MdNAC9 gene and application thereof in regulating and controlling low-temperature stress resistance of plants

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to an MdNAC9 gene and application of the MdNAC9 gene in regulation and control of low-temperature stress resistance of plants, the nucleotide sequence of the MdNAC9 gene is shown as SEQ ID NO.1, and the amino acid sequence of protein coded by the gene is shown as SEQ ID NO.2. By exploring the function of the MdNAC9 gene, it is proposed and verified for the first time that the MdNAC9 gene can regulate and control the low-temperature stress resistance of the plant, compared with a wild plant, the tolerance of the MdNAC9 transgenic plant to the low-temperature stress can be improved after overexpression of the MdNAC9 gene, and a theoretical basis is provided for subsequent cultivation of cold-resistant plant varieties and germplasm resource innovation.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering and specifically relates to MdNAC9 Genes and their applications in regulating plant tolerance to cold stress. Background Art

[0002] The winters in high-altitude areas are long and the temperatures are extremely low. In the summer, the temperature difference between day and night is large and the temperature fluctuates violently. Such harsh climatic conditions are not conducive to the growth and development of plants, and change the morphology and physiological and biochemical regulation of plants. Not only can they damage the structure and function of plant cell membranes and reduce the fluidity of cell membranes; they can also induce excessive accumulation of reactive oxygen in plant cells, thereby destroying cell components such as nucleotides, proteins, and lipids, affecting the normal metabolism of cells, causing abnormal plant development, and affecting the yield and quality of economic fruit crops. In severe cases, it can even lead to plant death.

[0003] As an important economic crop in my country, apple occupies an important position in agricultural economy and ecological construction. However, in high-altitude cold areas, low temperature stress has a serious impact on the growth, development, yield and quality of apples, becoming one of the main environmental factors restricting the sustainable development of the apple industry. Low temperature stress can lead to increased levels of lipid peroxidation in apple cell membranes, destroy the integrity of cell membranes, and thus affect the normal physiological functions of plants. Therefore, exploring the key genes of apple cold stress response is of great significance for improving the cold resistance of plants and cultivating new cold-resistant varieties. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a MdNAC9 Gene and its application in regulating plant resistance to low temperature stress, the present invention MdNAC9 Gene function was explored and overexpression was found MdNAC9 The gene can improve the plant's tolerance to low temperature stress.

[0005] To achieve the above purpose, the specific technical solutions of the present invention are as follows: A first aspect of the present invention provides a MdNAC9 Application of genes in regulating plant tolerance to low temperature stress, the MdNAC9 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0006] Furthermore, the application includes improving the tolerance of plants to low temperature stress.

[0007] Furthermore, the method for improving the tolerance of plants to low temperature stress is: improving the MdNAC9 The expression level of the gene can be increased to improve the plant's tolerance to low temperature stress.

[0008] The second aspect of the present invention provides an MdNAC9 application of the protein encoded by the gene in improving the low-temperature stress tolerance of plants, and the MdNAC9 nucleotide sequence of the gene is shown in SEQ ID NO.1; the MdNAC9 amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0009] The third aspect of the present invention provides an application of a recombinant expression vector containing the MdNAC9 gene in improving the low-temperature stress tolerance of plants, and the MdNAC9 nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0010] The fourth aspect of the present invention provides an application of an engineered bacterium containing the MdNAC9 gene in improving the low-temperature stress tolerance of plants, and the MdNAC9 nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0011] Further, the above-mentioned plant is tomato or apple.

[0012] The fifth aspect of the present invention provides a method for cultivating low-temperature-tolerant transgenic plants, comprising the following steps: Connect the above-mentioned MdNAC9 gene to an expression vector to construct a recombinant expression vector; Introduce the recombinant expression vector into the plant by the Agrobacterium-mediated transformation method to MdNAC9 overexpress the gene in the plant and obtain low-temperature-tolerant transgenic plants.

[0013] Further, the expression vector is pCAMBIA1300.

[0014] Further, the Agrobacterium is Agrobacterium tumefaciens GV3101.

[0015] Compared with the prior art, the beneficial effect of the present invention lies in: The present invention firstly proposes and verifies that the MdNAC9 gene can regulate the low-temperature stress tolerance of plants. Through transcriptome analysis in the early stage of the present invention, a candidate gene related to the cold hardiness of apples was found MdNAC9 , MdNAC9 the nucleotide sequence of the gene is shown in SEQ ID NO.1. By constructing a recombinant expression vector pCAMBIA1300- MdNAC9 containing the MdNAC9 gene, and then using the Agrobacterium-mediated transformation method to transfer the recombinant expression vector into the model plant tomato plants, MdNAC9 transgenic tomato plants were obtained. Experiments found that: compared with the wild-type control plants, MdNAC9The leaf wilting and water loss of transgenic tomato plants under low-temperature stress were improved, the activities of catalase, superoxide dismutase and peroxidase were all increased, and the contents of proline and soluble protein were significantly increased, indicating that MdNAC9 Transgenic tomato plants have stronger tolerance to low-temperature stress. The present invention provides a new theoretical basis and gene resources for the cultivation of cold-resistant plants, especially cold-resistant economic fruit tree varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0017] Figure 1 For MdNAC9 The relative expression levels in different tissues of apples. The lowercase letters a, b, and c indicate significant differences, and different lowercase letters indicate significant differences between groups.

[0018] Figure 2 For MdNAC9 The identification diagram of transgenic tomatoes. The sample corresponding to the WT band is the wild-type tomato plant, and the samples corresponding to the 1st, 2nd, and 3rd bands are the overexpressed MdNAC9 Transgenic tomato NAC9-OE1, NAC9-OE2, and NAC9-OE3. NAC9-OE1, NAC9-OE2, and NAC9-OE3 are MdNAC9 Three parallel samples of transgenic tomato plants.

[0019] Figure 3 For MdNAC9 The low-temperature tolerance phenotype of transgenic tomato plants; Figure 3 Figure A of Figure 3 is the control group treated at 25°C, Figure 3 Figure B of Figure 3 and Figure C of Figure 3 are both experimental groups treated at 4°C for 4 days. MdNAC9 Figure B is the top view of the plant, Figure C is the front view of the plant; WT represents the wild-type tomato plant, and NAC9-OE1, NAC9-OE2, and NAC9-OE3 are

[0020] Three parallel samples of transgenic tomato plants. Figure 4 For MdNAC9 The determination results of catalase activity in transgenic tomatoes and wild-type tomatoes. ** indicates that MdNAC9 There is a significant difference in the catalase activity levels between transgenic tomatoes and wild-type tomatoes.p <0.01.

[0021] Figure 5 is MdNAC9 The determination results of superoxide dismutase activity in transgenic tomatoes and wild-type tomatoes, ** indicates MdNAC9 There is a significant difference in the superoxide dismutase activity levels between transgenic tomatoes and wild-type tomatoes, p <0.01.

[0022] Figure 6 is MdNAC9 The determination results of peroxidase activity in transgenic tomatoes and wild-type tomatoes, ** indicates MdNAC9 There is a significant difference in the peroxidase activity levels between transgenic tomatoes and wild-type tomatoes, p <0.01.

[0023] Figure 7 is MdNAC9 The determination results of malondialdehyde content in transgenic tomatoes and wild-type tomatoes, ** indicates MdNAC9 There is a significant difference in the malondialdehyde content levels between transgenic tomatoes and wild-type tomatoes, p <0.01.

[0024] Figure 8 is MdNAC9 The determination results of proline content in transgenic tomatoes and wild-type tomatoes, ** indicates MdNAC9 There is a significant difference in the proline content levels between transgenic tomatoes and wild-type tomatoes, p <0.01.

[0025] Figure 9 is MdNAC9 The determination results of soluble protein content in transgenic tomatoes and wild-type tomatoes, ** indicates MdNAC9 There is a significant difference in the soluble protein content levels between transgenic tomatoes and wild-type tomatoes, p <0.01. Specific implementation manners

[0026] The specific implementation manners of the present invention will be described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific implementation manners. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0027] In the present invention: the all-round plant RNA extraction kit was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd., with the item number CW2598S; the reverse transcription kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd., with the item number AE311-02; the DNA product purification kit was purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd., with the item number CW2301M; the T5 cloning vector was purchased from Quanshijin Company, with the item number CT501; the Trans1-T1 competent cells were purchased from Quanshijin Company, with the item number CD501; and the Agrobacterium GV3101 competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd., with the item number AC1001.

[0028] As an important economic crop in my country, apple occupies an important position in agricultural economy and ecological construction. However, in high-altitude cold areas, low temperature stress has a serious impact on the growth, development, yield and quality of apples, becoming one of the main environmental factors restricting the sustainable development of the apple industry. Low temperature stress can lead to increased levels of lipid peroxidation in apple cell membranes, destroy the integrity of cell membranes, and thus affect the normal physiological functions of plants. Therefore, exploring the key genes of apple cold stress response is of great significance for improving the cold resistance of plants and cultivating new cold-resistant varieties.

[0029] The present invention provides a MdNAC9 Gene and its application in regulating plant tolerance to low temperature stress. In the early stage of the present invention, transcriptome analysis of apple genes was performed and a candidate gene related to apple cold resistance was found. MdNAC9 , MdNAC9 The nucleotide sequence of the gene is shown in SEQ ID NO.1. MdNAC9 The recombinant expression vector pCAMBIA1300- MdNAC9 Then, the recombinant expression vector was transformed into the model plant tomato plant using Agrobacterium transformation to obtain MdNAC9 Transgenic tomato plants. The experiment found that compared with wild-type control plants, MdNAC9 The transgenic tomato plants showed improved leaf wilting and water loss under low temperature stress, and their catalase, superoxide dismutase and peroxidase activities were increased, and their proline and soluble protein contents were significantly increased, indicating that MdNAC9 Transgenic tomato plants have a stronger tolerance to low temperature stress.

[0030] Embodiment 1: MdNAC9 Relative expression of genes in different tissues of apple The present invention screens candidate genes from the transcriptome database MdNAC9 , MdNAC9 The nucleotide sequence of the gene is shown in SEQ ID NO.1. MdNAC9The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0031] SEQ ID NO.1: Atgagcggcagcgtcgcacctgaagaaggagatgcaatgcgtgtgaatgtgagccgactacctgggttcggattctcccccactgatgagttactggtcagctactacctgaagaacaagatagaggggacggactcccatttccgccacgtcatccctgaaatcgatgtctgcaagtacgagccctgcgatattcctgcattcttcccagaagttcatgggaaggagtggttcttctttagccggctcgattacaagtacaacggcactcgctgcaaccggaccacgggtcaaggcttttacaagatcacaggaatggatcgtaagatcagggctgaagaatccaaagctgtgattgggaagaagaggattctgacattctacgaaggtcgtctgccgaaatcaaagaagaccaattgggtactccatgagtataatctcacagaaactaaggtgagttctaaacccaccaagcagatgaactttgtcctctggcgcctgaagaacatgtcagctagttataagaagccgaagggtgatccaatccacggcgaattagctgatacaggtgctacctcggaagattatcaagctgctgtaagtgacgtgattgcagagccagtggaacagctgggataa。

[0032] SEQ ID NO.2: MSGSVAPEEGDAMRVNVSRLPGFGFSPTDELLVSYYLKNKIEGTDSHFRHVIPEIDVCKYEPCDIPAFFPEVHGKEWFFFSRLDYKYNGTRCNRTTGQGFYKITGMDRKIRAEESKAVIGKKRILTFYEGRLPKSKKTNWVLHEYNLTETKVSSKPTKQMNFVLWRLKNMSASYKKPKGDPIHGELADTGATSEDYQAAVSDVIAEPVEQLG*。

[0033] To identify MdNAC9 the relative expression levels of genes in different tissues of apples, qRT-PCR was used to detect the expression levels of these genes in the roots, shoot tips, new leaves, mature leaves and fruits of apple plants. The specific methods are as follows: S1. The root, shoot tip, new leaf, mature leaf and fruit tissues of the cold-region small apple 'Jinhong' plants collected were placed in liquid nitrogen for quick freezing and then stored in an -80°C refrigerator for later use; the RNA of the above different tissues was extracted using an all-in-one plant RNA extraction kit.

[0034] S2. The extracted RNA was reverse transcribed into the first strand of cDNA using a reverse transcription kit.

[0035] S3. Design MdNAC9 the quantitative primer set for MdNAC9 The quantitative primer set for MdNAC9 contains a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4. qRT-PCR was performed according to the reaction system shown in Table 1 and the reaction conditions shown in Table 2, and the relative expression levels of

[0036] SEQ ID NO.3: 5’-CAAGTACAACGGCACTCGCT-3’; SEQ ID NO.4: 5’-TTGATTTCGGCAGACGACCT-3’.

[0037] Table 1 Reaction system Table 2 Reaction conditions Note: “-” in the table indicates no cycling.

[0038] MdNAC9 The results of the expression levels of Figure 1 genes in different tissues of apple plants are as MdNAC9 shown. It can be seen from the figure that MdNAC9 genes are expressed in different tissues of apple plants, MdNAC9 the expression level is the highest in new leaves, followed by

[0039] Example 2: Genetic transformation and positive identification of tomatoes Since it is very difficult to transform apple plants and the juvenile period of plants is long, while the model plant tomato has a fast growth cycle, the 'Little Tom' tomato was selected as the plant material in this invention to explore MdNAC9The specific biological functions of genes in plant tolerance to low temperature stress.

[0040] 1. Obtaining recombinant bacteria S1. PCR amplification: Using the cDNA synthesized in Example 1 as a template, design a specific primer combination for PCR amplification. The specific primer combination for PCR amplification includes MdNAC9-F and MdNAC9-R. The nucleotide sequence of MdNAC9-F is shown in SEQ ID NO.5, and the nucleotide sequence of MdNAC9-R is shown in SEQ ID NO.6. Perform PCR amplification according to the reaction system shown in Table 3 and the reaction conditions shown in Table 4 to obtain a PCR amplification product.

[0041] SEQ ID NO.5: 5’-ACGGGGGACGAGCTCGGTACCATGGCTGCTCCTACAACCCCAA-3’; SEQ ID NO.6: 5’-GGTGTCGACTCTAGAGGATCCCGGCCCATCCATGTTCC-3’.

[0042] Table 3 MdNAC9 Gene PCR reaction system Table 4 PCR reaction conditions Note: “-” in the table indicates no cycle.

[0043] S2. Electrophoresis and gel recovery: Perform agarose gel electrophoresis on the PCR amplification product using a 1g / 100mL agarose solution. Cut the bright single band and recover it. The gel recovery step is operated according to the instructions of the DNA product purification kit to obtain a gel-recovered DNA fragment.

[0044] S3. Ligation of DNA fragments: Add 2.5 μL of the DNA fragment obtained in step S2 and 1 μL of the T5 cloning vector to a PCR tube. Mix well and place it in a PCR instrument at 25 °C for 5 min to obtain a ligation product.

[0045] S4, Transformation and Cultivation: Add 3.5 μL of the ligation product obtained in step S3 to 50 μL of Trans1-T1 competent cells, then incubate on ice for 25 min, perform heat shock at 42 °C in a water bath for 30 s, and incubate on ice for 2 min. Do not shake the centrifuge tube during this process; add 250 μL of LB liquid medium to the centrifuge tube after transformation, mix well, and incubate at 37 °C and 200 rpm for 1 h to allow the cells to recover; then centrifuge at 15000 rpm for 1 min to collect the colonies, discard the supernatant, retain 100 μL of the cell suspension, resuspend the cells, and spread the cell suspension on an LB solid medium plate containing 0.05 mg / mL kanamycin and 0.05 mg / mL rifampicin, and incubate inverted overnight in a 37 °C constant temperature incubator.

[0046] S5, Detection of Positive Clones: Pick a white single colony from the overnight culture plate in step S4 into an LB liquid medium containing 0.05 mg / mL kanamycin and 0.05 mg / mL rifampicin, incubate at 37 °C and 200 rpm on a shaker for 4 h, and finally perform PCR verification and sequencing on the bacterial solution to screen for the DNA fragment successfully ligated to the promoter.

[0047] S6, Double Digestion: Perform double digestion on the pCAMBIA1300 vector and the DNA fragment ligated to the promoter respectively. The double digestion reaction system for the pCAMBIA1300 vector is shown in Table 5, and the double digestion reaction system for the DNA fragment ligated to the promoter is shown in Table 6. After digestion, place the reaction system in an 80 °C water bath for 15 min to inactivate the enzyme thermally, obtain the digested DNA fragment and the digested pCAMBIA1300 vector, and store them at 4 °C for standby.

[0048] Table 5 Double Digestion System for pCAMBIA1300 Vector Table 6 Double Digestion System for DNA Fragment Ligated to Promoter S7, Ligation: Ligate the digested DNA fragment and the digested pCAMBIA1300 vector at 22 °C according to the ligation system shown in Table 7.

[0049] Table 7 Ligation System for Digested Products S8, Verification: Use PCR technology to detect MdNAC9 whether it is ligated to the pCAMBIA1300 vector. When the size of the amplified target band is consistent with MdNAC9 the CDS size of MdNAC9 , it proves that the vector construction is successful, and finally screen for the recombinant plasmid pCAMBIA1300- MdNAC9 .

[0050] S9. Obtaining of recombinant bacteria: Add 6 μL of the recombinant expression vector pCAMBIA1300- MdNAC9 into a centrifuge tube containing 100 μL of Agrobacterium tumefaciens GV3101 competent cells. After mixing evenly, let it stand 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 successively; then add 700 μL of LB liquid medium into the tube and place it in a shaker at 28 °C for 2 h; after the culture is completed, centrifuge at 6000 rpm for 1 min; discard the centrifuged supernatant, retain 100 μL of the bacterial liquid with the bacterial pellet, gently resuspend the bacterial pellet with a pipette, and spread it on an LB solid medium containing 0.05 mg / mL kanamycin and 0.05 mg / mL rifampicin, and culture at 28 °C for 2 d; after single colonies grow on the medium, pick white monoclonal colonies into an LB liquid medium containing 0.05 mg / mL kanamycin and 0.05 mg / mL rifampicin, culture overnight at 28 °C and 180 rpm, and finally verify the obtained bacterial liquid by PCR to obtain recombinant bacteria.

[0051] 2. Tomato genetic transformation using the Agrobacterium-mediated transformation method S1. Seed disinfection and culture: Soak 0.5 g of "Little Tom" tomato seeds in sterile distilled water overnight for water absorption. The next day, first immerse all the seeds in 75% absolute ethanol in a laminar flow hood for 3 min, and then wash them 3 times with sterile distilled water to wash away the residual alcohol. Then add a sodium hypochlorite solution with an effective chlorine ion concentration of 2% to soak the seeds for 15 min, and finally wash them 3 times with sterile distilled water to remove the residual sodium hypochlorite solution. Sow the disinfected seeds in a 1 / 2MS medium for culture.

[0052] S2. Preparation of infection solution: Add the obtained recombinant bacteria into an LB liquid medium containing 0.05 mg / mL kanamycin and 0.05 mg / mL rifampicin, and activate and culture in a constant temperature shaker at 28 °C and 220 rpm for 16 h. Then resuspend the bacterial cells with MS liquid medium, and adjust the OD 600 value of the bacterial liquid to 0.5 to prepare the infection solution.

[0053] S3. Infection and co-culture: After the tomato seedlings grow in the medium for half a month, use scissors in a laminar flow hood to cut the tomato leaves into small pieces of 0.5 cm × 0.5 cm and the stems into small sections of 0.5 cm in length. Contact the wound with the pre-culture medium and evenly place it on the pre-culture medium for dark culture for 48 h. Then fully immerse the leaves and stem segments in the infection solution again, slowly shake for 15 min, then place them on sterile filter paper to absorb the residual infection solution, and transfer them to the co-culture medium for dark culture at 25 °C for 48 h.

[0054] S4. Screening and Bud Induction: Transfer the co-cultured materials to a screening medium for screening resistant buds, and culture them under the conditions of 25°C, 16 h light / 8 h dark for 2 weeks until resistant buds grow. Transfer the resistant buds to a bud elongation medium, and when the resistant buds grow to 2 cm under the conditions of 25°C, 16 h light / 8 h dark, transfer them to a rooting medium to induce root differentiation.

[0055] S5. Transplanting and Screening of Transgenic Seeds: When root differentiation is complete, transplant the seedlings into the soil for cultivation, harvest the transgenic tomato seeds, and screen the seeds using a seed screening medium to obtain stably transformed MdNAC9 transgenic tomatoes. Transplant them into nutrient pots for cultivation, and use them for stress treatment and determination of related indicators.

[0056] The media used in the tomato genetic transformation stage are shown in Table 8.

[0057] Table 8 Composition of Media in the Tomato Genetic Transformation Stage Example 3: MdNAC9 Identification of Transgenic Tomato Plants S1. According to the steps in the instruction manual of the all-purpose plant RNA extraction kit, extract the DNA of fresh leaves of the transgenic tomato plants prepared in Example 2, and at the same time use the leaves of non-transgenic tomato plants as a negative control. MdNAC9 S2. Use a reverse transcription kit to reverse transcribe the extracted RNA into the first strand of cDNA.

[0058] S3. According to the PCR amplification method in Example 2, perform PCR amplification on the cDNA obtained in step S2 of this example to obtain a PCR amplification product.

[0059] S4. Use a 1 g / 100 mL agarose solution to perform agarose gel electrophoresis on the PCR amplification product.

[0060] Observe the bands on the gel. As

[0061] shown, the transgenic plant samples show bands of the expected size, while the negative control shows no bands, proving that Figure 2 the gene has been successfully transferred into the 'Little Tom' tomato. MdNAC9

[0062] Example 4: MdNAC9 Effect of the Gene on the Low Temperature Stress Tolerance of Plants Transfer two-week-old wild-type tomato plants and MdNAC9 transgenic tomato plants to flower pots respectively, place them at 4°C for 4 days for low temperature stress treatment to simulate a low temperature stress environment, and the treatment conditions for the control group are 25°C for 4 days to explore MdNAC9The influence of genes on the physiological state and stress resistance of plants under low temperature stress.

[0063] Catalase, superoxide dismutase and peroxidase are key antioxidant enzymes that can scavenge reactive oxygen species and reduce oxidative damage; malondialdehyde is a product of lipid peroxidation, and its content reflects the degree of oxidative damage to cell membranes; proline and soluble proteins are important osmotic adjustment substances that can maintain the osmotic pressure and structural stability inside cells. After low temperature stress treatment, observe the leaf color and growth state of the plants, measure the activities of catalase, superoxide dismutase, peroxidase, the content of malondialdehyde, proline content and soluble protein content, and analyze MdNAC9 The influence of genes on the antioxidant capacity of plants.

[0064] 1. Test method The activity of catalase was measured by spectrophotometry. The specific measurement method can be referred to "Xu F, Liu S, Liu Y, et al. Effectiveness of lysozyme coatings and 1-MCP treatments on storage and preservation of kiwifruit[J]. Food Chem, 2019, 288: 201-207.".

[0065] The activity of superoxide dismutase was measured by nitroblue tetrazolium photoreduction method. The specific measurement method can be referred to "Li Zhongguang, Li Jianghong, Du Chaokun, et al. Simultaneous determination of five plant antioxidant enzymes in a single extraction system[J]. Journal of Yunnan Normal University, 2002, 22(6): 44-48.".

[0066] The activity of peroxidase was measured by guaiacol method. The specific measurement method can be referred to "Zhang L, Pei Y, Wang H, et al. Hydrogen sulfide alleviates cadmium-induced cell death through restraining ROS accumulation in roots of Brassica rapa L. ssp. Pekinensis[J]. Oxid Med Cell Longev, 2015, 1-11.".

[0067] The content of malondialdehyde was determined by spectrophotometric colorimetry. For the specific determination method, see "Huang Q, Qian X, Jiang T, et al. Effect of eugenol fumigation treatment on chilling injury and CBF gene expression in eggplant fruit during cold storage[J]. Food Chem, 2019, 4:48.".

[0068] The determination method of proline content can be found in "Li P, Zheng X, Liu Y, et al. Pre-storage application of oxalic acid alleviates chilling injury in mango fruit by modulating proline metabolism and energy status under chilling stress[J]. Food Chem, 2014, 142: 72-78.".

[0069] The determination method of soluble protein content can be found in "Karimi F, Hamidian Y, Behrouzifar F, et al. An applicable method for extraction of whole seeds protein and its determination through Bradford's method[J]. Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association, 2022, 164:113053.".

[0070] 2. Test Results As Figure 3 shown, after low-temperature treatment, the leaves of wild-type tomatoes showed wilting and water loss, while MdNAC9 the transgenic tomato leaves showed less wilting and water loss.

[0071] The wild-type and MdNAC9 transgenic tomatoes' antioxidant capacities are as Figures 4 - 9 shown. The wild-type and MdNAC9There were no significant differences in the physiological indices of transgenic tomatoes before low - temperature treatment. However, after 4 days of low - temperature treatment, compared with the wild - type tomatoes after low - temperature treatment, except for the decrease in malondialdehyde content, the activities of catalase, superoxide dismutase and peroxidase in transgenic tomatoes increased, and the contents of proline and soluble protein increased, indicating that MdNAC9 the level of cell - membrane lipid peroxidation in transgenic tomatoes was significantly lower than that of the control, reducing the oxidative damage caused by low - temperature stress and having a stronger tolerance to low - temperature stress.

[0072] It should be noted that when the present invention involves numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the step methods adopted are the same as those in the embodiments, to prevent repetition, the present invention describes the preferred embodiments. Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they know the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0073] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. MdNAC9 Use of a gene in regulating low-temperature stress tolerance of plants, characterized in that, The MdNAC9 nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. Use according to claim 1 MdNAC9 Application of the gene in regulating plant tolerance to low temperature stress, characterized in that The application includes enhancing the tolerance of plants to low-temperature stress; the method for enhancing the tolerance of plants to low-temperature stress is as follows: by means of transgenic technology or gene editing technology, the expression level of the MdNAC9 gene is increased to enhance the tolerance of plants to low-temperature stress.

3. An MdNAC9 application of a protein encoded by a gene in improving low temperature stress tolerance of plants, characterized in that The said MdNAC9 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the said MdNAC9 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

4. Use of a recombinant expression vector containing MdNAC9 gene in improving plant tolerance to low temperature stress, characterized in that The MdNAC9 nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. Use of a recombinant bacterium containing MdNAC9 gene in enhancing plant tolerance to low temperature stress, characterized in that The MdNAC9 nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. The application according to any one of claims 1 to 5, characterized in that The plant is tomato or apple.

7. A method for cultivating low-temperature tolerant transgenic plants, characterized in that, It includes the following steps: Connect the gene described in claim 1 MdNAC9 to an expression vector to construct a recombinant expression vector; The recombinant expression vector is introduced into a plant to overexpress the MdNAC9 gene in the plant, thereby obtaining a transgenic plant with cold tolerance.

8. The method for cultivating a low-temperature resistant transgenic plant according to claim 7, characterized in that, The expression vector is pCAMBIA1300.

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