Liriodendron hybrids LhDof6 gene as well as expression protein and application thereof

Abstract

The invention discloses a liriodendron hybrids LhDof6 gene as well as an expression protein and application thereof, and relates to the technical field of plant genetic engineering. The nucleotide sequence of the Liriodendron hybrids LhDof6 gene disclosed by the invention is shown as SEQ ID NO.1, and the amino acid sequence of the expression protein of the Liriodendron hybrids LhDof6 gene is shown as SEQ ID NO.2. After an expression vector of the hybrid liriodendron LhDof6 gene is constructed, the expression vector is transformed into the hybrid liriodendron; and cultivating and screening to obtain the transgenic hybrid liriodendron plants with obviously improved abiotic stress resistance. The LhDof6 gene disclosed by the invention promotes the extension of hypocotyl of somatic embryo seedlings, so that plant leaves are darker green, and the synthesis of chlorophyll of the plant leaves is promoted; the cold resistance of the hybrid liriodendron plants is improved, and the survival rate of the hybrid liriodendron in an extreme environment is improved; the anti-freezing capability of the plants is improved, and the death rate of the hybrid liriodendron plants after freezing injury is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and more specifically relates to a hybrid tulipwood LhDof6 gene and its expression protein and application. Background Art

[0002] Liriodendron belongs to the Magnoliaceae family of magnoliids and is a relict species of the Tertiary period. Currently, there are only two sister species of this genus, Liriodendron chinense and Liriodendron tμLipifera. Liriodendron chinense is naturally distributed from the eastern United States to southern Canada, showing a typical inter-regional discontinuous distribution from East Asia to Eastern North America. The earliest research hybridized Liriodendron chinense with Liriodendron tμLipifera to obtain a hybrid Liriodendron with significant hybrid vigor. While retaining the phenotype of the parents, it also shows stronger growth adaptability. The leaf shape has both the phenotype of the parents, and the flowers are bright and have a long flowering period. It is an excellent garden ornamental, road greening and fast-growing timber species. Later, some studies used somatic embryogenesis technology to achieve large-scale industrial reproduction of hybrid tulip trees. However, due to the limitations of the natural geographical distribution range, different clones have different adaptability to adversities such as high temperature, water stress, and drought, which has brought certain difficulties to the large-scale promotion and application of hybrid tulip trees. Therefore, it is very important to explore the stress resistance of tulip trees under 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 those related to the circadian clock). This association is crucial to 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 DOF (DNA-binding One Zinc Finger) gene family of plants is a family of genes encoding Dof transcription factors, which 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, which consists of a Cys2-Cys2 type zinc finger structure, which enables Dof protein to specifically bind to DNA and regulate the expression of downstream genes. The Dof gene family of plants plays an important role in a variety of biological processes, including photomorphogenesis, plant hormone signaling pathways, seed development and germination, flowering, etc. Since members of the Dof gene family have different expression patterns in different tissues and developmental stages, they are considered to be one of the important regulatory factors for regulating plant growth and development. In addition, some Dof genes are also involved in the response of plants to environmental stresses, such as stress responses under adverse conditions such as drought, salinity, high temperature and low temperature. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a hybrid tulipwood LhDof6 gene. Another technical problem to be solved by the present invention is to provide an expression protein of the hybrid tulipwood LhDof6 gene. Another technical problem to be solved by the present invention is to provide an application of the hybrid tulipwood LhDof6 gene for constructing a new plant germplasm with improved resistance to abiotic stress.

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

[0007] A hybrid Liriodendron chinense LhDof6 gene, the nucleotide sequence of which is shown as 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 the ability of plants to resist 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 expression vector of hybrid tulipwood LhDof6 gene into hybrid tulipwood;

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

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

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

[0019] Application of hybrid Liriodendron chinense 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 an amino acid sequence of its expressed protein as shown in SEQ ID NO.2. The present invention constructs an expression vector of the hybrid tulipwood LhDof6 gene; transforms the constructed hybrid tulipwood LhDof6 gene expression vector into hybrid tulipwood; cultivates, screens, and obtains transgenic hybrid tulipwood plants with significantly improved resistance to abiotic stress.

[0022] 2) The LhDof6 gene disclosed in the present invention promotes the elongation of the hypocotyl of somatic seedlings. In the LhDof6 gene overexpression strains, the hypocotyl lengths of the OE-1 strain and the OE-2 strain were longer than those of the wild type and the empty transgenic line. The results showed that the LhDof6 gene positively regulates the elongation of the hypocotyl.

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

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

[0025] 5) After cold stress, the H2O2 levels in both the wild type and overexpression lines increased, but the wild type had excessive accumulation of H2O2. 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 in both the wild type and overexpression lines increased, but the MDA content in the wild type plants increased more dramatically, indicating that overexpression of LhDof6 improved the stress resistance of the lines and protected the cell membrane of the transgenic plants from low temperature damage to a certain extent. After low temperature stress, the proline content in the overexpression lines 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 improves the survival rate of hybrid tulip trees in extreme environments.

[0026] 6) After the plants were placed in a -20℃ environment for 30 minutes to undergo freezing stress, they were transferred to room temperature (26℃) to recover for 1 day. Both the wild type and the LhDof6 overexpression lines showed varying degrees of wilting, but the degree of wilting in the wild type was greater than that in the overexpression lines. After 7 days of recovery, a large number of leaves in the wild type line died, and the leaves were severely chlorotic, while most of the LhDof6 overexpression lines were freed from the wilting state after 7 days, and the leaves were slightly chlorotic, and the number of dead leaves was small. Plant death is defined as the number of dead leaves of the whole plant exceeding 75% of the total leaves. In summary, overexpression of LhDof6 improves the frost resistance of the plant and reduces the mortality rate of hybrid tulipwood plants after freezing damage. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0028] Figure 2 The diagram is a callus diagram overexpressing the LhDof6 gene (A is a diagram of the callus during screening; B is a diagram of the callus after screening);

[0029] Figure 3 This is a validation 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 embryo seedlings (A is a hypocotyl length diagram of transgenic line OE-1; B is a hypocotyl length diagram of transgenic line OE-2; C is a hypocotyl length diagram of wild-type WT; D is a hypocotyl length diagram of empty-loaded EV; E is a statistical analysis diagram of hypocotyl lengths of different lines);

[0032] Figure 6 Leaf phenotypes and chlorophyll content analysis diagrams of wild-type and transgenic plants (A is the leaf phenotype diagram of transgenic strain OE-1; B is the leaf phenotype diagram of transgenic strain OE-2; C is the leaf phenotype diagram of wild-type WT; D is the leaf chlorophyll content analysis diagram of different strains);

[0033] Figure 7 The growth status diagrams of different strains after 4℃ stress (A is the growth status diagram after continuous cultivation in 4℃ environment for 14 days; B is the growth status diagram after continuous cultivation in 4℃ environment for 14 days and then transferred to 23℃ for recovery for 1 day);

[0034] Figure 8 This is a diagram showing the differences in physiological indicators of different strains after 4°C low temperature stress treatment;

[0035] Fig. 9 Figure 2 is a growth status diagram of different strains after -20℃ freezing stress treatment (A is a growth status diagram of wild-type hybrid tulipwood after -20℃ freezing stress treatment for 30 minutes; B is a growth status diagram of transgenic plants after -20℃ freezing stress treatment for 30 minutes; C is a growth status diagram of wild-type hybrid tulipwood after being transferred to a 26℃ environment for recovery for 1 day after -20℃ freezing stress treatment for 30 minutes; D is a growth status diagram of transgenic plants after being transferred to a 26℃ environment for recovery for 1 day after -20℃ freezing stress treatment for 30 minutes; E is a growth status diagram of wild-type hybrid tulipwood after being transferred to a 26℃ environment for recovery for 7 days after -20℃ freezing stress treatment for 30 minutes; F is a growth status diagram of transgenic plants after being transferred to a 26℃ environment for recovery for 7 days after -20℃ freezing stress treatment for 30 minutes). DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0037] The hybrid tulip tree material selected for this application was planted on the campus of Nanjing Forestry University.

[0038] Example 1

[0039] 1. Total RNA extraction and cDNA acquisition

[0040] use The total RNA of hybrid Liriodendron chinense was extracted using Super Total RNA Extraction Kit (Promega (Beijing) Biotechnology Co., Ltd.). The extracted total RNA was used as a template to perform reverse transcription experiments using the reverse transcription reagent HiScriptⅢ1st Strand cDNA Synthesis Kit (Nanjing Novogene Biotechnology Co., Ltd.) to obtain cDNA.

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

[0042] According to the genome sequence of Liriodendron chinense, primers were designed using snapgene software for PCR amplification. The primer sequences are as follows:

[0043] pLhDof6-F: 5'-GTCGAACTCGACCATATACGACCAAAGAC-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 site of the overexpression vector pBI121-p35S:GFP (the restriction 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 SalⅠ, and the digestion reaction system was: Cutsmart Buffer 2μL, SalⅠ 1μL, plasmid 10μL, ddH2O 6μL. The digestion reaction conditions were: 37℃1h, 65℃30min, 16℃∞.

[0052] The target gene fragment was recombined and ligated with the linearized vector fragment using ClonExpress II One Step Cloning Kit (Nanjing Novogene Biotech 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 infinity.

[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 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 antibiotic-free LB culture medium to the centrifuge tube, and place it on a shaker at 37°C and 150rpm for recovery for 45 minutes; use a centrifuge at 4000rpm, centrifuge for 2 minutes, and discard the supernatant; use a pipette to mix the remaining bacterial solution, take 100 μL of the bacterial solution and evenly apply it on the LB solid culture medium containing Amp antibiotics, put the culture dish upside down in a 37°C oven, and pick up the single clone with a white pipette tip after 14 hours and place it in a 1.5mL centrifuge tube. In each centrifuge tube, add 800 μL of LB culture medium containing Amp resistance, place it on a shaker at 220rpm, and amplify for 6 hours. Single colonies were verified by colony PCR, positive colonies were selected for expansion culture, plasmids were extracted and digested correctly, 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 EAH105. The specific steps are as follows:

[0058] Take out the Agrobacterium stored in the ultra-low temperature refrigerator and place it on ice to melt; add 10 μL of plasmid to each tube of Agrobacterium, invert and mix, 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 LB medium without antibiotics, place it on a shaker at 28°C and 200rpm for 2 to 3 hours; centrifuge at 4000rpm for 2 minutes to collect the bacterial solution; discard the supernatant, mix it by pipetting, and evenly apply the remaining bacterial solution on the LB solid medium containing Kan, and invert the culture dish in a 28°C incubator for 48 hours; pick a single colony and transfer it to a 1.5mL centrifuge tube, add 700 μL of LB medium containing Kan to each tube, and culture it on a shaker at 28°C and 220rpm for 6 hours, and then perform bacterial solution PCR verification. Select the PCR product with the correct size of the target band for sequencing, and save the correct bacterial solution after sequencing verification for future use.

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

[0060] In a 10mL centrifuge tube containing LB medium with Kan resistance, add 20μL of the correct bacterial solution after sequencing. Place it in 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 in 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 10min, 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 10min; infection results After the callus is collected with a sterilized cell sieve, and the excess bacterial liquid is absorbed with sterile filter paper; the callus is then transferred to M13 solid medium to co-culture the callus with Agrobacterium; the co-cultured callus is placed in a 100 mL conical flask, and is washed and sterilized with sterile water and M13 liquid medium alternately. After sterilization, the callus is collected with a cell sieve, and the callus is dried 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 and delicate milky white new callus grows ( Figure 2 ).

[0061] 3. Screening and identification of positive callus

[0062] The DNA of the new, delicate callus tissue regrown on the screening medium was extracted by the CTAB method, and PCR amplification was performed using 2×Rapid Taq Master Mix (Nanjing Novozyme Biotechnology 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] The wild type (WT), transgenic empty and 6 transgenic positive callus tissues were placed in a 250mL conical flask, 50mL M13 liquid medium was added, and the positive transgenic callus was liquid cultured to obtain a single cell suspension system; after 1 week, the M13 liquid medium was used for subculture; after two weeks of liquid culture, a sufficient amount of single cells were obtained, and the single cells of liquid culture were filtered with 150 mesh and 400 mesh cell sieves, and the single cells on the 400 mesh sieve were collected in a new 250mL conical flask, and 50mL Z14 hypertonic transition culture medium was added; after 1 day of transition culture, 1mL of cells were aspirated to count the number of cells under a microscope and calculate the cell density. The WT, empty and transgenic suspension cells were diluted to the appropriate concentration. Each suspension line was aspirated with a 1mL pipette and evenly spread on the Z36 somatic embryogenesis solid medium padded with filter paper to ensure that the number of cells on each dish of culture medium was consistent; finally, it was placed in a constant temperature dark incubator for culture to obtain regenerated plants.

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

[0066] use The Super total RNA extraction kit (Promega (Beijing) Biotechnology Co., Ltd.) was used to extract RNA from leaf tissues of wild-type, transgenic empty, and positive LhDof6-OE transgenic plants. The extracted total RNA was used as a template and reverse transcription was performed using the reverse transcription reagent HiScriptⅢ1st Strand cDNA Synthesis Kit (Nanjing Novogene Biotechnology Co., Ltd.) to obtain cDNA. The expression level of the LhDof6 gene in different strains was detected by qRT-PCR. The primer sequences of fluorescent quantitative PCR are as follows:

[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, compared with the control, the expression of LhDof6 gene in transgenic lines increased significantly. In transgenic lines, the expression of LhDof6 gene was from high to low in the order of OE-1, OE-2, and OE-3; therefore, OE-1 and OE-2 lines were selected for subsequent functional analysis.

[0072] Example 3

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

[0074] 1) The obtained overexpressed transgenic positive callus, empty transgenic positive callus and wild-type callus were suspended in M13 liquid medium, and then single cells were obtained by sieving, and transition cultured with Z36 transition medium. After one day, they were plated on Z14 somatic embryo induction medium. Before plating, the number of cells in each 10 μL transgenic line was counted, the cell density was calculated, and the initial number of single cells on each dish of culture medium was controlled to be consistent. Then, the hypocotyl length of mature somatic embryo seedlings was measured after dark culture in a constant temperature incubator for one month and light culture for two weeks.

[0075] The results are as follows Figure 5 As shown, overexpression of the LhDof6 gene promoted the elongation of the hypocotyls of somatic seedlings. In the LhDof6 gene overexpression lines, the hypocotyl lengths of the OE-1 and OE-2 lines were longer than those of the wild type and the empty transgenic lines. The results showed that the LhDof6 gene positively regulated the elongation of the hypocotyls.

[0076] 2) The mature somatic embryo seedlings are further cultured into plantlets, transferred to seedling selection medium for cultivation, and their phenotypes 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 LhDof6 gene promoted the synthesis of chlorophyll in plant leaves.

[0078] 2. Effect of overexpression of LhDof6 gene on 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 on the plants), the relative humidity was 70%-80%, the light time was 16h, and the dark time was 8h.

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

[0081] The plant leaves treated at 4°C were washed with distilled water, then dried with a paper towel, weighed, and quickly frozen with liquid nitrogen and stored at -80°C for later use. The frozen samples were ground into powder, 0.1 g of plant tissue powder was taken, and the tissue was homogenized with 9 times the volume of PBS solution to obtain a 10% plant tissue homogenate; the 10% plant tissue homogenate was centrifuged at 4000 rpm / min for 10 to 15 minutes, and the supernatant was taken for physiological index determination.

[0082] The results are as follows Figure 8 As shown in the figure, after cold stress, the H2O2 levels in both the wild type and overexpression lines increased, but the wild type had excessive accumulation of H2O2. Low temperature stress induced an increase in SOD enzyme activity in both the wild type and overexpression lines, but the SOD enzyme activity of the overexpression line was always greater than that of the wild type. After low temperature stress, the MDA content in both the wild type and overexpression lines increased, but the MDA content in the wild type plant increased more dramatically, indicating that overexpression of LhDof6 improved the stress resistance of the line and protected the cell membrane of the transgenic plant from low temperature damage to a certain extent. 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 plant.

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

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

[0085] Six-month-old hybrid tulip trees, wild hybrid tulip trees and empty transgenic lines with consistent and healthy growth were selected, placed in a -20°C environment for 30 minutes for freezing stress, and then transferred to room temperature (26°C) for recovery culture, with a light time of 16 hours and a dark time of 8 hours. The plant conditions were observed 1 day and 7 days after recovery.

[0086] The results are as follows Fig. 9As shown in the figure, after 1 day of recovery from stress, both the wild type and the overexpression LhDof6 lines showed different degrees of wilting, but the degree of wilting of the wild type was greater than that of the overexpression line; after 7 days of recovery, a large number of leaves of the wild type line died, and the leaves were seriously chlorotic, while most of the overexpression LhDof6 lines were freed from the wilting state after 7 days, and the leaves were slightly chlorotic, and the number of dead leaves was small. Plant death is defined as the number of dead leaves of the whole plant exceeding 75% of the total leaves. In summary, overexpression of LhDof6 improves the frost resistance of the plant and reduces the mortality rate of hybrid tulipwood plants after frost damage.

[0087] The above description is only illustrative rather than restrictive of the present invention. 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 will fall within the scope of protection of the present invention.

Claims

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

1.

2. The expressed protein of the hybrid Liriodendron chinense LhDof6 gene according to claim 1, whose amino acid sequence is shown in SEQ ID NO.

2.

3. A vector, recombinant bacteria or host cell containing the hybrid Liriodendron chinense LhDof6 gene according to claim 1.

4. Use of the hybrid Liriodendron chinense LhDof6 gene according to claim 1 in regulating the ability of plants to resist abiotic stress.

5. The use according to claim 4, characterized in that: The regulating the ability of the plant to resist abiotic stress is to improve the tolerance of the plant to low temperature stress.

6. The use according to claim 5, characterized in that: The low temperature stress is from -20°C to +4°C.

7. The use according to claim 4, characterized in that: include: 1) Construction of an expression vector for the hybrid Liriodendron chinense LhDof6 gene; 2) transforming the constructed expression vector of hybrid tulipwood LhDof6 gene into hybrid tulipwood; 3) Cultivate, screen and obtain transgenic hybrid tulipwood plants with significantly improved resistance to abiotic stresses.

8. Use of the hybrid Liriodendron chinense LhDof6 gene according to claim 1 in regulating the length of the hypocotyl of plant embryo seedlings.

9. Use of the hybrid Liriodendron chinense LhDof6 gene of claim 1 in regulating plant leaf color.

10. Use of the hybrid Liriodendron chinense LhDof6 gene of claim 1 in regulating the chlorophyll content of plant leaves.

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