Application of LkHY5 gene in regulation and control of somatic embryogenesis of hybrid larch

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to application of an LkHY5 gene in regulation and control of hybrid larch somatic embryogenesis. Results of the invention show that overexpression of the LkHY5 gene promotes somatic embryogenesis on a culture medium containing 30 mg / L of exogenous hormone ABA. The expression levels of LkGBSS1-3 and LkTAN1-1 are remarkably improved by overexpression of the LkHY5 gene. The overexpression of the LkHY5 gene significantly improves the contents of starch, maltose, glucose and soluble sugar in the callus. The overexpression of the LkHY5 gene significantly improves the stability of the callus microtubules.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of the LkHY5 gene in regulating the embryogenesis of hybrid larch. Background Technology

[0002] Larch (Larix spp.) is a tree belonging to the genus Larix in the family Pinaceae, and is one of the main afforestation tree species in Northeast my country. Interspecific hybridization of larch exhibits significant heterosis, mainly reflected in growth, resistance, and wood quality. Its wood is solid, rot-resistant, and has high compressive strength, making it valuable for industrial applications. The main propagation methods for larch are sowing and cuttings. However, conventional sowing is prone to variation, and cuttings have a low survival rate due to seasonal influences. Furthermore, the demand for germplasm is large, making large-scale propagation difficult.

[0003] The difficulty in rooting larch plants limits the application of conventional tissue culture and cutting techniques for rapid propagation. Therefore, somatic embryogenesis has become a primary approach for the efficient propagation of high-quality larch lines and the research and application of artificial seeds. Plant somatic embryogenesis refers to the process by which differentiated somatic cells, without sex cell fusion, directly form a complete plant through a process similar to zygotic embryo development. This approach is not only an important method for obtaining complete plants but also a manifestation of plant cell totipotency. Plant somatic embryogenesis involves the regulation of several key transcription factors. The somatic embryogenesis receptor-like kinase gene SERK plays a central role in promoting the conversion of somatic cells into embryogenic cells. Key plant embryogenesis genes, such as LEC1 and LEC2, are involved in embryo formation and maturation. The BBM (Baby Boom) gene can promote the formation of somatic embryos even without growth regulators. The WUS (WUSCHEL) gene participates in this process by regulating stem cell stability. The entire embryogenesis involves a complex transcriptional regulatory network, including the interaction of the auxin pathway and the collaborative efforts of transcription factors such as LEC, BBM, and WUS to promote somatic embryogenesis in plants.

[0004] Starch is a major storage substance in plants and is closely related to tissue differentiation and organ formation. No starch granules were found in non-embryonic callus of *Larix chinensis* in North China, while embryogenic callus began to accumulate starch after transformation in the culture medium, reaching its peak during the heart-shaped embryo stage. This demonstrates the important role of starch in tissue culture and morphogenesis. Studies have shown that after prolonged subculturing, the starch content and number of starch granules in callus cultures of certain species decrease, leading to a decline in regeneration capacity.

[0005] Studies have shown that successful embryogenesis depends on programmed cell death (PCD), with the cytoskeleton dominating cell morphogenesis. In somatic embryogenesis of the gymnosperm spruce, the formation of the apical-basal pattern of the spruce embryo requires the establishment of three main cell types: meristematic cells at one end of the embryonic mass and undifferentiated suspensory cells at the other, separated by tubular cells. From the embryonic body to the distal end of the suspensory cells, the microtubule and F-actin histological structure undergoes progressive changes: the microtubule array remains normal in the embryonic somatic cells, the microtubule network is partially disordered in the embryonic tubular cells, and the microtubule structure is completely destroyed in the septum cells. In the same embryo, the microtubule-associated protein MAP-65 binds only to ordered microtubules. Conversely, in cell lines where development is inhibited and a normal embryonic pattern cannot be formed, MAP-65 does not bind to cortical microtubules, a key indicator of the primordial embryonic mass (PEM) entering early embryonic development.

[0006] ELONGATED HYPOCOTYL 5 (HY5) is a bZIP-type transcription factor and a major regulator of light-mediated responses. HY5 activates the transcription of numerous light-responsive genes by directly binding to G-box (CACGTG) elements or ACGT-containing elements (ACEs) in its promoters. Therefore, HY5 coordinates adaptive changes throughout development, including inhibiting hypocotyl elongation, enhancing photoprotective pigment biosynthesis, and regulating the diurnal rhythm of photorhythm. Recent studies have also shown that HY5 plays a crucial role in regulating starch metabolism. In Arabidopsis, HY5 activates the transcription of starch synthase GBSS by binding to its promoter, promoting starch chain elongation and branching, thereby positively regulating starch synthesis. Furthermore, HY5 also regulates genes encoding microtubule-related proteins in Arabidopsis, negatively regulating the expression of the microtubule-stabilizing protein TPXL5, thus affecting cortical microtubule remodeling and consequently influencing lateral root initiation. In *Salix matsudana*, HY5 forms a complex with microtubule-associated protein SPR1, which participates in light-mediated microtubule elongation and cell expansion.

[0007] In larch regeneration systems, embryogenic callus tissue often loses its embryogenicity over long-term subculturing, leading to reduced somatic embryogenesis and low breeding efficiency. Optimizing the larch in vitro regeneration system can significantly increase propagation speed, enabling rapid propagation and preservation of superior germplasm resources. Currently, research on forest tree somatic embryogenesis largely focuses on optimizing and improving somatic embryogenesis conditions, with relatively little research on its molecular mechanisms. Strengthening research on the regulatory mechanisms of somatic embryogenesis will not only help provide reference genes for plants with low somatic embryogenesis capacity but also improve the capacity and efficiency of somatic embryogenesis. Hybrid larch, as a plant with enormous potential economic value, suffers from low seed yield and declining quality, seriously affecting the quality of larch seedling cultivation and afforestation. Research on improving its somatic embryogenesis and plant regeneration efficiency is of great significance for the preservation, genetic improvement, and rapid propagation of its germplasm resources. Summary of the Invention

[0008] To address the aforementioned issues, this invention provides the application of the LkHY5 gene in regulating somatic embryogenesis in hybrid larch (in this invention, the hybrid larch is obtained through artificial hybridization, using Japanese larch as the female parent and Dahurian larch as the male parent, resulting in a Japanese 5 × Dahurian 9 hybrid larch). Overexpression of the LkHY5 gene in hybrid larch is beneficial for improving the somatic embryogenesis efficiency of hybrid larch.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] This invention provides the application of the LkHY5 gene in regulating the embryogenesis of hybrid larch.

[0011] Preferably, overexpression of the LkHY5 gene positively regulates embryogenesis in hybrid larch.

[0012] Preferably, the nucleotide sequence of the LkHY5 gene is shown in SEQ ID No. 1.

[0013] Preferably, the amino acid sequence of the protein encoded by the LkHY5 gene is shown in SEQ ID No. 2.

[0014] Preferably, overexpression of the LkHY5 gene positively regulates hybrid larch embryogenesis by increasing the expression of genes related to hybrid larch embryogenesis.

[0015] Preferably, the hybrid larch embryogenesis-related genes include the LkGBSS1-3 gene and / or the LkTAN1-1 gene.

[0016] This invention also provides the application of overexpression of the LkHY5 gene in increasing the sugar content in hybrid larch callus.

[0017] Preferably, the carbohydrate includes one or more of starch, maltose, soluble sugars and glucose.

[0018] This invention also provides the application of overexpression of the LkHY5 gene in improving the microtubule stability of hybrid larch callus.

[0019] The beneficial effects of this invention are:

[0020] 1) This invention aims to promote somatic embryogenesis in hybrid larch by increasing the expression of the LkHY5 gene, the nucleotide sequence of which is shown in SEQ ID No. 1; constructing an overexpression vector for the LkHY5 gene of hybrid larch; transforming the constructed overexpression vector into callus tissue of hybrid larch; and screening and cultivating transgenic hybrid larch embryogenic cell lines with significantly improved somatic embryogenesis efficiency.

[0021] 2) The results of this invention show that overexpression of the LkHY5 gene promotes somatic embryogenesis on a culture medium containing 30 mg / L of exogenous hormone ABA. Compared with the control group, the number of somatic embryos in the overexpression lines (LkHY5-OE#14, LkHY5-OE#20) was significantly increased.

[0022] 3) The results of this invention show that overexpression of the LkHY5 gene significantly increases the expression levels of LkGBSS1-3 and LkTAN1-1.

[0023] 4) The results of this invention show that overexpression of the LkHY5 gene significantly increases the content of starch, maltose, glucose and soluble sugar in callus tissue.

[0024] 5) The results of this invention show that overexpression of the LkHY5 gene significantly improves the stability of microtubules in callus tissue.

[0025] 6) The results of this invention show that disrupting microtubule stability significantly affects the sugar content of callus tissue. Attached Figure Description

[0026] 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.

[0027] Figure 1 Image showing positive identification of LkHY5 transgenic callus;

[0028] Figure 2 Image showing the RT-qPCR results of LkHY5 transgenic callus;

[0029] Figure 3The diagram shows the induction of somatic embryogenesis. a) Somatic embryos induced from wild-type callus, b) Somatic embryos induced from 35S:LkHY5 transgenic callus, and c) Statistical results of somatic embryo induction data.

[0030] Figure 4 This diagram illustrates the effect of LkHY5 on the expression levels of somatic embryogenesis-related genes LkGBSS1-3 and LkTAN1-1.

[0031] Figure 5 The graph shows the sugar content of LkHY5 transgenic callus, from left to right: starch content, maltose content, glucose content, and soluble sugar content.

[0032] Figure 6 Fluorescence imaging of microtubules in LkHY5 transgenic callus, a) wild-type callus microtubules, b) 35S:LkHY5 transgenic callus microtubules;

[0033] Figure 7 Fluorescence imaging of microtubules in LkHY5 transgenic callus after application of the plant microtubule inhibitor Oryzalin. a) Microtubules of wild-type callus, b) Microtubules of 35S:LkHY5 transgenic callus.

[0034] Figure 8 The graph shows the sugar content of LkHY5 transgenic callus after the application of the plant microtubule inhibitor Oryzalin. From left to right, the graph shows the starch content, soluble sugar content, maltose content, and glucose content. Detailed Implementation

[0035] This invention provides the application of the LkHY5 gene in regulating hybrid larch serosa embryogenesis. In this invention, overexpression of the LkHY5 gene preferably positively regulates hybrid larch serosa embryogenesis. In this invention, the nucleotide sequence of the LkHY5 gene is shown in SEQ ID No. 1. In this invention, the amino acid sequence of the protein encoded by the LkHY5 gene is shown in SEQ ID No. 2.

[0036] SEQ ID No. 1:

[0037] ATGCAGGACACTGCTGCCTCCACATCTACACAACATCAGTCAACAAGTGAAAAGTCTTCTAGTTCAGCAGCACCAGCGCCATTTAGACAAGCCAAAGATGCAATTGAGAGCGATGATGATATCAGGAGGGTTCCTGAAATGGGAGGAATGCAAGCAGGACCATCTTCGTGTGCCGATGAGGTTAGACAATCCCCAACCCACCACAGGCGTTGTTACCCACAGGAAGAGAGGGAGAGCCCCTG CAGACAAGGAACACAAGCGTCTTAAAAGATTGCTTAGGAACAGAGTGTCCGCCCAACAGGCAAGAGAAAGAAAGAAAGCGTACTTGAATGATTTAGAAACAAGGGTTAAGGAGATTGAGCTCAAAAACTCTGAGTTAGAGGAAAGGCTTTCCACTTTGCAGAATGAAAATCATATGCTTAGACAGATTTTGAAGAATACAACAATGAAAAAAAAGGGATCCGGTAACTCAGGAACAGAAACCTAA

[0038] SEQ ID No. 2:

[0039] MQDTAASTSTQHQSTSEKSSSSAAPAPFRQAKDAIESDDDIRRVPEMGGMQAGPSSCVPMRLDNPQPTTGVVTHRKRGRAPADKEHKRLKRLLRNRVSAQQARERKKAYLNDLETRVKEIELKNSELEERLSTLQNENHMLRQILKNTTMKKKGSGNSGTET*

[0040] In this invention, overexpression of the LkHY5 gene preferably positively regulates hybrid larch embryogenesis by increasing the expression of hybrid larch embryogenesis-related genes. In this invention, the hybrid larch embryogenesis-related genes preferably include the LkGBSS1-3 gene and / or the LkTAN1-1 gene.

[0041] This invention also provides the application of overexpression of the LkHY5 gene in increasing the carbohydrate content in hybrid larch callus. In this invention, the carbohydrates preferably include one or more of starch, maltose, soluble sugars, and glucose.

[0042] This invention also provides the application of overexpression of the LkHY5 gene in improving the microtubule stability of hybrid larch callus.

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the materials, reagents, enzymes, competent cells, and plasmids used are all commercially available.

[0044] The material used in this application is embryogenic callus induced from immature embryos, which is cultured in a constant temperature incubator at 22°C in the dark.

[0045] The pCAMBIA1300-35S-sGFP used in this application was preserved by the National Key Laboratory of Forest Genetics and Breeding, Northeast Forestry University.

[0046] The reagents used in this application are as follows:

[0047] (1) 0.5 M EDTA: Weigh 18.6 g of EDTA powder, dissolve it in ddH2O, adjust the pH value to 8.0 with NaOH, make up to 100 mL, autoclave and store at room temperature.

[0048] (2) CTAB extract (RNA): Weigh 0.381 g of borax and dissolve it in 10 mL of deionized water. Adjust the pH value to 8.5 with HCl. Then add 1 mL of EDTA (0.5 M), 2 g of CTAB and 9.36 g of NaCl in sequence. Place the solution in a 65℃ water bath to aid dissolution. After complete dissolution, add 100 μL of 0.1% DEPC water and add deionized water to make up to 100 mL. Mix thoroughly and incubate at 37℃ for 12 h. After autoclaving, store at room temperature.

[0049] (3) 0.1% DEPC water: Measure 100 mL of deionized water, add 100 μL of DEPC solution, mix well, incubate at 37℃ for 12 h, autoclave and store at room temperature.

[0050] (4) 8 M LiCl: Weigh 33.91 g LiCl and dissolve it in 100 mL ddH2O. Incubate at 37℃ for 12 h, autoclave, and store at room temperature.

[0051] (5) CTAB extraction solution (DNA): Weigh 5 g CTAB and 20.47 g NaCl and dissolve them in ddH2O. Then add 25 mL Tris-HCl (1 M) and 10 mL EDTA (0.5 M) and bring the volume up to 250 mL. After autoclaving, store at room temperature.

[0052] (6) 3 M NaAc: Weigh 40.8 g NaAc, dissolve it in ddH2O, add glacial acetic acid to adjust the pH to 5.2, and make up to 100 mL. Store at room temperature.

[0053] The culture medium used in this application is shown below:

[0054] (1) DCR basic medium: 340.0 mg / L KNO3+556.0 mg / L Ca(NO3)2·4H2O+400.0 mg / LNH4NO3+170.0mg / L KH2PO4+370.0mg / L MgSO4·6H2O+85.0mg / L CaCL2·2H2O+6.2mg / L H3BO3+8.6 mg / L ZnSO4·7H2O+22.3 mg / L MnSO4·H2O+0.25 mg / L NaMoO4·2H2O+0.83mg / L KI+0.25mg / L CuSO4·5H2O+0.025mg / L CoCL2·6H2O+0.025mg / L NiCL·6H2O+13.93mg / LFeSO4·7H2O+18.65mg / L EDTA-Na2+2.0 mg / L Glycine+0.5 mg / L Nicotinic acid+1.0 mg / L Thiamine HCl+0.5 mg / L Tocopherol+2.0 mg / L Mgo-inositol, adjust the pH to 5.8-6.0.

[0055] (2) Embryogenic callus induction medium: DCR basal medium + 30 g / L sucrose + 9 g / L agar + 2.25 μM 2,4-D + 2.2 μM BA, adjust the pH to 5.8-6.0.

[0056] (3) Callus subculture: DCR basal medium + 30 g / L sucrose + 9 g / L agar + 9 μM 2,4-D + 2.2 μM BA, adjust the pH to 5.8-6.0.

[0057] (4) Somatic embryo induction medium: DCR basal medium + 60 g / L sucrose + 9 g / L agar + 30 mg / L ABA, adjust the pH to 5.8-6.0.

[0058] (5) LB liquid culture medium: Yeast extract (5g / L) + Tryptone (10g / L) + NaCl (10g / L).

[0059] 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.

[0060] Example 1

[0061] 1. Total RNA extraction

[0062] Fresh hybrid larch callus tissue (approximately 0.5 g) was placed in a mortar pre-cooled with liquid nitrogen and ground thoroughly. Liquid nitrogen was added and grinding was repeated until the sample was powdered. A suitable amount of the ground sample powder was quickly placed into a 1.5 mL centrifuge tube containing 750 μL CTAB and shaken thoroughly for 2 min. The mixture was then incubated in a 65°C water bath for 10 min, mixing thoroughly 5 times during this period. After the water bath, the mixture was quickly placed on ice to cool for 2 min, then centrifuged at 4°C and 12000 rpm for 1 min. The supernatant was collected, and pre-cooled 350 μL chloroform and 350 μL water-saturated phenol were added. The mixture was shaken thoroughly and centrifuged at 4°C and 12000 rpm for 1 min. This step was repeated once. The supernatant was then collected, and pre-cooled 700 μL chloroform was added. The mixture was shaken thoroughly and centrifuged at 4°C and 12000 rpm for 1 min. This step was repeated once. The supernatant was gently aspirated and placed in a pre-cooled container containing 300 μL anhydrous ethanol and 300 μL LiCl (8). In a 1.5 mL centrifuge tube (M), vortex to mix and place on ice for 20 min; centrifuge at 4°C, 12000 rpm for 20 min; slowly discard the supernatant, gently wash the RNA with pre-prepared and chilled 75% ethanol, discard the ethanol, and centrifuge again at 4°C, 12000 rpm for 30 s, gently aspirating any residual ethanol with a pipette; dissolve the RNA in 30 μL of 1‰ DEPC water; take 1.5 μL of RNA sample and perform electrophoresis on a 1% agarose gel at 100 V, and determine the concentration using a nucleic acid analyzer. Observe the electrophoretic bands to analyze the RNA quality. After confirming that the quality meets the requirements, quickly freeze in liquid nitrogen at -80°C for later use.

[0063] 2. cDNA Acquisition

[0064] Using extracted total RNA as a template, reverse transcription experiments were performed using an RNA reverse transcription kit (Vazyme) to obtain cDNA. The product was stored at -20°C to minimize the number of freeze-thaw cycles.

[0065] 3. Cloning of the LkHY5 gene

[0066] Using the CDS sequence of the LkHY5 gene (derived from Japanese larch) as a reference sequence, primer sequences for LkHY5 gene cloning were designed. The sequence information is shown below:

[0067] LkHY5-CDS-F (SEQ ID No.3): 5'-ATGCAGGACACTGCTGCCTCC-3',

[0068] LkHY5-CDS-R (SEQ ID No. 4): 5'-TTAGGTTTCTGTTCCTGAGTT-3'.

[0069] Using cDNA from hybrid larch embryogenic callus as a template, a premixed reaction system was prepared according to a total reaction volume of 200 μL high-fidelity enzyme (KOD Fx). All operations were performed on ice, and each component was thoroughly mixed and centrifuged to the bottom of the tube.

[0070] The PCR reaction system consisted of: 34 μL ddH2O, 100 μL 2×PCR Buffer for KOD Fx, 40 μL dNTP (25 μM), 6 μL LkHY5-CDS-F (10 μM), 6 μL LkHY5-CDS-R (10 μM), 4 μL KOD Fx, and 10 μL cDNA.

[0071] The reaction program was as follows: 95℃ for 2 min; 98℃ for 30 s, 56℃ for 30 s, 72℃ for 1 min, for a total of 32 cycles; 68℃ for 2 min; then cooled to 4℃ or immediately placed on ice.

[0072] The PCR amplification products showed clear and bright bands. The PCR amplification products were purified using the EZ-10 column PCR product purification kit (B518141) from Sangon Biotech. The purified PCR products were then ligated into the pCE2 TA / Blunt-Zero Vector intermediate vector.

[0073] The ligation reaction mixture consisted of: 1 μL of 5 × TA / Blunt-Zero Cloning Mix, 3 μL of PCR purified product, and 1 μL of ddH2O. Gently tap the bottom of the tube to mix, then centrifuge briefly at low speed to collect all liquid at the bottom of the centrifuge tube.

[0074] The ligation reaction procedure was as follows: PCR instrument temperature controlled at 25°C for 5 min. After the reaction, the centrifuge tubes were placed on ice.

[0075] The intermediate vector containing the target fragment was transformed into DH5α *E. coli*. The bacterial culture was validated by PCR. Cultures with correct PCR results were sent to the company for sequencing. Successful sequencing yielded the LkHY5 gene CDS target fragment. Plasmids containing the LkHY5 gene CDS target fragment were extracted from the DH5α *E. coli* using the Sangon Biotech DiaSpin column-based plasmid DNA mini-extraction kit (B110091). The prepared intermediate vector plasmid containing the target fragment was stored at -20°C.

[0076] The final cloned CDS sequence of the LkHY5 gene is shown in SEQ ID No. 1, and the amino acid sequence of its expressed protein is shown in SEQ ID No. 2.

[0077] Example 2

[0078] 1. Constructing overexpression vectors

[0079] Homologous arms are added to the target sequence, and the primer sequences for the homologous arms of the vector are shown below:

[0080] LkHY5-pCAMBIA1300-F (SEQ ID No.5):

[0081] 5'-acccggggatcctctagagtcgacATGCAGGACACTGCTGCC-3',

[0082] LkHY5-pCAMBIA1300-R (SEQ ID No. 6):

[0083] 5'-ctcgcccttgctcaccatgtcgacGGTTTCTGTTCCTGAGTT-3';

[0084] The pCAMBIA1300-35S-sGFP vector plasmid was digested with a single enzyme, SalI. The vector digestion product was recovered using a product purification kit.

[0085] The enzyme digestion reaction system was as follows: 5 μl plasmid DNA, 10×LabFD TM Buffer 5 μL, LabFD TM SalI 5 μL, ddH2O 35 μL.

[0086] The enzyme digestion reaction procedure is as follows: 37℃ for 15 min, 80℃ for 20 min; cool to 4℃ or immediately place on ice to cool.

[0087] Homologous recombination was performed on the linearized vector and the target gene fragment recovered from gel excision using a PCR product purification kit.

[0088] The homologous recombination ligation reaction system consisted of: 4 μL of 5×CE II Buffer, 2 μL of Exnase II, 4 μL of linearized vector, 2 μL of target fragment, and 8 μL of ddH2O.

[0089] The homologous recombination ligation reaction procedure is as follows: 37°C for 30 min; then cool to 4°C or immediately place on ice to cool.

[0090] The homologous recombination ligation product was transformed into DH5α *E. coli*, and the bacterial culture was verified by PCR. The correctly sequenced DH5α *E. coli* cultures were then sent to the company for sequencing. Plasmids were extracted from the sequenced DH5α *E. coli* cultures using a bioengineering plasmid mini-prep kit and transformed into *Agrobacterium*. The specific steps are as follows:

[0091] Take competent Agrobacterium cells stored at -80℃ and allow them to partially thaw at room temperature or in your palm. While in an ice-water mixture, insert them into ice. Add 0.01-1 μg of plasmid DNA to every 100 μl of competent cells, gently stir to mix, and incubate sequentially on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37℃ water bath for 5 minutes, and in an ice bath for 5 minutes. Add 700 μl of antibiotic-free LB broth and incubate at 28℃ with shaking for 2-3 hours. Centrifuge at 6000 rpm for one minute to collect the bacteria. Retain approximately 100 μl of supernatant, gently pipette to resuspend the bacterial cells, and spread them onto LB agar plates containing the appropriate antibiotics. Invert the plates and incubate at 28℃ for 2-3 days. Single colonies can then be picked for further experiments.

[0092] 2. Transformation and screening of hybrid larch positive callus

[0093] 1) Preparation of Agrobacterium-containing bacterial solution

[0094] Single colonies of Agrobacterium that have grown on a medium containing kanamycin (Kan) were picked. These colonies were inoculated into 20 ml of liquid LB medium containing Kan (50 mg / L) and incubated at 28°C and 220 rpm for 10–12 h with shaking. The bacterial culture was analyzed using a UV spectrophotometer, maintaining an OD value between 0.6 and 0.8. After confirming the positive result by bacterial PCR, the culture was centrifuged at 4000 rpm for 5 min at room temperature, the supernatant was discarded, and the bacterial cells were collected. The cells were resuspended in a certain volume of DCR liquid medium until the OD value reached 0.3. The infection solution was prepared for later use.

[0095] 2) Infection and co-culture

[0096] Two g of hybrid larch callus, pre-cultured for 10 days, was placed in a 150 mL Erlenmeyer flask. The flask was gently shaken to ensure the callus was fully in contact with the infection liquid, resulting in a flocculent dispersion. The flask was then placed on a shaker at 100 rpm for 30 min for inoculation. Subsequently, the flask was transferred to sterile filter paper, and a Buchner funnel was used to create a vacuum to remove excess bacterial solution.

[0097] 3) Sterilization and screening of positive callus tissue

[0098] Hybrid larch callus, after co-culturing, was washed 3-6 times with DCR liquid medium supplemented with 200 mg / L cephalosporin, each wash lasting 1 minute. After washing, excess liquid was removed using a Buchner funnel until the callus surface was dry. The callus was then transferred to DCR medium supplemented with 200 mg / L cephalosporin and 100 mg / L termethin for further culture. After 7 days, the medium was changed to DCR medium supplemented with 200 mg / L cephalosporin, 100 mg / L termethin, and 4 mg / L hygromycin for selection. The medium was changed every 14 days until yellowish-white callus appeared, at which point the callus was propagated.

[0099] 4) Identification of transgenic positive callus

[0100] DNA was extracted from the selected overexpressing transgenic lines, and positive identification of transgenic callus was performed by PCR, with wild-type hybrid callus (WT) as a control. Figure 1 Subsequently, RNA was extracted from the above-mentioned positive callus lines, reverse transcribed into cDNA, and its expression level was detected by RT-qPCR. The results are as follows: Figure 2 As shown, the expression level of the LkHY5 gene in the transgenic lines was significantly higher than that in the wild-type callus. WT represents wild-type callus, while LkHY5-OE#14 and LkHY5-OE#20 are two independent transgenic callus cell lines.

[0101] 3. Induction of transgenic positive somatic embryos

[0102] After screening and proliferation, the callus tissue was divided into tissue blocks approximately 0.6 cm in diameter and transferred to DCR basal medium for a one-week transitional dark culture to ensure complete removal of residual hormones within the callus cells. Next, the clear portions of newly proliferating callus tissue were selected and inoculated into somatic embryo induction medium. After approximately 6 weeks, the callus surface was largely covered by pale yellow somatic embryos.

[0103] Example 3

[0104] 1. Effect of LkHY5 on the efficiency of somatic embryo induction

[0105] Using transgenic line LkHY5-OE and control group callus tissue as experimental materials, the effect of LkHY5 on somatic embryogenesis was further investigated by inducing somatic embryogenesis. Results are as follows: Figure 3 As shown, overexpression of LkHY5 promotes somatic embryogenesis on a culture medium containing 30 mg / L ABA exogenous hormone. LkHY5-OE#14 and LkHY5-OE#20 are two independent transgenic callus cell lines.

[0106] 2. Effects of LkHY5 on genes related to microtubule dynamics and starch and soluble sugar metabolism

[0107] Using transgenic callus tissue overexpressing LkHY5 as experimental material, RNA was extracted and reversed into cDNA. RT-qPCR was then used to detect the expression of LkGBSS1-1, LkGBSS1-2, LkGBSS1-3, LkSS3, LkSS4-1, LkSS4-2, LkTAN1-1, and LkTAN1-2 genes. The results are as follows: Figure 4 As shown, overexpression of LkHY5 significantly increased the expression levels of LkGBSS1-3 and LkTAN1-1, promoting amylose synthesis and maintaining microtubule stability.

[0108] 3. Effect of LkHY5 on the carbohydrate content of callus tissue

[0109] Using LkHY5-OE and control callus tissues as experimental materials, the starch content kit (G0507F), maltose content kit (G0566F), glucose content kit (G0504F), and soluble sugar content kit (G0501F) of Greens Biotechnology Co., Ltd. were used to determine the starch, maltose, glucose, and soluble sugar contents of the callus tissues, further investigating the effect of LkHY5 on somatic embryogenesis. Results are as follows: Figure 5 As shown, overexpression of LkHY5 increased carbohydrate content.

[0110] 4. Effect of LkHY5 on the stability of callus microtubules

[0111] Using LkHY5-OE and control callus tissues as experimental materials, the microtubules in the callus tissues were fluorescently labeled using the Tubulin-Tracker Red antibody-based microtubule fluorescent probe (YP0054) from UYLandi Biotechnology Co., Ltd. The effect of LkHY5 on somatic embryogenesis was further investigated. Results are as follows: Figure 6 As shown, overexpression of LkHY5 improves microtubule stability.

[0112] Example 4

[0113] 1. Effects of the microtubule inhibitor Oryzalin on callus microtubules

[0114] Using LkHY5-overexpressing transgenic positive callus as experimental material, the callus was cultured for 7 days in DCR medium supplemented with 0 μm and 10 μm microtubule inhibitors, respectively. Microtubules in the callus were fluorescently labeled using the Tubulin-Tracker Red antibody method from UYLandi Biotechnology Co., Ltd. (YP0054). The effect of the microtubule inhibitor Oryzalin on the callus was investigated. Results are as follows: Figure 7 As shown, the microtubule inhibitor Oryzalin disrupted the microtubule stability of LkHY5-OE callus.

[0115] 2. The effect of microtubule stability on the carbohydrate content of callus tissue

[0116] Using transgenic positive callus tissue overexpressing LkHY5 as experimental material, it was cultured for 7 days in DCR medium supplemented with 0 μm and 10 μm microtubule inhibitors, respectively. The effects of microtubule stability on callus carbohydrate content were investigated by measuring the starch, maltose, glucose, and soluble sugar contents of the callus tissue, further exploring the role of LkHY5 in somatic embryogenesis. Results are as follows: Figure 8 As shown, the use of the microtubule inhibitor Oryzalin significantly reduced the sugar content of LkHY5-OE callus.

[0117] 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. Application of LkHY5 gene in regulating hybrid larch somatic embryogenesis, characterized in that, The nucleotide sequence of the LkHY5 gene is shown as SEQ ID No.

1.

2. Use according to claim 1, characterized in that, The overexpression of the LkHY5 gene positively regulates hybrid larch somatic embryogenesis.

3. Use according to claim 2, characterized in that, The overexpression of the LkHY5 gene increases the expression amount of LkGBSS1-3 gene and / or LkTAN1-1 gene.

4. Application of the overexpression of the LkHY5 gene in increasing the content of saccharides in hybrid larch callus.

5. Use according to claim 4, characterized in that, The saccharides include one or more of starch, maltose, soluble sugar and glucose.

6. Application of the overexpression of the LkHY5 gene in increasing the microtubule stability of hybrid larch callus.

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