Liriodendron hybrids LhCOLD1 gene as well as expression protein and application thereof

Abstract

The invention discloses a liriodendron hybrids LhCOLD1 gene as well as an expression protein and application thereof, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the LhCOLD1 gene disclosed by the invention is shown as SEQ ID NO. 1, and the amino acid sequence of the expression protein of the LhCOLD1 gene is shown as SEQ ID NO. 2. The method comprises the following steps: constructing an overexpression vector of a hybrid liriodendron LhCOLD1 gene, and transforming the overexpression vector into arabidopsis thaliana and hybrid liriodendron calluses; and cultivating and screening to obtain a transgenic line with improved low-temperature stress resistance. After the LhCOLD1 transgenic arabidopsis thaliana plant is subjected to low-temperature treatment at-10 DEG C for 3 hours and the LhCOLD1 transgenic arabidopsis thaliana plant recovers to grow at room temperature for two weeks, the growth of the LhCOLD1 transgenic After the LhCOLD1 transgenic hybrid liriodendron tulipifera strain is treated at the low temperature of-10 DEG C for 1.5 hours, the influence of low-temperature stress on the growth is small. The result shows that the LhCOLD1 gene enhances the tolerance of the plant to low-temperature stress.

Description

Technical Field

[0001] The invention belongs to the technical field of plant gene engineering, and more specifically relates to a hybrid Liriodendron tulipifera LhCOLD1 gene and its expression protein and application. Background Art

[0002] Liriodendron is an ancient relict plant of the genus Liriodendron in the Magnoliaceae family, mainly distributed in the area south of the Yangtze River Basin in my country. my country began to introduce Liriodendron tulipifera in the 1930s, and successfully obtained a hybrid variety of Liriodendron tulipifera and Liriodendron tulipifera in 1963, namely Hybrid Liriodendron tulipifera. Hybrid Liriodendron tulipifera has the characteristics of rapid growth, upright trunk, bright color, etc., and is often used for landscaping and mountain afforestation.

[0003] Hybrid tulip tree is often affected by low temperature and cold damage in northern my country, which greatly limits its regional introduction and promotion. When plants suffer from low temperature during growth and development, their normal metabolic reaction physiological processes are inhibited, causing slow growth or even death of the plants. Studies have found that some low temperature response genes in plants play an important role in enhancing their low temperature tolerance, such as ICE1 and CBF. The COLD1 gene was reported to be a type of cold receptor, which was first discovered and named in rice. COLD1 encodes a GPCR-type G protein (GTGs) with nine transmembrane domains, which is located on the plasma membrane and endoplasmic reticulum membrane. It plays a role in regulating plant growth and development, stress resistance and other functions in crops such as rice, wheat and corn.

[0004] In summary, the regulation of cold tolerance by the LhCOLD1 gene of hybrid tulipwood has not been studied. Cloning the LhCOLD1 gene of hybrid tulipwood and studying its cold tolerance have important theoretical significance and potential practical value for understanding the mechanism of hybrid tulipwood in coping with cold stress and breeding resistant varieties. 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 LhCOLD1 gene. Another technical problem to be solved by the present invention is to provide an expression protein of the hybrid tulipwood LhCOLD1 gene. Another technical problem to be solved by the present invention is to provide an application of the hybrid tulipwood LhCOLD1 gene for regulating plant stress resistance.

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

[0007] The nucleotide sequence of the hybrid Liriodendron chinense LhCOLD1 gene is shown in SEQ ID NO:1.

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

[0009] Vectors and recombinant bacteria containing the hybrid Liriodendron chinense LhCOLD1 gene.

[0010] Application of hybrid Liriodendron chinense LhCOLD1 gene in regulating stress resistance of Arabidopsis thaliana.

[0011] The regulation of Arabidopsis stress resistance is to promote the improvement of Arabidopsis' ability to resist low temperature stress.

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

[0013] Application of hybrid Liriodendron chinense LhCOLD1 gene in regulating stress resistance of Arabidopsis thaliana, including:

[0014] 1) Construction of an overexpression vector of the hybrid Liriodendron chinense LhCOLD1 gene;

[0015] 2) Transform the constructed hybrid Liriodendron chinense LhCOLD1 gene overexpression vector into Arabidopsis thaliana;

[0016] 3) Cultivate, screen and obtain transgenic Arabidopsis strains with improved resistance to low temperature stress.

[0017] Application of hybrid Liriodendron truncatum LhCOLD1 gene in regulating stress resistance of hybrid Liriodendron truncatum.

[0018] The method for regulating the stress resistance of hybrid tulip tree is to promote the improvement of the ability of hybrid tulip tree to resist low temperature stress.

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

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

[0021] 1) The present invention discloses for the first time that the LhCOLD1 gene is cloned from the leaves of hybrid Liriodendron chinense, 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) The present invention constructs an overexpression vector of the hybrid tulipwood LhCOLD1 gene; transforms the constructed overexpression vector of the hybrid tulipwood LhCOLD1 gene into Arabidopsis; cultivates, screens and obtains transgenic Arabidopsis strains with improved resistance to low temperature stress. The function of the LhCOLD1 gene in regulating plant stress resistance was revealed for the first time. The strains overexpressing the LhCOLD1 gene were treated at -10℃ for 3h and then resumed growth at room temperature for one week. The growth of the LhCOLD1 transgenic plants was less affected by low temperature stress, while the growth of the wild-type plants was inhibited and the leaves wilted. The results showed that the hybrid tulipwood LhCOLD1 gene enhanced the tolerance of Arabidopsis to low temperature stress.

[0023] 3) The present invention constructs an overexpression vector of the hybrid tulipwood LhCOLD1 gene; transforms the constructed overexpression vector of the hybrid tulipwood LhCOLD1 gene into the hybrid tulipwood callus; cultivates, screens and obtains transgenic hybrid tulipwood callus strains with improved resistance to low temperature stress. The function of the LhCOLD1 gene in regulating plant stress resistance was revealed for the first time. The growth of the LhCOLD1 transgenic strain was less affected by low temperature stress after being treated at -10℃ for 1.5h, while the growth of the wild-type plant was inhibited and the leaves wilted. The results showed that the LhCOLD1 gene enhanced the tolerance of hybrid tulipwood to low temperature stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the image of PCR amplification product of LhCOLD1 gene (M is Marker DL2000, 1 is PCR product);

[0025] Figure 2 The figure is the result of PCR electrophoresis of the bacterial solution of positive transformants in LhCOLD1 gene cloning (M is Marker DL2000; 1-8 are the PCR products of the bacterial solution);

[0026] Figure 3 This is the pRI101-6xFlag plasmid map;

[0027] Figure 4 The phenotypes of wild-type Arabidopsis and transgenic Arabidopsis with LhCOLD1 gene treated at -10℃ for 3 hours and then recovered and grown at room temperature for one week;

[0028] Figure 5 The phenotypes of hybrid Liriodendron chinense carrying an empty vector and hybrid Liriodendron chinense carrying a LhCOLD1 gene treated under -10℃ cold stress for 1.5h. DETAILED DESCRIPTION

[0029] The present invention is further described below in conjunction with specific examples, but the examples do not limit the form of the present invention. Without departing from the spirit and essence of the present invention, the modification or replacement of the method, steps or conditions of the present invention all belong to the scope of the present invention. The experimental methods and reagents without specifying the specific conditions in the examples are all in accordance with the conventional conditions in the art.

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

[0031] Example 1

[0032] 1. LhCOLD1 gene cloning

[0033] Using Vazyme's FastPure®Plant total RNA extraction kit, extract RNA from hybrid Liriodendron leaf according to the kit instructions. Using the extracted total RNA as a template, Vazyme's reverse transcription reagent HiScript Ⅲ 1stStrand cDNA Synthesis Kit was used for reverse transcription experiments. The COLD1 protein sequences of the model plants Arabidopsis thaliana and rice were downloaded from the NCBI website. In Arabidopsis thaliana, they are GTG1 and GTG2, respectively, and in rice, they are OsCOLD1. Using BLASTP for comparison, the COLD1 gene in Liriodendron thaliana was identified for cloning. Primers were designed using Primer 5 software and sent to Qingke Biotechnology Company for synthesis. The primer sequences are as follows:

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

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

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

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

[0038] The PCR product was subjected to 1% agarose gel electrophoresis. The electrophoresis detection 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 about 1400 bp ( Figure 1 ), and the DNA gel recovery kit of Qingke Biotechnology Co., Ltd. was used for fragment recovery. The recovered product target DNA was connected to the pClone007B intermediate vector (Beijing Qingke Biotechnology Co., Ltd.) and reacted at 25°C for 5 minutes. The ligation product was transformed into Escherichia coli DH5α competent cells. After amplification, the positive bacterial solution was sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The bacterial test results showed that the size of the bacterial solution PCR product was basically consistent with the size of the target fragment, and there was no mixed band. It was initially identified as a recombinant. LhCOLD1 is a DNA fragment of about 1500bp ( Figure 2 The No. 1 positive bacterial solution was expanded and cultured, and the plasmid was extracted and purified using the small extraction kit of TIANGEN Company. The extracted plasmid was sent to Nanjing Qingke Biological Company for sequencing.

[0039] The TA clone sequencing results showed that there was a base difference between the TA clone sequence of LhCOLD1 gene No. 1 and the reference sequence, and the amino acid of clone No. 1 was consistent with the reference sequence after translation. After confirmation, clone No. 1 was used to construct an overexpression vector. The CDS sequence of 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. Construction of LhCOLD1 gene overexpression vector

[0041] According to the pRI101-6xFlag plasmid map ( Figure 3 ), using the CDS sequence of the LhCOLD1 gene (excluding the stop codon) as a template. The software was used to search for restriction sites in the LhCOLD1 gene ORF, and the results showed that there were no XbaI or EcoRI restriction sites in the CDS sequence of the LhCOLD1 gene. The XbaI and EcoRI double restriction primers were designed using Snap Gene software, and the primer sequences are as follows:

[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 restriction enzymes, and the selected vector restriction enzyme sites were XbaI and EcoRI.

[0047] The enzyme digestion reaction system is: Cutsmart Buffer 2μL, XbaI 1μL, EcoRI 1μL, plasmid 10μL, ddH 2 O 5μL.

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

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

[0050] The ligation reaction system is: 5×CE II Buffer 4μL, Exnase® II 2μL, linearized vector XμL, target fragment YμL, ddH 2 O Up to 20μL.

[0051] The ligation reaction program was: 30°C for 30 min, 4°C for ∞.

[0052] The ligated product was transformed into TIANGEN's E. coli DH5α, and the bacterial solution was verified by PCR amplification. The bacterial solution with the correct detection band size was selected, and the No. 1 positive bacterial solution with the correct size was expanded and cultured to extract the plasmid. The extracted plasmid was sent to Nanjing Qingke Company for sequencing. The verification primer sequence is as follows:

[0053] 35S seq-F:

[0054] 5'-CACAATCCCACCCCTACTCC-3',

[0055] M13-R:

[0056] 5′-CAGGAAACAGCTATGACC-3′.

[0057] The sequencing results showed that the sequencing sequence of the constructed overexpression vector No. 1 clone 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 nutrient soil, perlite and vermiculite in a ratio of 3:1:1 and put them in a flower pot. Take an appropriate amount of Arabidopsis and sprinkle it on the soil surface, seal it with plastic wrap, and place it in a light incubator for cultivation. After the seeds germinate, remove the plastic wrap. When they grow to 4 leaves, transplant them into a single-family division pot, pour in an appropriate amount of nutrient solution, and place them in a tissue culture box (temperature 24℃, light 16h, dark 8h) for cultivation.

[0061] 2. Freeze-thaw method to transfer Agrobacterium

[0062] Take the GV3101 Agrobacterium competent cells stored at -80 and melt them. When they are in the ice-water mixing state, insert them into ice. Add 0.01-1μg plasmid DNA to every 100μL competent cells, stir the bottom of the tube by hand to mix, and then stand on ice for 5min, liquid nitrogen for 5min, 37 degrees water bath for 5min, and ice bath for 5min. Add 700μL LB medium without antibiotics and shake and culture at 28 degrees for 2-3 hours. Collect the bacteria at 4000rpm for 2min, take about 100μL of supernatant and gently blow to resuspend the bacteria, spread it on a plate containing 50 mg / L kanamycin and 20 mg / L rifampicin, and invert and culture at 28 degrees for 48h.

[0063] 3. Preparation of Agrobacterium infection solution

[0064] Pick a single clone from the plate containing two antibiotics, and culture it in 1mL LB liquid medium containing two antibiotics (2mL centrifuge tube) at 28℃ for 16-24h. Perform bacterial liquid PCR and electrophoresis detection. The correct band size indicates that the vector plasmid has been transferred into Agrobacterium. Shake the bacterial liquid: Take the positive bacterial liquid in the previous step, add it to 2mL of LB containing two antibiotics (10mL centrifuge tube) at 1:100, and culture it in a shaker at 28 degrees and 220rpm for 12-16h. Expand the bacterial liquid culture: Add the bacterial liquid in the previous step to 50mLLB (250mL conical flask) containing two antibiotics at 1:100, and culture it in a shaker at 28 degrees and 220rpm. The OD value reaches 0.8-1.2. Centrifuge at 5000rpm at room temperature for 10min, collect the bacterial precipitate, and resuspend the bacteria with the prepared 5% (w / v) sucrose solution. Resuspend the Agrobacterium and add Silwet L-77 to a concentration of 0.02%.

[0065] 4. Agrobacterium infection

[0066] Soak each Arabidopsis in the above Agrobacterium resuspension for 1 minute, seal with film to maintain humidity > 90%, and incubate in the dark at 24°C for 24 hours. Remove the plastic wrap, put the Arabidopsis back into the incubator, and grow normally for one month. Stop watering after one month 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 pH to 5.8, and then add 8.5 g / L agar.

[0069] Washing of Arabidopsis seeds: Take an appropriate amount of seeds and put them into a sterilized centrifuge tube, wash them once with sterile water, centrifuge and discard the filtrate; add 75% to wash for 4-5 minutes, centrifuge and discard the filtrate; add 10% sodium hypochlorite disinfectant and disinfect for 4-5 minutes. After washing with clean water three times, take an appropriate amount of Arabidopsis seeds and distribute them on the culture medium with antibiotics. When they grow to a clear phenotype (dark green cotyledons and well-developed root system), they can be transplanted into the soil and wait for verification.

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

[0071] Positive plants harvesting: Plants identified as positive continue to grow in the incubator until T3 seeds are received.

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

[0073] The T3 generation Arabidopsis thaliana with 4 to 5 leaves growing well in 1 / 2MS solid culture medium was transplanted into nutrient soil and then grown for 10 days before cold stress treatment. The LhCOLD1 transgenic Arabidopsis thaliana OE4, OE5, OE9 strains and wild-type Arabidopsis thaliana were placed in a -10°C refrigerator at the same time and treated at low temperature for 3 hours. After recovery and growth at room temperature for two weeks, the phenotypes of the transgenic plants and Arabidopsis thaliana plants were observed by photographing.

[0074] The results are as follows Figure 4 As shown in the figure, 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 showed that the hybrid tulipwood LhCOLD1 gene enhanced the tolerance of Arabidopsis to low temperature stress.

[0075] Example 3

[0076] 1. Preparation of Agrobacterium infection solution

[0077] The constructed overexpression vector pRI101-6xFlag-COLD1 plasmid was transferred into Agrobacterium EHA105 and a single clone was picked. The bacterial solution was subjected to PCR and electrophoresis detection. The correct band size indicated that the vector plasmid had been transferred into EHA105 Agrobacterium.

[0078] Shake the bacterial solution: Take 40 μL of the bacterial solution and add it to 2 mL of LB containing kana (10 mL centrifuge tube). Incubate in a shaker at 28 degrees and 220 rpm in the dark for 12-16 hours.

[0079] Expand the culture of bacterial liquid: take 2mL of the shaken bacterial liquid into a 250mL conical flask, and add 50mL LB (50μL kana+100μL AS), and culture it in a shaker at 28 degrees and 220rpm in the dark for 4-6 hours. After 4 hours, take 2mL of the bacterial liquid at regular intervals to measure the OD value (0.6-1.0), which is good for infection.

[0080] 2. Agrobacterium infection, transformation and co-cultivation of hybrid Liriodendron chinense callus

[0081] Take 40mL of the expanded culture solution and place it in a 50mL centrifuge tube. Centrifuge at 4 degrees and 5000rpm for 10min. Discard the supernatant and add an appropriate amount of M13 liquid suspension solution to adjust the OD value to 0.8. Take three dishes of hybrid Liriodendron tulipifera callus with genotype 166302 in a 100mL conical flask, add an appropriate amount of bacterial solution and infect for about 10min. During this period, shake the conical flask from time to time, sieve with a 400-mesh sieve, and use a sterile paper towel to absorb the residual bacterial solution on the sieve. Then transfer the callus to M13 (AS) solid medium with filter paper and culture for 36-48h.

[0082] 3. Degerming and screening

[0083] Degerming: Place the co-cultivated callus in a 100 mL conical flask and elute with sterile water and M13 liquid culture medium. The elution order is water + cef / M13 + cef / water / M13 + cef / water / M13. The total time should not exceed 10 min. Sieve with a 400-mesh sieve, dry the liquid on the sieve with a sterile paper towel, and culture in M13 (cef) solid culture medium.

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

[0085] 4. Detection of transgenic callus

[0086] The CTAB method was used to extract DNA from fresh and delicate callus newly grown on the screening medium, and 2×Rapid Taq Master Mix from Vazyme was used for PCR amplification to screen positive overexpression callus.

[0087] 5. Obtaining regenerated plants through somatic embryogenesis

[0088] The obtained positive embryonic callus was used as material to establish a suspension system and induce somatic embryo plant regeneration. The specific steps are as follows:

[0089] Place the transgenic empty vector (EV) and positive overexpression callus tissue in a 250mL conical flask, add 50mL M13 liquid medium for liquid culture, and obtain a single cell suspension system. Subculture with M13 liquid medium after 1 week. Use 150 mesh and 400 mesh cells to screen out single cells that have been liquid cultured for two weeks, add 50mL Z14 hypertonic transition medium for transition culture for 2 days. Pipette 1mL of single cells to count the number of cells under a microscope and calculate the cell density. Dilute the suspension cells to an appropriate concentration. Use a de-pointed pipette tip to absorb each suspension line, evenly spread it on the Z36 somatic embryogenesis solid medium padded with filter paper, and culture it in a constant temperature dark incubator.

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

[0091] The three-month-old regenerated plants obtained through somatic embryogenesis were transferred to nutrient soil and grown for one month before cold stress treatment. The LhCOLD1 transgenic and empty-transgenic hybrid tulipwood were placed in a -10℃ refrigerator at the same time for 1.5h, and the phenotypic characteristics of the transgenic and empty-transgenic plants were observed by taking photos.

[0092] The results are as follows Figure 5 As shown in the figure, the growth of LhCOLD1 transgenic lines was less affected by low temperature stress after being treated at -10℃ for 1.5h, while the growth of wild-type plants was inhibited and the leaves wilted. The results showed that the LhCOLD1 gene enhanced the tolerance of hybrid tulipwood to low temperature stress.

[0093] The embodiments described above are merely 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 LhCOLD1 gene, the nucleotide sequence of which is shown in SEQ ID NO:

1.

2. The expressed protein of the hybrid Liriodendron chinense LhCOLD1 gene according to claim 1, whose amino acid sequence is shown in SEQ ID NO:

2.

3. A vector and a recombinant bacterium containing the hybrid Liriodendron chinense LhCOLD1 gene according to claim 1.

4. Use of the hybrid Liriodendron chinense LhCOLD1 gene according to claim 1 in regulating stress resistance of Arabidopsis thaliana.

5. The use according to claim 4, characterized in that: The regulation of Arabidopsis stress resistance is to promote the improvement of Arabidopsis' ability to resist low temperature stress.

6. The use according to claim 5, characterized in that: The low temperature stress is -10°C.

7. The use according to claim 3, characterized in that: include: 1) Construction of an overexpression vector of the hybrid Liriodendron chinense LhCOLD1 gene; 2) Transform the constructed hybrid Liriodendron chinense LhCOLD1 gene overexpression vector into Arabidopsis thaliana; 3) Cultivate, screen and obtain transgenic Arabidopsis strains with improved resistance to low temperature stress.

8. Use of the hybrid tulipwood LhCOLD1 gene according to claim 1 in regulating the stress resistance of hybrid tulipwood.

9. The use according to claim 8, characterized in that: The method for regulating the stress resistance of hybrid tulip tree is to promote the improvement of the ability of hybrid tulip tree to resist low temperature stress.

10. The use according to claim 9, characterized in that: The low temperature stress is -10°C.

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