored at 4 degrees Celsius and -20 degrees Celsius grew well, which further proves that the vitality of Masson pine seeds stored at room temperature is the lowest.

[0057] In summary, the present invention achieves this through... PmACAA1 The relative expression levels of genes were used to successfully determine the predicted viability of Masson pine seeds.

[0058] Example 2 PmACAA1 Relative expression levels of Pinus massoniana seeds at different aging times: 1) Experimental materials and treatment: Masson pine seeds stored for one year in a seed storage box at 4°C were used as the seed aging treatment material. An artificial aging chamber (Top Cloud Agriculture, model LH-150) was used to create a high-temperature and high-humidity environment (45±0.5°C, 95±5% relative humidity) for accelerated artificial aging treatment of the Masson pine seeds. Uniformly sized and matured Masson pine seeds were placed in mesh bags, evenly spread, and then placed in the pre-treated artificial aging chamber. Four biological replicates were set up. After 2, 4, 6, 8, and 10 days of treatment, seeds were sampled at 10:00 AM on the corresponding aging time points. The extracted seeds were placed at room temperature for 24 hours to allow moisture equilibration until they returned to their original weight. Masson pine seeds treated for different aging times and restored to their original weight were used for index determination.

[0059] 2) Amplification process: This embodiment uses PmACAA1 The primer pairs and sequences of the internal reference genes (18S rRNA and UBC gene) are shown in Table 1.

[0060] RNA extraction: Masson pine seeds were soaked in distilled water at room temperature (26°C) for 24 hours. The softened seeds were then peeled to remove the seed coat. Total RNA was collected from the samples using the RNAprepPure polysaccharide and polyphenol plant total RNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd. The purity of the extracted RNA was determined by measuring the ratio of the extracted RNA quality using 1% agarose gel electrophoresis. A ratio of approximately 2.0 indicates high RNA sample purity.

[0061] Reverse transcription of total RNA from samples: The RNA obtained from RNA extraction was reverse transcribed using the Fastking one-step genomic cDNA first-strand synthesis kit from Tiangen Biotech (Beijing) Co., Ltd. The reverse transcription system is shown in Table 2.

[0062] Add the above system sequentially to RNase-free PCR tubes and mix gently. Place the tubes in a PCR instrument for reverse transcription. Set the program to incubate at 42°C for 15 min and heat at 95°C for 3 min. Store the final cDNA product in a -20°C freezer.

[0063] 3) Real-time quantitative PCR verification: The cDNA prepared in this invention was diluted to 100 ng / µL and used as a template for quantitative real-time PCR amplification using the primer pairs described above. The quantitative real-time PCR amplification reaction system, in 10 µL increments, included: 5 µL of 2×SuperRealPreMixPlus, 1 µL of cDNA template, 0.5 µL of 100 µM upstream primer, 0.5 µL of 100 µM downstream primer, and 3 µL of RNase-free water. The quantitative real-time PCR amplification reaction program was: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 5 s, 55℃ annealing for 30 s, for 40 cycles. The instrument used for quantitative real-time PCR amplification was a BIO-RadCFX96; the software used for gene expression data analysis was Bio-RadCFXManager. Results are as follows: Figure 4~5 As shown in Table 3.

[0064] Table 3. Determination of Vitality-Related Indicators of Aged Seeds

[0065] according to Figure 4~5 As can be seen from Table 3, with the extension of aging time, PmACAA1 The relative expression level of the gene increased significantly, while the germination, staining, and vigor index of Masson pine seeds all indicated a gradual decrease in vigor, proving that... PmACAA1 The relative expression level of genes is significantly correlated with the viability of Masson pine seeds. This invention utilizes... PmACAA1 The relative expression level of genes can be a good predictor of the viability of Masson pine seeds.

[0066] As can be seen from the above embodiments, PmACAA1 An upregulation of expression is a sign of low seed vigor; PmACAA1 Downregulation is a sign of high vigor in Masson pine seeds.

[0067] The embodiments of the present invention have been described above; however, these embodiments are merely illustrative and not intended to limit the scope of the invention. Although various embodiments have been described above, this does not mean that the measures in the embodiments cannot be used advantageously in combination. Various substitutions and modifications can be made by those skilled in the art without departing from the scope of the invention, and all such substitutions and modifications should fall within the scope of the invention.

Claims

1. A kind PmACAA1 The application of genes in determining the vigor of Masson pine seeds is characterized by, The PmACAA1 The base sequence of the gene is shown as SEQ ID NO.

1.

2. The method of claim 1 PmACAA1 genes encoding PmACAA1 application of the protein in determining the seed viability of Pinus massoniana, characterized in that, The PmACAA1 The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A primer pair for amplifying a gene as described in the above scheme. PmACAA1 gene.

4. The primer pair according to claim 3, characterized in that, The primer pair includes a forward primer and a reverse primer, the nucleotide sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.

5. A kit characterized in that, The kit includes the primer pairs and PCR amplification reagents described in the above protocol.

6. The kit of claim 5, wherein The kit also includes a cDNA template and RNase-free water.

7. The kit of claim 5, wherein The kit also comprises primers for a reference gene; the reference gene is 18S rRNA gene and UBC gene.

8. The use of the primer pair according to any one of claims 3 to 4 or the kit according to any one of claims 5 to 7 in determining the viability of Masson pine seeds.

9. A method for determining the viability of Pinus massoniana seeds, characterized by, Includes the following steps: 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 the above scheme are used for fluorescence quantitative PCR amplification, and the expression amounts of the genes in the sample to be tested and the reference sample are counted respectively PmACAA1 the expression amount of the gene; When the sample to be tested contains PmACAA1 The gene expression level was lower than that in the reference sample. PmACAA1 When the gene expression level is measured, the viability of the test sample is determined to be higher than that of the reference sample.

10. The method of claim 9, wherein, The reaction system for the quantitative PCR amplification is 10 µL and includes: 5 µL of 2× SuperReal PreMix Plus, 1 µL of cDNA template, 0.5 µL of 100 µM upstream primer, 0.5 µL of 100 µM downstream primer, and 3 µL of RNase-free water.

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