Hybrid liriodendron tulipifera lhcold1 gene, expression protein and application thereof

Abstract

This invention discloses the LhCOLD1 gene of hybrid tulip tree, its expressed protein, and its applications, belonging to the field of plant genetic engineering technology. The nucleotide sequence of the LhCOLD1 gene disclosed in this invention is shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.2. This invention constructs an overexpression vector of the LhCOLD1 gene of hybrid tulip tree and transforms it into callus tissues of Arabidopsis thaliana and hybrid tulip tree; transgenic lines with enhanced resistance to low-temperature stress are cultivated, screened, and obtained. After being treated at -10℃ for 3 hours and then allowed to recover at room temperature for two weeks, the growth of LhCOLD1 transgenic Arabidopsis thaliana plants was less affected by low-temperature stress; the growth of LhCOLD1 transgenic hybrid tulip tree lines was less affected by low-temperature stress after being treated at -10℃ for 1.5 hours. The results indicate that the LhCOLD1 gene enhances the plant's tolerance to low-temperature stress.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to the LhCOLD1 gene of hybrid tulip tree, its expressed protein, and its applications. Background Technology

[0002] The tulip tree (Liriodendron chinense) is an ancient relict plant belonging to the genus Liriodendron in the family Magnoliaceae, mainly distributed in areas south of the Yangtze River in my country. my country began introducing North American tulip trees in the 1930s, and in 1963 successfully obtained a hybrid variety of tulip tree and North American tulip tree, known as the hybrid tulip tree. The hybrid tulip tree is characterized by rapid growth, upright trunk, and vibrant flower colors, and is commonly used for landscaping and afforestation in mountainous areas.

[0003] Hybrid tulip trees are frequently affected by low-temperature damage in northern my country, severely limiting their regional introduction and promotion. Low temperatures during plant growth and development primarily inhibit normal metabolic processes, leading to slow growth or even death. Research has found that some low-temperature response genes in plants play important roles in enhancing their cold tolerance, such as ICE1 and CBF. The COLD1 gene, reported as a type of cold receptor, was first discovered and named in rice. COLD1 encodes a GPCR-type G protein (GTG) with nine transmembrane domains, located on the plasma membrane and endoplasmic reticulum membrane. It plays a role in regulating plant growth and development, and stress resistance in crops such as rice, wheat, and maize.

[0004] In summary, the regulation of cold tolerance by the LhCOLD1 gene in hybrid tulip trees has not yet been studied. Cloning the LhCOLD1 gene in hybrid tulip trees and studying its cold tolerance are of great theoretical significance and potential practical value for understanding the mechanism by which hybrid tulip trees cope with cold stress and for breeding stress-resistant varieties. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, the technical problem this invention aims to solve is to provide the LhCOLD1 gene of hybrid tulip tree. Another technical problem this invention aims to solve is to provide the expression protein of the LhCOLD1 gene of hybrid tulip tree. A further technical problem this invention aims to solve is to provide applications for the LhCOLD1 gene of hybrid tulip tree for regulating plant stress resistance.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] The LhCOLD1 gene of hybrid tulip tree has the nucleotide sequence shown in SEQ ID NO:1.

[0008] The amino acid sequence of the expressed protein of the hybrid tulip tree LhCOLD1 gene is shown in SEQ ID NO:2.

[0009] Vectors and recombinant bacteria containing the LhCOLD1 gene from hybrid tulip tree.

[0010] Application of the LhCOLD1 gene from hybrid tulip tree in regulating stress resistance in Arabidopsis thaliana.

[0011] The regulation of Arabidopsis stress resistance aims to enhance its ability to resist low-temperature stress.

[0012] The low-temperature stress is -10°C.

[0013] The application of the hybrid tulip tree LhCOLD1 gene in regulating Arabidopsis stress resistance includes:

[0014] 1) Construct an overexpression vector for the LhCOLD1 gene in hybrid tulip tree;

[0015] 2) The constructed overexpression vector of the hybrid tulip tree LhCOLD1 gene was transformed into Arabidopsis thaliana;

[0016] 3) Breed, screen and obtain transgenic Arabidopsis strains with enhanced resistance to low-temperature stress.

[0017] Application of the LhCOLD1 gene in regulating the stress resistance of hybrid tulip trees.

[0018] The regulation of stress resistance in hybrid tulip trees aims to enhance their ability to resist low-temperature stress.

[0019] The low-temperature stress is -10°C.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] 1) This invention discloses for the first time the LhCOLD1 gene cloned from the leaves of hybrid tulip tree, the nucleotide sequence of which is shown in SEQ ID NO. 1, and the amino acid sequence of the expressed protein is shown in SEQ ID NO. 2.

[0022] 2) This invention constructs an overexpression vector for the LhCOLD1 gene of hybrid tulip tree; transforms the constructed overexpression vector into Arabidopsis thaliana; and cultivates, screens, and obtains transgenic Arabidopsis lines with enhanced resistance to low-temperature stress. The function of the LhCOLD1 gene in regulating plant stress resistance is revealed for the first time. After being treated at -10℃ for 3 hours and then allowed to recover at room temperature for one week, the LhCOLD1 transgenic plants showed less impact from low-temperature stress, while the wild-type plants exhibited inhibited growth and leaf wilting. The results indicate that the LhCOLD1 gene in hybrid tulip tree enhances the tolerance of Arabidopsis thaliana to low-temperature stress.

[0023] 3) This invention constructs an overexpression vector for the LhCOLD1 gene in hybrid tulip tree; transforms the constructed LhCOLD1 gene overexpression vector into callus tissue of hybrid tulip tree; and cultivates, screens, and obtains transgenic hybrid tulip tree callus lines with enhanced resistance to low-temperature stress. The function of the LhCOLD1 gene in regulating plant stress resistance is revealed for the first time. After treatment at -10℃ for 1.5 h, the growth of the LhCOLD1 transgenic lines was less affected by low-temperature stress, while the growth of wild-type plants was inhibited, and leaves showed wilting. The results indicate that the LhCOLD1 gene enhances the tolerance of hybrid tulip tree to low-temperature stress. Attached Figure Description

[0024] Figure 1 Image of LhCOLD1 gene PCR amplification products (M is Marker DL2000, 1 is PCR product).

[0025] Figure 2 Image showing the PCR electrophoresis results of positive transformants in the LhCOLD1 gene clone (M is Marker DL2000; 1-8 are PCR products of the bacterial culture).

[0026] Figure 3 Image of pRI101-6xFlag plasmid;

[0027] Figure 4 Phenotypic images of wild-type Arabidopsis thaliana and LhCOLD1 transgenic Arabidopsis thaliana after being treated with -10℃ cold stress for 3 h and then recovering at room temperature for one week;

[0028] Figure 5 Phenotypic diagrams of *Liriodendron tulipifera* hybrids with empty vector and hybrids with LhCOLD1 gene treated with -10℃ cold stress for 1.5 h. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments, but the embodiments do not limit the form of the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the invention are within the scope of the present invention. Experimental methods and reagents not specified in the embodiments are performed according to conventional conditions in the art.

[0030] The plant materials used in this application are hybrid tulip tree seedlings and callus tissue, which are preserved in the Molecular Laboratory of Nanjing Forestry University.

[0031] Example 1

[0032] 1. Cloning of the LhCOLD1 gene

[0033] RNA was extracted from leaves of hybrid tulip trees using the Vazyme FastPure® Plant Total RNA Extraction Kit according to the kit instructions. Using the extracted total RNA as a template, reverse transcription experiments were performed using Vazyme's HiScript III 1st Strand cDNA Synthesis Kit. The COLD1 protein sequences from the model plants Arabidopsis thaliana and rice were downloaded from the NCBI website. In Arabidopsis, these sequences were identified as GTG1 and GTG2, while in rice, they were identified as OsCOLD1. The COLD1 gene in tulip trees was identified and cloned using BLASTP alignment. Primers were designed using Primer 5 software and synthesized at Qingke Biotechnology Co., Ltd. The primer sequences are shown below:

[0034] LhCOLD1-F: 5'-ATGGGGTGGGGATGGG-3',

[0035] LhCOLD1-R: 5'-TCAATCAATGGGGTGCTTGTC-3'.

[0036] The PCR reaction system consisted of 2 μL each of LhCOLD1-F and LhCOLD1-R, 2 μL of cDNA, and 25 μL of 2×PhantaMaxBuffer. a High-fidelity enzyme (purchased from Vazyme), 1 μL dNTP Mix, 1 μL Phanta Max Super-Fidelity DNA Polymerase, ddH2O to 50 μL.

[0037] PCR reaction program: 95℃ for 3 min; 95℃ for 15 s, 56~72℃ for 15 s, 72℃ for 30-60 s, 25-35 cycles; 72℃ for 5 min.

[0038] The PCR products were subjected to 1% agarose gel electrophoresis. The electrophoresis results showed that a single band was successfully amplified, and the size was consistent with the expected size compared with the marker. The amplified LhCOLD1 was a DNA fragment of approximately 1400 bp. Figure 1 Fragment recovery was performed using the DNA gel recovery kit from Qingke Biotechnology Co., Ltd. The recovered target DNA was ligated into the pClone007B intermediate vector (Beijing Qingke Biotechnology Co., Ltd.) and reacted at 25°C for 5 min. The ligation product was then transformed into *E. coli* DH5α competent cells. After amplification culture, the positive bacterial suspension was sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. Bacterial analysis showed that the size of the PCR product was basically consistent with the size of the target fragment, and there were no extraneous bands. Preliminary identification indicated a recombinant, with LhCOLD1 being a DNA fragment of approximately 1500 bp. Figure 2 The positive bacterial culture of No. 1 was expanded, and plasmids were extracted and purified using a small-sample preparation kit from TIANGEN. The extracted plasmids were then sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing.

[0039] TA cloning and sequencing results showed that the LhCOLD1 gene TA clone 1 differed from the reference sequence by one base. The amino acid sequence of clone 1 after translation was identical to the reference sequence. After confirmation, clone 1 was used for overexpression vector construction. The CDS sequence of the LhCOLD1 gene is shown in SEQ ID NO.1, and the amino acid sequence of its expressed protein is shown in SEQ ID NO.2.

[0040] 2. Construct an overexpression vector for the LhCOLD1 gene.

[0041] Based on the pRI101-6xFlag plasmid map ( Figure 3 Using the LhCOLD1 gene CDS sequence (with stop codon removed) as a template, the LhCOLD1 gene ORF restriction site search was performed using software. The results showed that there were no XbaI or EcoRI restriction sites within the LhCOLD1 gene CDS sequence. XbaI and EcoRI double digestion primers were designed using Snap Gene software, and the primer sequences are shown below:

[0042] pRI101-6xFlag-COLD1-F:

[0043] 5'-gagaacacgggggactctagaATGGGGTGGGGATGGGCG-3',

[0044] pRI101-6xFlag-COLD1-R:

[0045] 5'-tttgtaatcatcgatgaattcATCAATGGGGTGCTTGTCAATT-3'.

[0046] The pRI101-6xFlag vector stored in the laboratory was digested with enzymes, and the selected restriction sites were XbaI and EcoRI.

[0047] The enzyme digestion reaction system consisted of: Cutsmart Buffer 2 μL, XbaI 1 μL, EcoRI 1 μL, plasmid 10 μL, and ddH2O 5 μL.

[0048] The enzyme digestion reaction program was: 37℃ for 1 h, 65℃ for 30 min, and 4℃ for ∞.

[0049] The recovered target fragment and the enzyme digestion vector fragment were recombined and ligated using the Vazyme ClonExpress II OneStep Cloning Kit.

[0050] The ligation reaction system consisted of: 4 μL of 5×CE II Buffer, 2 μL of Exnase® II, X μL of linearized carrier, Y μL of target fragment, and ddH2O up to 20 μL.

[0051] The connection reaction program is: 30℃ for 30 min, 4℃ for ∞.

[0052] The ligation product was transformed into *E. coli* DH5α from TIANGEN, and the bacterial culture was verified by PCR amplification. The culture with the correct band size was selected, and the positive culture (number 1) matching the band size was expanded and plasmid extracted. The extracted plasmid was sent to Nanjing Qingke Company for sequencing. The verification primer sequences are shown below:

[0053] 35S seq-F:

[0054] 5'-CACAATCCCACCCCTACTCC-3',

[0055] M13-R:

[0056] 5'-CAGGAAACAGCTATGACC-3'.

[0057] Sequencing results showed that the sequence of clone 1 of the overexpression vector was consistent with the sequence shown in SEQ ID NO.1, indicating that the target gene had been inserted into the vector and the expression vector pRI101-6xFlag-COLD1 was successfully constructed.

[0058] Example 2

[0059] 1. Cultivation of Arabidopsis thaliana

[0060] Prepare a mixture of potting soil, perlite, and vermiculite in a 3:1:1 ratio and fill it with water into a flowerpot. Sprinkle an appropriate amount of Arabidopsis thaliana on the soil surface, seal with plastic wrap, and place in a light incubator. Once the seeds germinate, remove the plastic wrap. When the seeds have grown to the size of four leaves, transplant them individually into separate pots, add an appropriate amount of nutrient solution, and place them in a tissue culture box (temperature 24℃, 16 hours of light, 8 hours of darkness) for further cultivation.

[0061] 2. Transfer of Agrobacterium using the freeze-thaw method

[0062] Thaw competent Agrobacterium GV3101 cells stored at -80°C and place them in ice while they are in an ice-water mixture. Add 0.01-1 μg of plasmid DNA to each 100 μL of competent cells, mix by hand by tapping the bottom of the tube, and incubate sequentially on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. Add 700 μL of antibiotic-free LB medium and incubate at 28°C with shaking for 2-3 hours. Harvest the bacteria at 4000 rpm for 2 min, and resuspend approximately 100 μL of the supernatant by gently pipetting. Spread the resuspended culture onto agar plates containing 50 mg / L kanamycin and 20 mg / L rifampin, and incubate upside down at 28°C for 48 hours.

[0063] 3. Preparation of Agrobacterium infection solution

[0064] Pick single colonies from plates containing two antibiotics and incubate them in 1 mL of LB broth containing both antibiotics (in a 2 mL centrifuge tube) at 28°C for 16-24 h. Perform PCR and electrophoresis on the bacterial culture; correct band size indicates that the vector plasmid has been successfully transferred into Agrobacterium. Large-scale culture: Take the positive bacterial culture from the previous step and add it to 2 mL of LB broth containing both antibiotics (in a 10 mL centrifuge tube) at a 1:100 ratio. Incubate in the dark at 28°C and 220 rpm for 12-16 h. Large-scale culture: Add the bacterial culture from the previous step to 50 mL of LB broth containing both antibiotics (in a 250 mL Erlenmeyer flask) at a 1:100 ratio. Incubate in the dark at 28°C and 220 rpm until the OD value reaches 0.8-1.2. Centrifuge at 5000 rpm at room temperature for 10 min, collect the bacterial precipitate, and resuspend the cells in freshly prepared 5% (w / v) sucrose solution. Add Silwet L-77 to the Agrobacterium resuspension to a concentration of 0.02%.

[0065] 4. Agrobacterium infection

[0066] Immerse each Arabidopsis inflorescence in the Agrobacterium resuspension solution described above for 1 minute, seal with plastic wrap to maintain humidity >90%, and incubate in the dark at 24°C for 24 hours. Remove the plastic wrap and return the Arabidopsis to the incubator for normal growth for one month. After one month, stop watering and wait for the siliques to mature.

[0067] 5. Screening and identification of positive Arabidopsis seedlings

[0068] Preparation of screening medium: Use 50 mg / L kanamycin as the screening concentration, add 4.4 g / L MS medium powder and 20 g / L sucrose, adjust the pH to 5.8, and then add 8.5 g / L agar.

[0069] Cleaning Arabidopsis seeds: Take an appropriate amount of seeds and place them in a sterile centrifuge tube. Wash them once with sterile water, centrifuge, and discard the filtrate. Add 75% sodium hypochlorite solution and wash for 4-5 minutes, then centrifuge and discard the filtrate. Add 10% sodium hypochlorite disinfectant solution and disinfect for 4-5 minutes. Afterward, wash them three times with clean water. Take an appropriate amount of Arabidopsis seeds and spread them on a culture medium containing antibiotics. Once they grow to a clear phenotype (dark green cotyledons and well-developed root system), they can be transplanted into the soil for further evaluation.

[0070] Identification of positive plants: Take leaves of 1-3 cm in size and extract plant DNA using the CTAB method.

[0071] Positive plant seed collection: Plants identified as positive continue to grow in the incubator until T3 seeds are collected.

[0072] 6. Phenotypic characteristics of overexpressing plants under cold stress

[0073] T3 generation Arabidopsis thaliana plants with 4-5 leaves that were growing well in 1 / 2MS solid medium were transplanted into nutrient soil and grown for 10 days before being subjected to cold stress. LhCOLD1 transgenic Arabidopsis thaliana OE4, OE5, and OE9 lines and wild-type Arabidopsis thaliana were placed in a -10℃ refrigerator for 3 hours. After two weeks of recovery at room temperature, the transgenic plants and Arabidopsis thaliana plants were photographed and their phenotypic characteristics were observed.

[0074] The results are as follows Figure 4 As shown, the growth of LhCOLD1 transgenic plants was less affected by low-temperature stress, while the growth of wild-type plants was inhibited, and the leaves wilted. The results indicate that the LhCOLD1 gene in hybrid tulip trees enhances the tolerance of Arabidopsis thaliana to low-temperature stress.

[0075] Example 3

[0076] 1. Preparation of Agrobacterium infection solution

[0077] After transforming the constructed overexpression vector pRI101-6xFlag-COLD1 plasmid into Agrobacterium EHA105, single clones were picked, and the bacterial culture was subjected to PCR and electrophoresis. The correct band size indicated that the vector plasmid had been transformed into Agrobacterium EHA105.

[0078] Large-scale shaking of bacterial culture: Take 40 μL of bacterial culture into 2 mL of LB containing kana (10 mL centrifuge tube), and incubate in the dark at 28 degrees Celsius and 220 rpm for 12-16 h.

[0079] Large-scale bacterial culture: Take 2 mL of the shaken bacterial culture into a 250 mL Erlenmeyer flask, add 50 mL LB (50 μL kana + 100 μL AS), and incubate in the dark at 28 degrees Celsius and 220 rpm for 4-6 hours. After 4 hours, take 2 mL of the bacterial culture every so often to measure the OD value (0.6-1.0 is better) and it is ready for infection.

[0080] 2. Agrobacterium infection and transformation of hybrid tulip tree callus and co-culture

[0081] Take 40 mL of the expanded bacterial culture and place it in a 50 mL centrifuge tube. Centrifuge at 5000 rpm for 10 min at 4 degrees Celsius. Discard the supernatant and add an appropriate amount of M13 liquid suspension to adjust the OD value to 0.8. Take three plates of hybrid tulip tree callus with genotype 166302 into a 100 mL Erlenmeyer flask. Add an appropriate amount of bacterial suspension and infect for about 10 min, shaking the Erlenmeyer flask occasionally. Sieve the callus through a 400-mesh sieve and blot dry the residual bacterial suspension on the sieve with a sterile paper towel. Then transfer the callus to M13 (AS) solid medium with filter paper and incubate for 36-48 h.

[0082] 3. Sterilization and screening

[0083] Sterilization: Place the co-cultured callus in a 100mL Erlenmeyer flask and elute with sterile water and M13 liquid medium. The elution order is water + cef / M13 + cef / water / M13 + cef / water / M13, with a total time not exceeding 10min. Sieve through a 400-mesh sieve, blot dry the liquid on the sieve with sterile paper towels, and culture in M13 (cef) solid medium.

[0084] Screening: After about 7 days of culture, the callus was transferred to M13 (G418, cef) solid medium for screening. After several subculture cycles, the cef concentration could be appropriately reduced according to the callus status until fresh and delicate callus tissue grew.

[0085] 4. Detection of transgenic callus tissue

[0086] DNA was extracted from fresh, delicate callus tissue newly grown on the selection medium using the CTAB method, and PCR amplification was performed using Vazyme's 2×Rapid Taq Master Mix to screen for positively overexpressing callus tissue.

[0087] 5. Obtaining regenerated plantlets from somatic embryogenesis

[0088] Using the obtained positive embryogenic callus as material, a suspension system was established to induce somatic embryonic plant regeneration. The specific steps are as follows:

[0089] Transgenic empty vector (EV) and positively overexpressing callus tissue were placed in 250 mL Erlenmeyer flasks, and 50 mL of M13 liquid medium was added for liquid culture to obtain single-cell suspension lines. After one week, the cells were subcultured in M13 liquid medium. Single cells that had undergone two weeks of liquid culture were screened using 150-mesh and 400-mesh cells, and then added to 50 mL of Z14 hypertonic transition medium for transition culture for 2 days. 1 mL of single cells was aspirated and counted under a microscope to calculate the cell density. The suspension cells were diluted to an appropriate concentration. Each suspension line was aspirated using a de-pointed pipette tip and evenly spread on Z36 somatic embryogenesis solid medium lined with filter paper, and then cultured in a constant temperature dark incubator.

[0090] 6. Phenotypic characteristics of overexpressing plants under cold stress

[0091] Three-month-old regenerated plants obtained through somatic embryogenesis were transplanted into nutrient soil and grown for one month before being subjected to cold stress treatment. LhCOLD1 transgenic plants and empty-gene hybrid tulip trees were simultaneously placed in a -10°C freezer for 1.5 hours, and the phenotypic characteristics of the transgenic and empty-gene plants were observed by photographing.

[0092] The results are as follows Figure 5 As shown, the LhCOLD1 transgenic line showed less impact from low-temperature stress after being treated at -10℃ for 1.5 h, while the growth of wild-type plants was inhibited and the leaves wilted. The results indicate that the LhCOLD1 gene enhances the tolerance of hybrid tulip trees to low-temperature stress.

[0093] The embodiments described above are merely illustrative of the present invention and not restrictive. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. The LhCOLD1 gene of hybrid tulip tree, the nucleotide sequence of which is shown in SEQ ID NO:

1.

2. The expressed protein of the hybrid tulip tree LhCOLD1 gene according to claim 1, the amino acid sequence of which is as shown in SEQ ID NO:2 As shown.

3. A vector and recombinant bacteria containing the LhCOLD1 gene of the hybrid tulip tree as described in claim 1.

4. The application of the hybrid tulip tree LhCOLD1 gene as described in claim 1 in enhancing the ability of Arabidopsis thaliana to resist low-temperature stress.

5. The application according to claim 4, characterized in that, The low-temperature stress is -10°C.

6. The application according to claim 4, characterized in that, include: 1) Construct an overexpression vector for the LhCOLD1 gene in hybrid tulip tree; 2) The constructed overexpression vector of the hybrid tulip tree LhCOLD1 gene was transformed into Arabidopsis thaliana; 3) Breed, screen and obtain transgenic Arabidopsis strains with enhanced resistance to low-temperature stress.

7. The application of the LhCOLD1 gene of hybrid tulip tree as described in claim 1 in promoting the ability of hybrid tulip tree to resist low temperature stress.

8. The application according to claim 7, characterized in that, The low-temperature stress is -10℃.

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