A hybrid tulip tree LhDof6 gene, expression protein and application thereof

Abstract

The application discloses a hybrid tulip tree LhDof6 gene and an expression protein and application thereof, and relates to the technical field of plant genetic engineering.The hybrid tulip tree LhDof6 gene disclosed by the application has a nucleotide sequence as shown in SEQ ID NO.1, and the amino acid sequence of the expression protein of the hybrid tulip tree LhDof6 gene is as shown in SEQ ID NO.2.After an expression vector of the hybrid tulip tree LhDof6 gene is constructed, the expression vector is transformed into the hybrid tulip tree; and a transgenic hybrid tulip tree plant with obviously improved non-biological stress resistance is obtained through cultivation and screening.The LhDof6 gene disclosed by the application promotes the hypocotyl elongation of the somatic embryo seedling, makes the color of the plant leaves darker green, promotes the synthesis of chlorophyll of the plant leaves, improves the cold resistance of the hybrid tulip tree plant, improves the survival rate of the hybrid tulip tree plant in an extreme environment, improves the anti-freezing capacity of the plant, and reduces the mortality rate of the hybrid tulip tree plant after experiencing freezing damage.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and more particularly relates to a hybrid Liriodendron tulipifera LhDof6 gene, an expression protein thereof and an application thereof. Background Art

[0002] The genus Liriodendron (Liriodendron chinense) belongs to the Magnoliaceae family (magnoliids) and is a relict species from the Tertiary period. Currently, the only extant species in this genus are Liriodendron chinense and Liriodendron tμLipifera. Liriodendron tμLipifera, a sister species, are naturally distributed from the eastern United States to southern Canada, exhibiting a typical East Asian-Eastern North American interspersed distribution. Early studies hybridized Liriodendron chinense with Liriodendron tμLipifera, yielding hybrids with significant heterosis. These hybrids retain the phenotypes of their parents while exhibiting enhanced growth adaptability, leaf morphology similar to that of their parents, and showy, long-lived flowers, making them excellent ornamental trees for landscape gardening, roadside landscaping, and fast-growing timber. Later, studies using somatic embryogenesis achieved large-scale industrial propagation of hybrid tulip trees. However, due to the limitations of their natural geographical distribution, different clones exhibit varying adaptability to stresses such as high temperatures, water stress, and drought, which has hindered the widespread application of hybrid tulip trees. Therefore, it is crucial to explore the resistance of tulip trees to stress.

[0003] Research in recent years has revealed a complex and flexible transcriptional regulatory network in response to cold stress. In response to the key signaling pathways of cold stress, researchers have conducted a lot of research on the transcriptional regulatory network and the role of transcription factors in post-translational regulation. The cold stress signaling pathway is closely related to other signaling pathways (such as signaling pathways related to the circadian clock). This association is crucial for understanding the interactions and balance between them, and a large amount of research data has provided relevant information. Although a large amount of data on the interactions between these signaling pathways has been accumulated, there are still some unsolved mysteries. For example, we still don’t know how plants perceive and transmit cold stress signals. Research in this area is crucial to revealing the mechanisms by which plants perceive environmental changes and regulate responses to cold stress.

[0004] The plant DOF (DNA-binding One Zinc Finger) gene family encodes transcription factors (Dof) that play a key role in plant growth, development, and response to environmental stress. Dof transcription factors contain a DNA-binding domain, called the DOF domain, composed of a Cys2-Cys2 zinc finger structure. This allows the Dof protein to specifically bind to DNA and regulate the expression of downstream genes. The plant Dof gene family plays an important role in a variety of biological processes, including photomorphogenesis, plant hormone signaling, seed development and germination, and flowering. Because members of the Dof gene family exhibit distinct expression patterns in different tissues and developmental stages, they are considered to be important regulatory factors in plant growth and development. Furthermore, some Dof genes are involved in plant responses to environmental stresses, such as drought, salinity, high temperature, and low temperature. Summary of the Invention

[0005] In response to the aforementioned problems in the prior art, the present invention aims to provide a hybrid tulipwood LhDof6 gene. Another technical problem addressed by the present invention is to provide an expression protein of the hybrid tulipwood LhDof6 gene. A further technical problem addressed by the present invention is to provide the use of the hybrid tulipwood LhDof6 gene for constructing new plant germplasm with enhanced resistance to abiotic stress.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] A hybrid Liriodendron chinense LhDof6 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] The amino acid sequence of the expressed protein of the hybrid Liriodendron chinense LhDof6 gene is shown in SEQ ID NO.2.

[0009] A vector, recombinant bacteria or host cell containing the hybrid Liriodendron chinense LhDof6 gene.

[0010] Application of hybrid Liriodendron chinense LhDof6 gene in regulating plant resistance to abiotic stress.

[0011] The regulating the ability of the plant to resist abiotic stress is to improve the tolerance of the plant to low temperature stress.

[0012] The low temperature stress is from -20°C to +4°C.

[0013] The applications include:

[0014] 1) Construction of an expression vector for the hybrid Liriodendron chinense LhDof6 gene;

[0015] 2) transforming the constructed hybrid tulipwood LhDof6 gene expression vector into hybrid tulipwood;

[0016] 3) Cultivate, screen and obtain transgenic hybrid tulipwood plants with significantly improved resistance to abiotic stresses.

[0017] Application of the hybrid Liriodendron chinense LhDof6 gene in regulating the hypocotyl length of plant somatic seedlings.

[0018] Application of the hybrid Liriodendron chinense LhDof6 gene in regulating plant leaf color.

[0019] Application of hybrid Liriodendron tulipifera LhDof6 gene in regulating chlorophyll content in plant leaves.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1) The hybrid tulipwood LhDof6 gene disclosed in the present invention has a nucleotide sequence as shown in SEQ ID NO. 1, and the amino acid sequence of its expressed protein is shown in SEQ ID NO. 2. The present invention constructs an expression vector for the hybrid tulipwood LhDof6 gene; transforms the constructed hybrid tulipwood LhDof6 gene expression vector into hybrid tulipwood; and cultivates, screens, and obtains transgenic hybrid tulipwood plants with significantly improved abiotic stress resistance.

[0022] 2) The LhDof6 gene disclosed in this invention promotes hypocotyl elongation in somatic embryos. Among LhDof6-overexpressing strains, OE-1 and OE-2 showed longer hypocotyl lengths than wild-type and empty transgenic lines. These results suggest that LhDof6 positively regulates hypocotyl elongation.

[0023] 3) The LhDof6 gene disclosed in the present invention makes the color of plant leaves darker green than that of the wild type; the results of chlorophyll content determination show that overexpression of the LhDof6 gene promotes the synthesis of chlorophyll in plant leaves.

[0024] 4) After the plants were cultured at 4°C for 14 days, they were transferred to a normal room temperature (23°C) for 1 day of recovery. The young leaves of the LhDof6-overexpressing strain were almost completely free of wilting, while the wild type and the empty vector showed a weak recovery.

[0025] 5) After cold stress, H2O2 levels increased in both the wild-type and overexpression lines, but the wild-type experienced excessive H2O2 accumulation. Low temperature stress induced an increase in SOD enzyme activity in both the wild-type and overexpression lines, but the SOD enzyme activity in the overexpression line was always greater than that in the wild-type. After low temperature stress, the MDA content increased in both the wild-type and overexpression lines, but the increase in MDA content in the wild-type plants was more dramatic, indicating that overexpression of LhDof6 improved the strain's stress resistance and, to a certain extent, protected the cell membranes of the transgenic plants from low temperature damage. After low temperature stress, the proline content in the overexpression line increased significantly, which was significantly different from the proline content in the wild-type plants. In summary, the LhDof6 gene improves the cold tolerance of hybrid tulip trees and increases the survival rate of hybrid tulip trees in extreme environments.

[0026] 6) After freezing stress at -20°C for 30 minutes and then recovering at room temperature (26°C) for 1 day, both the wild-type and LhDof6-overexpressing strains showed varying degrees of wilting, but the wild-type strain wilted more severely than the overexpressing strain. Seven days after recovery, the wild-type strain experienced a large number of leaf dieback and severe chlorosis, while the LhDof6-overexpressing strains mostly recovered from the wilting state after 7 days, with only slight chlorosis and a smaller number of dieback leaves. Plant death is defined as the death of more than 75% of the total leaves on the plant. In summary, overexpressing LhDof6 improves the plant's freeze tolerance and reduces the mortality of hybrid tulipwood plants after freezing. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a map of the pBI121-35S:LhDof6 overexpression vector;

[0028] Figure 2 The diagrams are for callus tissues overexpressing the LhDof6 gene (A is the diagram for callus tissues during screening; B is the diagram for callus tissues after screening);

[0029] Figure 3 This is a verification diagram of positive callus tissue overexpressing the LhDof6 gene (+ is a positive control; - is a negative control);

[0030] Figure 4 This is the relative expression level of LhDof6 gene in wild type, empty vector and transgenic callus;

[0031] Figure 5 Statistical analysis of hypocotyl lengths of wild-type, empty-loaded, and transgenic somatic embryos (A is the hypocotyl length of transgenic line OE-1; B is the hypocotyl length of transgenic line OE-2; C is the hypocotyl length of wild-type WT; D is the hypocotyl length of empty-loaded EV; E is the statistical analysis of hypocotyl lengths of different lines);

[0032] Figure 6 Figure 1 is a chart of leaf phenotype and chlorophyll content analysis of wild type and transgenic plants (A is a chart of leaf phenotype of transgenic line OE-1; B is a chart of leaf phenotype of transgenic line OE-2; C is a chart of leaf phenotype of wild type WT; D is a chart of leaf chlorophyll content analysis of different lines);

[0033] Figure 7 Figure 2 is a chart of growth state of different lines after 4℃ stress (A is a chart of growth state after 14d of continuous culture in 4℃ environment; B is a chart of growth state after 1d of recovery in 23℃ environment after 14d of continuous culture in 4℃ environment);

[0034] Figure 8 Figure 3 is a chart of physiological index difference of different lines after 4℃ low temperature stress treatment;

[0035] Figure 9 Figure 4 is a chart of growth state of different lines after -20℃ freezing stress treatment (A is a chart of growth state of wild type Liriodendron hybrid after 30min of -20℃ freezing stress treatment; B is a chart of growth state of transgenic plants after 30min of -20℃ freezing stress treatment; C is a chart of growth state of wild type Liriodendron hybrid after 1d of recovery in 26℃ environment after 30min of -20℃ freezing stress treatment; D is a chart of growth state of transgenic plants after 1d of recovery in 26℃ environment after 30min of -20℃ freezing stress treatment; E is a chart of growth state of wild type Liriodendron hybrid after 7d of recovery in 26℃ environment after 30min of -20℃ freezing stress treatment; F is a chart of growth state of transgenic plants after 7d of recovery in 26℃ environment after 30min of -20℃ freezing stress treatment); DETAILED DESCRIPTION

[0036] In order to make the objects, technical solutions and advantages of the present application clearer, the present application will be further described below in combination with specific examples. In the following examples, if no detailed description is given, the technical means used are all conventional means well known to those skilled in the art.

[0037] The Liriodendron hybrid material selected in the present application is planted in the campus of Nanjing Forestry University.

[0038] Example 1

[0039] 1. Total RNA extraction and cDNA acquisition

[0040] Use Total RNA from Liriodendron hybridum was extracted using the Super Total RNA Extraction Kit (Promega Biotechnology Co., Ltd., Beijing). cDNA was obtained by reverse transcription using the HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novozymes Biotechnology Co., Ltd.) using the extracted total RNA as a template.

[0041] 2. CDS sequence amplification of LhDof6 gene in hybrid Liriodendron chinense

[0042] Based on the genome sequence of Liriodendron chinense, primers were designed using snapgene software for PCR amplification. The primer sequences are shown below:

[0043] pLhDof6-F: 5'-GTCGAACTCGACCATATACGACCAAGAC-3',

[0044] pLhDof6-R: 5'-CTTCCCGTTTCGGATTTTTCGGGAAGAAGAA-3'.

[0045] PCR reaction system: 2×Phanta Max Buffer 25μL; dNTP Mix (10mM each) 1μL; cDNA template 2μL; LhDof6-F (10μM) 2μL; LhDof6-R (10μM) 2μL; Phanta Max Super-Fidelity DNA Polymerase 1μL; make up to 50μL with sterile deionized water.

[0046] PCR amplification program: 95°C for 5 min; 95°C for 30 s, 57°C for 30 s, 72°C for 30-60 s / kb, 35 cycles; 72°C for 5 min; 4°C∞.

[0047] The CDS sequence of the LhDof6 gene was obtained by PCR, and the nucleotide sequence of the hybrid Liriodendron chinense LhDof6 gene was finally obtained by sequencing as shown in SEQ ID NO.1, and the amino acid sequence of the expressed protein was shown in SEQ ID NO.2.

[0048] Example 2

[0049] 1. Construction of hybrid Liriodendron chinense LhDof6 gene overexpression vector

[0050] According to the CDS sequence of the LhDof6 gene and the restriction enzyme cutting site of the overexpression vector pBI121-p35S:GFP (the restriction enzyme cutting site is SalⅠ), the amplification primers were designed: 35s-Dof6-F: 5'-gagagaacacgggggactATGCCAGAGACCAGAGACCCTGCG-3', 35s-Dof6-R: 5'-gatcggggaaattcgagctTTACGTGCTCTCCTGGAAGTTAAGTGACCGTGAC-3', and the target fragment of the LhDof6 gene was amplified by PCR.

[0051] The pBI121-p35S:GFP vector was digested with Sal I. The digestion reaction system was: 2 μL Cutsmart Buffer, 1 μL Sal I, 10 μL plasmid, and 6 μL ddH2O. The digestion conditions were: 37°C for 1 hour, 65°C for 30 minutes, and 16°C for ∞.

[0052] The target gene fragment was recombined and ligated with the linearized vector fragment using the ClonExpress II One Step Cloning Kit (Nanjing Novozymes Biotechnology Co., Ltd.).

[0053] The ligation reaction system is: 5×CE II Buffer 4μL, II 2μL, linearized vector XμL, target fragment YμL, ddH2O up to 20μL.

[0054] The ligation reaction conditions were: 30°C for 30 min, 4°C for ∞.

[0055] Take out the competent state from the ultra-low temperature refrigerator and place it on ice. When the competent state is in a semi-thawed state, add 10 μL of the ligation product to the centrifuge tube, mix gently, and place it on ice for 30 minutes; place the centrifuge tube in a 42°C water bath for heat shock for 90 seconds, and then quickly place it on ice to cool for 3 minutes; add 900 μL of LB medium without antibiotics to the centrifuge tube, and place it on a shaker at 37°C and 150 rpm to recover for 45 minutes; use a centrifuge at 4000 rpm, centrifuge for 2 minutes, and discard the supernatant; use a pipette to mix the remaining bacterial liquid, take 100 μL of the bacterial liquid and evenly spread it on the LB solid medium containing Amp antibiotics, turn the culture dish upside down and place it in a 37°C oven. After 14 hours, use a white pipette tip to pick up the single clone and place it in a 1.5 mL centrifuge tube. Add 800 μL of LB medium containing Amp resistance to each centrifuge tube, place it on a shaker at 220 rpm, and amplify for 6 hours. Single colonies were verified by colony PCR, and positive colonies were selected for expansion culture. Plasmids were extracted and digested to verify the correctness and then sequenced. The correct sequence was the pBI121-35S:LhDof6 overexpression vector ( Figure 1 ).

[0056] 2. Agrobacterium infection of Arabidopsis

[0057] The correctly sequenced plasmid was transformed into Agrobacterium tumefaciens EAH105. The specific steps are as follows:

[0058] Remove the Agrobacterium from the ultra-low temperature freezer and place on ice to thaw. Add 10 μL of plasmid to each tube of Agrobacterium, mix thoroughly by inversion, and place on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and on ice for 5 minutes. Add 900 μL of antibiotic-free LB medium and incubate at 28°C at 200 rpm on a shaker for 2-3 hours. Centrifuge at 4000 rpm for 2 minutes to collect the culture. Discard the supernatant, pipette to mix thoroughly, and spread the remaining culture evenly on LB solid medium containing Kan. Incubate the culture dish upside down at 28°C in an incubator for 48 hours. Pick a single colony and transfer it to a 1.5 mL centrifuge tube. Add 700 μL of LB medium containing Kan to each tube and incubate at 28°C at 220 rpm on a shaker for 6 hours. Verify the culture by PCR. Select PCR products with the correct target band size for sequencing. Once the culture is verified to be correct, store it for future use.

[0059] The embryonic callus of hybrid Liriodendron chinense was infected by Agrobacterium-mediated method, and the specific steps were as follows:

[0060] In a 10mL centrifuge tube containing Kan-resistant LB medium, add 20μL of the correct bacterial solution after sequencing. Place it on a shaker at 28℃, 220rpm for overnight culture; transfer the cultured bacterial solution to a 250mL Erlenmeyer flask, add 50mL of LB medium containing Kan resistance, and culture it on a shaker at 28℃, 220rpm for several hours; measure the OD600 value of the bacterial solution, stop shaking the bacteria when the OD600 value is between 0.6 and 1.0, place it in a 50mL centrifuge tube, centrifuge at 12000rpm, 4℃, for 10 minutes, and remove the supernatant; resuspend the bacterial solution with M13 liquid culture medium; take 3 dishes of callus tissue and place them in a 100mL Erlenmeyer flask, add the resuspended bacterial solution, and gently shake for infection for 10 minutes; infection results After the bundle is bundled, the callus tissue is collected with a sterilized cell sieve and the excess bacterial liquid is absorbed with sterile filter paper; the callus tissue is then transferred to M13 solid medium to co-cultivate the callus tissue with Agrobacterium; the co-cultivated callus tissue is placed in a 100 mL triangular flask and washed and sterilized with sterile water and M13 liquid medium alternately. After sterilization, the callus tissue is collected with a cell sieve, the callus tissue is absorbed dry with sterile filter paper, and then placed on M13 medium containing Cef for recovery culture; the recovered callus is transferred to M13 solid medium supplemented with antibiotics for screening and culture until fresh, delicate, milky white new callus tissue grows ( Figure 2 ).

[0061] 3. Screening and identification of positive callus

[0062] The DNA of the new, delicate callus regrown on the screening medium was extracted by CTAB method, and PCR amplification was performed using 2× Rapid Taq Master Mix (Nanjing Novozymes Biotech Co., Ltd.). The amplified target bands were sent to the company for sequencing to obtain transgenic positive callus ( Figure 3 ).

[0063] 4. Obtaining regenerated plants through somatic embryogenesis

[0064] Wild-type (WT), transgenic empty-cell, and six transgenic-positive calli were placed in a 250mL Erlenmeyer flask and 50mL of M13 liquid medium was added. The positive transgenic calli were liquid-cultured to obtain single-cell suspension lines. After one week, the calli were subcultured with M13 liquid medium. After two weeks of liquid culture, sufficient single cells were obtained. The liquid-cultured single cells were filtered through 150-mesh and 400-mesh cell sieves. The single cells on the 400-mesh sieve were collected and placed in a new 250mL Erlenmeyer flask, and 50mL of Z14 hypertonic transition culture medium was added. After one day of transition culture, 1mL of cells was aspirated and counted under a microscope to calculate the cell density. The WT, empty-cell, and transgenic suspension cell lines were diluted to the appropriate concentration. Using a 1mL pipette, the suspension cells of each line were evenly plated onto Z36 somatic embryogenesis solid medium lined with filter paper, ensuring a consistent number of cells per dish. Finally, the suspension cells were cultured in a constant-temperature dark incubator to obtain regenerated plants.

[0065] 5. Relative expression of the LhDof6 gene in transgenic plants

[0066] use RNA was extracted from leaf tissue of wild-type, untransgenic, and LhDof6-OE transgenic plants using the Super Total RNA Extraction Kit (Promega Biotechnology Co., Ltd., Beijing). Reverse transcription was performed using the HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Novozymes Biotechnology Co., Ltd.) using the extracted total RNA as a template to generate cDNA. qRT-PCR was used to examine the expression of the LhDof6 gene in the different lines. The primer sequences for quantitative PCR are shown below:

[0067] qLhDof6-F: 5'-GAACAAGAACTCCGCCTCACA-3',

[0068] qLhDof6-R: 5'-AAGACAGATGCCATGATTC-3';

[0069] 18S-F: 5'-ATTTCTGCCCTATCAACTTTCG-3',

[0070] 18S-R: 5'-ATTTATTGTCACTACCTCCCCG-3'.

[0071] The results are as follows Figure 4As shown in the figure, compared with the control, the expression level of the LhDof6 gene in the transgenic lines was significantly increased. Among the transgenic lines, the expression level of the LhDof6 gene was ranked from high to low as follows: OE-1, OE-2, and OE-3; therefore, the OE-1 and OE-2 lines were selected for subsequent functional analysis.

[0072] Example 3

[0073] 1. Effects of overexpression of the LhDof6 gene on the phenotype and growth traits of transgenic plants

[0074] 1) Overexpression-positive transgenic calli, empty transgenic calli, and wild-type calli were suspended in M13 liquid medium. Single cells were then screened and transitioned to Z36 transition medium. One day later, these cells were plated onto Z14 somatic embryo induction medium. Before plating, the number of cells per 10 μL of transgenic line was counted and the cell density was calculated to ensure a consistent initial single cell count per dish. The cells were then placed in a constant-temperature incubator for one month in the dark and two weeks in the light. Hypocotyl length of mature somatic embryos was then measured.

[0075] The results are as follows Figure 5 As shown, overexpression of LhDof6 promoted hypocotyl elongation in somatic seedlings. Among the LhDof6-overexpressing lines, OE-1 and OE-2 had longer hypocotyl lengths than the wild-type and empty transgenic lines. These results indicate that LhDof6 positively regulates hypocotyl elongation.

[0076] 2) The mature somatic seedlings are further cultured into plantlets, which are then transferred to a seedling selection medium for cultivation. The phenotypes of the plantlets are observed, and the chlorophyll content of the leaves is determined.

[0077] The results are as follows Figure 6 As shown, the leaf color of the overexpression line is darker green than that of the wild type ( Figure 6 AC); The results of chlorophyll content determination showed that overexpression of the LhDof6 gene promoted the synthesis of chlorophyll in plant leaves.

[0078] 2. Effect of overexpression of the LhDof6 gene on the cold tolerance of transgenic plants

[0079] Three-month-old hybrid tulipwood, wild-type hybrid tulipwood and empty transgenic lines with consistent and healthy growth were selected and cultured in a Climacell climate incubator for 14 days. The temperature was set to 4°C (simulating the low temperature stress suffered by plants), the relative humidity was 70%-80%, the light time was 16 hours, and the dark time was 8 hours.

[0080] The results are as follows Figure 7As shown in the results, after 14 days of continuous cultivation at 4°C, all strains almost stopped growing and their leaves wilted to varying degrees. All strains were then transferred to a normal room temperature (23°C) for 1 day of recovery. The young leaves of the LhDof6-overexpressing strain almost completely recovered from the wilting state, while the wild-type and empty-carrier strains showed a slight recovery.

[0081] After treatment at 4°C, plant leaves were washed with distilled water, dried with a paper towel, weighed, and quickly frozen in liquid nitrogen at -80°C until use. The frozen samples were ground into a powder. 0.1 g of plant tissue powder was homogenized with 9 volumes of PBS to obtain a 10% plant tissue homogenate. The 10% plant tissue homogenate was centrifuged at 4000 rpm / min for 10-15 minutes, and the supernatant was collected for physiological parameter measurements.

[0082] The results are as follows Figure 8 As shown, after cold stress, H2O2 levels increased in both wild-type and overexpressing lines, but the wild-type experienced excessive H2O2 accumulation. Low temperature stress induced an increase in SOD activity in both wild-type and overexpressing lines, but SOD activity in the overexpressing line was consistently higher than that in the wild-type. MDA content increased in both wild-type and overexpressing lines after cold stress, but the increase was more dramatic in the wild-type, suggesting that overexpression of LhDof6 enhanced the strain's stress tolerance and, to some extent, protected the cell membranes of the transgenic plants from cold damage. After cold stress, proline content in the overexpressing lines increased significantly, differing significantly from that in the wild-type plants.

[0083] In summary, the LhDof6 gene improves the cold tolerance of hybrid tulip trees and increases the survival rate of hybrid tulip trees in extreme environments.

[0084] 3. Effect of overexpression of the LhDof6 gene on the frost resistance of transgenic plants

[0085] Six-month-old hybrid tulipwood plants, wild-type hybrid tulipwood plants, and untransgenic lines with consistent growth and health were selected and placed in a -20°C environment for 30 minutes to induce freezing stress. The plants were then transferred to room temperature (26°C) for recovery under a 16-hour light cycle and an 8-hour dark cycle. The plants were observed 1 and 7 days after recovery.

[0086] The results are as follows Figure 9As shown, after one day of recovery from stress, both the wild-type and LhDof6-overexpressing lines showed varying degrees of wilting, but the wild-type showed greater wilting than the overexpressing lines. Seven days after recovery, the wild-type experienced a large number of leaf dieback and severe chlorosis, while the LhDof6-overexpressing lines had mostly resolved the wilting state after seven days, with only slight chlorosis and a smaller number of dieback leaves. Plant death is defined as the death of more than 75% of the total leaves on the plant. In summary, overexpressing LhDof6 improves plant tolerance to freezing and reduces mortality in hybrid tulipwood plants after freezing.

[0087] The above description is only illustrative of the present invention and not restrictive. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all of them will fall within the scope of protection of the present invention.

Claims

1. Overexpression of hybrid Liriodendron chinense LhDof6 Application of a gene in promoting the elongation of the hypocotyl of hybrid tulipwood somatic embryo seedlings, the hybrid tulipwood LhDof6 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. Overexpression of hybrid Liriodendron chinense LhDof6 Application of genes in promoting the darkening of leaf color of hybrid tulipwood, the hybrid tulipwood LhDof6 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. Overexpression of hybrid Liriodendron chinense LhDof6 Application of genes in promoting the increase of chlorophyll content in leaves of hybrid tulipwood, the hybrid tulipwood LhDof6 The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

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