Pinus koraiensis PkDof5.6 gene and application thereof in regulating somatic embryogenesis of pinus koraiensis

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a pinus koraiensis PkDof5.6 gene and application of the pinus koraiensis PkDof5.6 gene to regulation and control of pinus koraiensis somatic embryogenesis. The nucleotide sequence of the PkDof5.6 gene of the Korean pine is as shown in SEQ ID No. 1 (Sequence Identifier Number 1). The Korean pine PkDof5.6 gene provided by the invention regulates Korean pine somatic embryogenesis, and overexpression of the Korean pine PkDof5.6 gene significantly improves the yield and quality of the Korean pine somatic embryogenesis, and promotes the growth of Korean pine somatic embryogenesis seedlings. The method has important significance in optimizing a Korean pine somatic embryogenesis system and realizing efficient large-scale propagation.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to a red pine. PkDof5.6 Genes and their application in regulating embryogenesis in Korean pine. Background Technology

[0002] As an important economic and ecological tree species, Korean pine's somatic embryogenesis technology is of great significance for the preservation, propagation, and genetic improvement of germplasm resources. With the rapid development of biotechnology, transgenic technology has provided new research methods and application prospects for Korean pine somatic embryogenesis. However, due to the low transformation efficiency of coniferous species and the weak integration of conventional and molecular breeding, the promotion and application of transgenic plants are not widespread. It is necessary to effectively combine transgenic technology with research on key genes and molecular regulatory mechanisms in the somatic embryogenesis process of coniferous species, and further utilize other cutting-edge technologies such as gene editing to cultivate new varieties with fast growth, high yield, and strong resistance. The application of transgenic technology in the regulation of Korean pine somatic embryogenesis has significant value. By precisely regulating the expression of target genes, it can effectively shorten the breeding cycle and improve breeding efficiency, which is crucial for promoting the efficient and large-scale propagation of superior Korean pine germplasm resources. Dof Transcription factors can regulate plant growth and development and are widely involved in processes such as plant biotic and abiotic stress responses, playing an important role in plant growth and development. Summary of the Invention

[0003] To solve the above problems, the present invention provides a red pine PkDof5.6 Genes and their application in regulating somatic embryogenesis in Korean pine, and the Korean pine provided by this invention PkDof5.6 Gene regulation of somatic embryogenesis in Korean pine, overexpression of Korean pine PkDof5.6 The gene significantly improves the yield and quality of Korean pine embryos and promotes the growth of Korean pine seedlings.

[0004] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a red pine PkDof5.6 Genes, the red pine PkDof5.6 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0005] Preferably, the red pine PkDof5.6 The amino acid sequence encoded by the gene is shown in SEQ ID No. 2.

[0006] The present invention also provides the red pine described in the above technical solution. PkDof5.6 Application of genes in regulating embryogenesis in Korean pine.

[0007] Preferred, overexpressing red pine PkDof5.6 Genes positively regulate embryogenesis in Korean pine.

[0008] Preferred, overexpressing red pine PkDof5.6 Genes improve the yield and quality of red pine embryos.

[0009] Preferred, overexpressing red pine PkDof5.6 Genes promote the growth of Korean pine seedling embryos.

[0010] Preferred, overexpressing red pine PkDof5.6 Genes increase the content of soluble sugars and soluble proteins in the embryos of Korean pine trees.

[0011] This invention also provides an overexpression vector for the red pine described in the above technical solution. PkDof5.6 The gene was ligated into the pCambia1300-35s-sGFP vector to obtain the overexpression vector.

[0012] Preferably, the nucleotide sequence of the pCambia1300-35s-sGFP vector is shown in SEQ ID No. 3.

[0013] This invention also provides a method for improving the yield and quality of red pine embryos, comprising the following steps: 1) The overexpression vector described in the above technical solution is transformed into Agrobacterium to obtain the transformed bacteria; 2) Infect the embryonic callus tissue of red pine with the transforming bacteria described in step 1).

[0014] The beneficial effects of this invention are: The red pine provided by this invention PkDof5.6 Gene regulation of somatic embryogenesis in Korean pine, overexpression of Korean pine PkDof5.6 The gene significantly improves the yield and quality of Korean pine embryogenesis and promotes the growth of Korean pine embryonic seedlings. This invention is of great significance for optimizing the Korean pine embryogenesis system and achieving efficient large-scale propagation. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0016] Figure 1 Is it a display clone? PkDof5.6 Electrophoresis diagram of the gene; M: DL 2000 DNA Marker; 1: Amplified DNA. PkDof5.6 sequence; Figure 2 This displays a diagram of the pCambia1300-35s-sGFP plasmid. Figure 3 It displays the p1300- build. PkDof5.6 - Electrophoresis diagram of the GFP vector; M: DL 2000 DNA Marker; 1-5: p1300-PkDof5.6 -sGFP bacterial culture PCR product; N: ddH2O; Figure 4 It is a display PkDof5.6 Genetically modified callus tissue; A: PkDof5.6 -A3 Genetically modified callus; B: PkDof5.6 -A8 genetically modified wound healing; Figure 5 It shows that in transgenic callus tissue PkDof5.6 Relative gene expression levels; relative expression levels are the mean and standard deviation A of each treatment group. PkDof5.6 -A3 Transgenic Callus Identification; B: PkDof5.6 -A8 Identification of genetically modified callus; Compared to the wild type P <0.05; Figure 6 It shows the somatic embryo of wild-type red pine and PkDof5.6 Transgenic embryo; A: Wild-type A3 mature material; B: Wild-type A3 embryo; C: Wild-type A8 embryo; D: Wild-type A8 embryo; E: PkDof5.6 -A3-OE mature material; F: PkDof5.6 -A3-OE embryo; G: PkDof5.6 -A8-OE mature material; H: PkDof5.6 -A8-OE embryo; Figure 7 This displays the number of cotyledonous somatic embryos in wild-type and transgenic Korean pine cell lines; the number of somatic embryos is the mean and standard deviation for each treatment group; A: Number of cotyledonous somatic embryos obtained from callus subcultured for 8 months; B: Number of cotyledonous somatic embryos obtained from callus subcultured for 9 months; C: Number of cotyledonous somatic embryos obtained from callus subcultured for 10 months; different lowercase letters indicate... p The difference was statistically significant at the <0.05 level; Figure 8 This displays the EC and SE nutrient content of different wild-type and transgenic Korean pine strains; the nutrient content is the mean and standard deviation of each treatment group; A: soluble sugar content; B: soluble protein content; C: starch content; different capitalization letters indicate... p The difference was significant at the <0.05 level.

[0017] Figure 9 These are somatic embryos of different wild-type and transgenic Korean pine lines; A: One-month somatic embryo of wild-type A3; B: PkDof5.6 - A3-OE 1-month somatic embryos; C: Wild-type A8 1-month somatic embryos; D: PkDof5.6 -A8-OE 1-month somatic embryos; E: wild-type A3 2-month somatic embryos; F: PkDof5.6-A3-OE 2-month somatic embryos; G: Wild-type A8 2-month somatic embryos; H: PkDof5.6 -A8-OE 2-month somatic embryos; Figure 10 This shows the growth status of Korean pine seedlings one month after transplanting; 1: Wild-type A3 seedlings one month after transplanting; 2-3: PkDof5.6 - One month after transplanting A3-OE embryonic seedlings; 4: One month after transplanting wild-type A8 embryonic seedlings; 5-6: PkDof5.6 -A8-OE embryo seedlings 1 month after transplanting; bar=1cm. Detailed Implementation

[0018] This invention provides a red pine PkDof5.6 Genes, the red pine PkDof5.6 The nucleotide sequence of the gene is shown in SEQ ID No. 1, as follows: SEQ ID No. 1:

[0019] In this invention, the red pine PkDof5.6 The amino acid sequence encoded by the gene is shown in SEQ ID No. 2, as follows: MTRFMGASSIQVCCMDYIQCPQEKVAMDHKDMMGCAPSLLERGGLKPQPEKALKCPRCESTNTKFCYYNNYSLSQPRYFCKTCRRYWTKGGTLRNVPVGGSCRKNKRMKRSPPDQSLNARESELTSTTTHGEDHNNNNNFRSCCTLDSDNIASLSNSMYYGPNNNTNDIIHKIFPRIQEAVGDGFTFTNCSNSD ILGPTSGGRPSLSNYLSPMSSLANVWAANSTPLRPISSSTFPAPGFNDKSLKWVDHEAVLSPADPNSIVESHQQISQPGGVQNFASFTMDSGLNRVHSDRLQWRLQEQHPESEGHGIIQLEGQTVEHPCRQSKALEDFMSSFGCNKGGLGEKEWQNPSEQSNCETVKDSIYWNGRSWPYLPNYASSSVSPLI.

[0020] The present invention also provides the red pine described in the above technical solution. PkDof5.6 Application of genes in regulating somatic embryogenesis in Korean pine. In this invention, overexpression of Korean pine genes is used. PkDof5.6 Gene selection positively regulates somatic embryogenesis in Korean pine. In this invention, overexpression of Korean pine... PkDof5.6 Gene optimization improves the yield and quality of Korean pine embryos. In this invention, overexpression of Korean pine embryos is used. PkDof5.6 Gene selection promotes the growth of Korean pine seedling embryos. In this invention, overexpression of Korean pine... PkDof5.6 Genetic optimization increases the content of soluble sugars and soluble proteins in the embryos of Korean pine trees.

[0021] This invention also provides an overexpression vector for the red pine described in the above technical solution. PkDof5.6 The gene was ligated into the pCambia1300-35s-sGFP vector to obtain an overexpression vector. In this invention, the nucleotide sequence of the pCambia1300-35s-sGFP vector is shown in SEQ ID No. 3, and is as follows: GGATCC TCTAGA GTCG ACATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGG CCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACC ACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACC CCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTT CAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTG AAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCT ATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGT GCAGCTCGCCGACCACTACCAGCAGAACACCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTG AGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCG CCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

[0022] This invention also provides a method for improving the yield and quality of red pine embryos, comprising the following steps: 1) The overexpression vector described in the above technical solution is transformed into Agrobacterium to obtain the transformed bacteria; 2) Infect the embryonic callus tissue of red pine with the transforming bacteria described in step 1).

[0023] The overexpression vector described in the above technical solution is transformed into Agrobacterium to obtain the transformed bacteria. This invention does not specifically limit the method for transforming the overexpression vector into Agrobacterium; conventional methods can be used by those skilled in the art.

[0024] This invention describes the use of the transformed bacteria to infect Korean pine embryonic callus. The invention does not specifically limit the method of infection; conventional methods can be used by those skilled in the art.

[0025] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0026] Example 1 PkDof5.6 Cloned genes 1. Korean pine embryogenic callus tissue was selected from solid proliferation medium, and total RNA was extracted and reverse transcribed to synthesize cDNA. The proliferation medium was mLV supplemented with 30 g / L sucrose, 2 mg / L 2,4-D, 0.5 mg / L 6-BA, 0.5 g / L glutamine, 0.5 g / L acid-hydrolyzed casein, 4 g / L gellan gum, and pH 5.8.

[0027] Table 1 RNA reverse transcription procedure 2. Utilize PkDof5.6 Gene sequence was used to design specific primers, which are shown in Table 2.

[0028] Table 2 PkDof5.6 Cloning primer sequences 3. Obtained by PCR amplification PkDof5.6 The full-length sequence of the coding region (CDS) of a gene. PkDof5.6 The nucleotide sequence is shown in SEQ ID No. 1, and the amino acid sequence of the protein it encodes is shown in SEQ ID No. 2. The PCR amplification products were detected by 1.2% agarose gel electrophoresis, as shown below. Figure 1 As shown, the target band was excised and purified using gel extraction, and the resulting DNA fragment was preserved.

[0029] Table 3 PkDof5.6Amplification System Table 4 PkDof5.6 Gene amplification program Example 2 Construction of plant overexpression vectors 1. The pCambia1300-35s-sGFP plant expression vector used in this invention was preserved in our laboratory. For plasmid information, please refer to [link / reference needed]. Figure 2 .

[0030] The nucleotide sequence of pCambia1300-35s-sGFP is shown in SEQ ID No. 3.

[0031] 2. Required reagents and culture media (1) LB liquid medium: 10 g / L tryptic peptone, 5 g / L yeast extract, 5 g / L NaCl, pH=7.

[0032] (2) LB solid medium: The preparation method is the same as LB liquid medium, but 15 g / L agar is added before sterilization.

[0033] (3) Kanamycin (Kan): To prepare 50 mg / mL Kan, weigh 5 g Kan powder in a clean bench, dissolve it in an appropriate amount of ddH2O, stir until fully dissolved, bring the volume to 100 mL, filter through a 0.22 μm filter membrane for sterilization, dispense into containers and store at -20℃.

[0034] (4) 1×TAE buffer: Take 20 mL of 50× TAE electrophoresis buffer, add 980 mL of deionized water, mix well, and store at room temperature.

[0035] 3. The material obtained in Example 1 PkDof5.6 The target gene fragment was ligated into the pTOPO TA / Blunt intermediate vector. The reaction system was prepared according to Table 5 at room temperature, and ligation was carried out for 30 min. 100 μL of *E. coli* DH5α competent cells were thawed on ice, and 10 μL of the ligation product was added and gently mixed. The mixture was incubated on ice for 25 min, followed by heat shock at 42°C for 45 s, and immediately incubated on ice for 2 min. 500 μL of LB liquid medium was added, and the mixture was incubated at 37°C with shaking at 200 rpm for 1 h. The mixture was centrifuged at 3500 rpm for 3 min, and most of the supernatant was discarded. The remaining bacterial culture was spread onto LB agar plates containing 50 mg / L Kan and incubated upside down at 37°C for 12–16 h.

[0036] Table 5 Connection Reaction System 4. Select single colonies and inoculate them into LB liquid medium containing 50 mg / L Kan. Incubate at 37°C for 12–16 h, and perform colony PCR verification using the pTOPO TA / Blunt Vector universal primers M13-F / R (Table 6). After PCR product detection by 1.2% agarose gel electrophoresis, select bacterial solutions with the correct band size for sequencing. Mix the sequencing results of the correctly aligned bacterial solutions with 50% glycerol at a 1:1 volume ratio and store at -80°C for long-term storage. Inoculate successfully aligned E. coli colonies into LB liquid medium containing Kan and incubate at 37°C with shaking for 12–16 h, then extract plasmids.

[0037] Table 6. Primer sequences for the pTOPO TA / Blunt vector 5. Take 10 μL of laboratory-preserved overexpression vector p1300-35s-GFP bacterial culture, inoculate it into 20 mL of LB liquid medium containing 50 mg / L Kan, and culture overnight at 37℃ and 200 rpm with shaking. After extracting the plasmid, store it at -20℃ for later use. PkDof5.6 The CDS sequence and p1300-35s-GFP vector sequence were obtained, and BamHI was selected as the restriction enzyme site for primer design. The p1300-35s-GFP vector was digested with BamHI QuickCut restriction enzyme. After the reaction, 5 μL of 10× Loading Buffer was added, and the target band was extracted and purified by agarose gel electrophoresis. PkDof5.6 Add BamH I restriction sites to the upstream and downstream sequences of CDS to enable pTOPO TA / Blunt- PkDof5.6 PCR amplification was performed using plasmids as templates; primer sequences are shown in Table 7. After verification by agarose gel electrophoresis, the target band was excised and purified using gel electrophoresis.

[0038] Table 7 p1300- PkDof5.6 cloning primers for -sGFP restriction enzyme site and p1300-sGFP vector primer sequences 6. The purified PCR product was ligated into the linearized p1300-sGFP vector using a seamless cloning kit. The ligation product was transformed into DH5α competent cells, and positive clones were screened on Kan-resistant LB plates. Colony PCR was performed using universal primers for the p1300-sGFP vector, and the results were confirmed by agarose gel electrophoresis. Figure 3 As shown. After confirming the sequence was correct through sequencing, the plasmid was extracted and named p1300- PkDof5.6 -GFP.

[0039] Example 3 Pk Dof5.6 Functional verification of genes in Korean pine 1. Required reagents and culture media (1) Co-culture medium: 2 mg / L 2,4-D, 0.5 mg / L 6-BA, 30 g / L sucrose, 0.5 g / L CH, 0.5 g / L glutamine (Gln), 100 µM acetylsuccinone (AS), 4 g / L gellan gum, MLV medium at pH 5.2.

[0040] (2) Recovery medium: 2 mg / L 2,4-D, 0.5 mg / L 6-BA, 30 g / L sucrose, 0.5 g / L CH, 0.5 g / L Gln, 200 mg / L cephalosporin (Cef), 4 g / L gellan gum, MLV medium at pH 5.8.

[0041] (3) Screening medium: 2 mg / L 2,4-D, 0.5 mg / L 6-BA, 30 g / L sucrose, 0.5 g / L CH, 0.5 g / L Gln, 200 mg / L Cef, 20 mg / L hygromycin (Hyg), 4 g / L gellan gum, MLV medium with pH=5.8.

[0042] (4) Maturation medium: mLV with 1 g / L activated carbon, 68.4 g / L sucrose, 12 g / L gellan gum, 80 μmol / LABA, 0.5 g / L Gln and 0.5 g / L acid hydrolyzed casein, pH 5.8.

[0043] (5) Resuspension: MLV medium containing 100 µM AS, pH=5.8.

[0044] (6) Rif: To prepare 50 mg / mL Rif, weigh 5 g of Rif powder in a clean bench, add DMSO to dissolve it, stir until completely clear, and then bring the volume to 100 mL. Filter through a 0.22 μm filter membrane for sterilization, dispense into containers, and store at -20℃ protected from light.

[0045] (7) Acetyleugenol (AS): To prepare 100 mM AS, weigh 1.962 g AS in a clean bench, add DMSO to dissolve it completely, bring the volume up to 100 mL, filter it through a 0.22 μm filter membrane for sterilization, dispense it into aliquots and store it at -20℃ in the dark.

[0046] (8) Cephalosporin (Cef): To prepare 200 mg / mL Cef, weigh 20 g of Cef powder in a clean bench, add ddH2O and stir until completely dissolved, bring the volume up to 100 mL, filter through a 0.22 μm filter membrane for sterilization, dispense and store at -20℃.

[0047] (9) Hygromycin (Hyg): To prepare 50 mg / mL Hyg, weigh 5 g Hyg powder in a clean bench, add ddH2O to dissolve it completely, bring the volume up to 100 mL, filter it through a 0.22 μm filter membrane for sterilization, dispense it into individual containers, and store it at -20℃ in the dark. 2. Place Agrobacterium GV3101 on ice, and add the above 5 µL p1300- PkDof5.6 Add the GFP plasmid to competent cells and mix gently. Perform the following treatments sequentially: ice bath for 5 min, liquid nitrogen treatment for 5 min, 28°C metal bath for 5 min, and ice bath again for 5 min. Add 700 μL of antibiotic-free LB liquid medium and incubate at 28°C with shaking at 200 rpm for 2-3 hours. Centrifuge at 6000 rpm for 1 min, discard part of the supernatant, retain approximately 150 μL of bacterial culture, resuspend and mix well, and spread onto LB plates containing Kan and Rif resistance. Incubate at 28°C for 2-3 days. Pick single colonies and expand them in LB liquid medium containing Kan and Rif resistance. Perform colony PCR verification using p1300-sGFP vector primers. Sequencing the PCR product with the correct band size. Mix the sequencing-verified bacterial culture with 50% glycerol at a 1:1 volume ratio and store at -80°C for long-term storage.

[0048] 3. (The last part is incomplete and likely refers to a specific product or service.) PkDof5.6 -GFP Agrobacterium tumefaciens bacterial suspension was spread onto LB solid medium containing 50 mg / L Kan and 20 mg / L Rif, and incubated at 28°C for 2 days to screen for single colonies. Two to three single colonies were picked and inoculated into LB liquid medium containing the same antibiotic, and incubated at 28°C with shaking at 200 rpm until... Prepare for later use. Centrifuge the above bacterial suspension at 4000 rpm for 10 min at 4°C, discard the supernatant, and collect the bacterial cells. Resuspend the bacterial cells in 100 μM acetylsylcholine resuspension solution to ensure the bacterial suspension is ready. The solution obtained at this point is the infection solution. Two red pine embryogenic callus cell lines (A3 and A8) that had been proliferated and subcultured for 6 months were selected. 2g of callus tissue was placed in 30-40 mL of infection solution for 10 min at 25℃ and 120 rpm. After infection, the mixture was dried using a vacuum filtration sterilization device. The callus tissue was then transferred to a co-culture medium containing 100 μM AS and co-cultured at 25±2℃ in the dark for 2 days. After sterilization, the tissue was washed three times with a sterilization solution containing 200 mg / L CEF for 5 min each time. The sterilization solution was then dried using a vacuum filtration device. The material was placed in a recovery medium containing 200 mg / L CEF and cultured for 7 days. Subsequently, the material was transferred to a selection medium containing 20 mg / L Hyg and selected three times, each time for 21 days. Figure 4 As shown in the figure. The selected transgenic callus tissue was transferred to a proliferation medium for propagation.

[0049] 4. The callus tissue grown on the selection medium was proliferated and subcultured. RNA was extracted from WT and transgenic callus tissues and reverse transcribed into cDNA for use in transgenic callus tissues. PkDof5.6 qRT-PCR validation, such as Figure 5 As shown, and in PkDof5.6 The callus tissues with the highest gene expression levels in the overexpression transgenic callus tissues were selected and named as follows: PkDof5.6-A3-OE and PkDof5.6-A8-OE .

[0050] 5. PkDof5.6 -A3-OE and PkDof5.6 Transgenic callus from A8-OE and wild-type materials A3 and A8, which were subcultured for 8 months at the same time, were used for somatic embryo maturation experiments. After 8 weeks of somatic embryo maturation culture, the number, morphology, and size of somatic embryos formed from transgenic and wild-type callus from Korean pine were observed and statistically analyzed. Figure 6 , Figure 7 The results showed that the somatic embryo yield of wild-type callus decreased with prolonged culture time, and wild-type A8 was superior to A3. PkDof5.6 The number of cotyledonous somatic embryos obtained from transgenic callus under different culture time conditions in transgenic cell lines was higher than that of wild-type cells, indicating that overexpression... PkDof5.6 It can improve the yield and quality of embryos.

[0051] 6. The nutrient content of wild-type and transgenic callus tissues and somatic embryos was determined, such as... Figure 8 As shown, compared with WT, overexpression PkDof5.6 The contents of soluble sugars and soluble proteins were significantly increased in the cell lines. The results showed that... PkDof5.6 Overexpression of this substance promotes the accumulation of nutrients, which in turn facilitates the formation of the embryo.

[0052] 7. After 8 weeks of somatic embryo maturation, cotyledonary embryos were inoculated into six-well cell plates and cultured at 4°C in the dark for one week. They were then inoculated into germination medium and cultured in the dark at 25°C for one week, followed by 8 hours of dark culture and 16 hours of light culture daily. After one month of germination culture… Figure 9 The growth status of somatic embryos from different cell lines was compared, and the lengths of cotyledons, hypocotyls, and radicles were statistically analyzed. The significance of these results is shown in Table 8. The results indicate that the transgenic cell lines have significant advantages in somatic embryo growth, which may be related to the inserted genes, thereby improving growth efficiency and adaptability.

[0053] Table 8. Length of different parts of wild-type and transgenic cell line seedlings of Korean pine 8. Transplant the 1-month-old somatic embryos to a rooting medium for 1 month, and observe the growth of wild-type and transgenic somatic embryos for 2 months. Figure 9 The results showed that the number of cotyledons in the transgenic seedlings gradually increased, and the root development was more complete.

[0054] 9. Transplant the 2-month-old seedlings into the substrate (peat moss: perlite = 1:1), water regularly, and cover with plastic wrap, leaving a small opening to ensure air circulation. Cultivate under 16 hours of light per day and 25℃. After one week, remove the plastic wrap. After one month of cultivation, regenerated plants will be obtained. Observe their growth status. Figure 10 As shown in the figure. The results indicate that wild-type A3 and A8 have shorter roots, while transgenic lines generally have more and larger cotyledons, longer and darker roots, showing stronger developmental capabilities.

[0055] Experiments have shown that the present invention PkDof5.6 Gene introduction into Korean pine callus tissue can significantly improve the yield and quality of Korean pine embryogenesis.

[0056] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A type of red pine PkDof5.6 Genes, characterized by, The red pine PkDof5.6 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. The red pine according to claim 1 PkDof5.6 Genes, characterized by, The red pine PkDof5.6 The amino acid sequence encoded by the gene is shown in SEQ ID No.

2.

3. The Korean pine as described in claim 1 PkDof5.6 Application of genes in regulating embryogenesis in Korean pine.

4. The application according to claim 3, characterized in that, Overexpression of red pine PkDof5.6 Genes positively regulate embryogenesis in Korean pine.

5. The application according to claim 3, characterized in that, Overexpression of red pine PkDof5.6 Genes improve the yield and quality of red pine embryos.

6. The application according to claim 3, characterized in that, Overexpression of red pine PkDof5.6 Genes promote the growth of Korean pine seedling embryos.

7. The application according to claim 3, characterized in that, Overexpression of red pine PkDof5.6 Genes increase the content of soluble sugars and soluble proteins in the embryos of Korean pine trees.

8. An overexpression vector, characterized in that, The red pine described in claim 1 PkDof5.6 The gene was ligated into the pCambia1300-35s-sGFP vector to obtain an overexpression vector.

9. The overexpression vector according to claim 8, characterized in that, The nucleotide sequence of the pCambia1300-35s-sGFP vector is shown in SEQ ID No.

3.

10. A method for improving the yield and quality of red pine embryos, characterized in that, Includes the following steps: 1) Transform the overexpression vector according to claim 8 or 9 into Agrobacterium to obtain the transformed bacteria; 2) Infect the embryonic callus tissue of red pine with the transforming bacteria described in step 1).

Images