Application of AhMFT1 gene in improving waterlogging tolerance of plants

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of an AhMFT1 gene in improving waterlogging tolerance of plants, the nucleotide sequence of the AhMFT1 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. The invention further discloses a preparation method of the AhMFT1 gene. The method comprises the following steps: firstly, constructing a recombinant expression vector pCAMBIA1307-AhMFT1 containing an AhMFT1 gene, and then transferring the AhMFT1 gene into a plant by utilizing agrobacterium tumefaciens-mediated genetic transformation, so as to obtain a transgenic plant. A flooding treatment experiment shows that the tolerance of the plant to waterlogging stress can be improved by overexpressing the AhMFT1 gene, and the waterlogging tolerance of the plant can be subjected to genetic improvement by utilizing the AhMFT1 gene.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering technology and specifically relates to AhMFT1 Application of genes in improving waterlogging tolerance in plants. Background Art

[0002] Peanuts are an important economic crop with high nutritional value and a wide range of uses. In regions where plum rains are prevalent, the uneven temporal and spatial distribution of precipitation caused by the monsoon, coupled with the terrain's high west and low east, makes the peanut growing season extremely vulnerable to flooding. Peanuts produce fruit underground, and pod development requires a balanced balance of water and air in the soil. Research has shown that peanuts have a medium-to-low water requirement among crops, and waterlogging or excessively wet soils can affect peanut development. Flooding exposes peanut roots to prolonged submergence, leading to oxygen deprivation, increased respiration, and increased energy consumption. This alters some metabolic pathways, triggering physiological and biochemical reactions that affect pod development, resulting in blighted pods and, in severe cases, a complete peanut harvest.

[0003] Therefore, breeding peanut varieties with waterlogging tolerance can ensure high and stable peanut yields in waterlogged areas and is also the most economical, safe, and effective method. However, conventional breeding methods are time-consuming and labor-intensive, and it is difficult to improve target traits in a targeted manner. With the continuous deepening of research in functional genomics and molecular-assisted breeding technology, discovering key genes for waterlogging tolerance in peanuts and combining these discovered peanut waterlogging tolerance genes with conventional breeding methods is an effective way to efficiently, accurately, and quickly breed new waterlogging-tolerant varieties.

[0004] Therefore, exploring the key genes for waterlogging tolerance in peanuts is of great significance for improving the waterlogging tolerance of plants and cultivating new waterlogging-resistant varieties. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a AhMFT1 The present invention is to use the gene in improving the waterlogging resistance of plants. AhMFT1 Gene function was explored and overexpression was found AhMFT1 The gene can improve the plant's tolerance to low temperature stress.

[0006] 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 AhMFT1 Application of genes in improving waterlogging tolerance of plants, the AhMFT1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0007] Furthermore, the application method is as follows: using transgenic technology or gene editing technology to improve the AhMFT1The expression level of the gene can be increased to improve the plant's tolerance to waterlogging stress.

[0008] Furthermore, the plant is tobacco or peanut.

[0009] The second aspect of the present invention provides an application of AhMFT1 protein in improving plant waterlogging tolerance, wherein the AhMFT1 protein is composed of the above-mentioned AhMFT1 Genetic coding.

[0010] Furthermore, the plant is tobacco or peanut.

[0011] The third aspect of the present invention provides an application of a recombinant expression vector in improving waterlogging tolerance of plants, wherein the recombinant expression vector contains the above-mentioned AhMFT1 Gene.

[0012] Furthermore, the plant is tobacco or peanut.

[0013] The fourth aspect of the present invention provides a use of a recombinant bacterium in improving plant waterlogging tolerance, wherein the recombinant bacterium contains the AhMFT1 Gene.

[0014] Furthermore, the plant is tobacco or peanut.

[0015] A fifth aspect of the present invention provides a method for cultivating waterlogging-tolerant transgenic plants, comprising the following steps: The above-mentioned AhMFT1 The gene is connected to the expression vector to construct a recombinant expression vector; The recombinant expression vector is introduced into plants through Agrobacterium transformation. AhMFT1 The gene is overexpressed in plants to obtain waterlogging-tolerant transgenic plants.

[0016] Furthermore, the expression vector is pCAMBIA1307.

[0017] Furthermore, the Agrobacterium is EHA105.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The present invention first proposed and verified AhMFT1 Genes can regulate plant waterlogging tolerance. This study conducted transcriptome and metabolome analyses on samples of the waterlogging-tolerant peanut variety 'Changhua 18' and the waterlogging-sensitive peanut variety 'Huayu 39', and discovered a candidate gene related to plant waterlogging tolerance. AhMFT1 , by constructing a AhMFT1 The recombinant expression vector pCAMBIA1307- AhMFT1Then, the recombinant expression vector was transferred into the plant by Agrobacterium-mediated genetic transformation to obtain transgenic plants. AhMFT1 Genes can improve plant tolerance to waterlogging stress. AhMFT1 Genes can be used to genetically improve plant waterlogging tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0020] Figure 1 The map of expression vector pCAMBIA1307, * indicates the expression vector pCAMBIA1307 BsaB The I restriction site is blocked by Dam methylation.

[0021] Figure 2 The electrophoresis diagram for the identification of T0 generation positive seedlings of transgenic tobacco for 6 candidate genes; Figure 2 Figure A, Figure 2 Figure B, Figure 2 Figure C, Figure 2 Figure D, Figure 2 Figure E, Figure 2 The samples of the F graph are AhMFT1 genetically modified tobacco, AhMFT5 genetically modified tobacco, AhMFT6 genetically modified tobacco, AhMFT8 genetically modified tobacco, AhMFT10 genetically modified tobacco, AhMFT14 genetically modified tobacco; Figure 2 In the electrophoresis diagram, WT represents wild-type tobacco, and the Arabic numerals represent different samples of transgenic tobacco.

[0022] Figure 3 The phenotypic results of transgenic tobacco and wild-type tobacco of 6 candidate genes under waterlogging stress; Figure 3 Figure A is AhMFT1 genetically modified tobacco, AhMFT5 genetically modified tobacco, AhMFT6 Phenotypes of transgenic tobacco and wild-type tobacco under waterlogging stress, Figure 3 The (a) row of Figure A is the picture before flooding. Figure 3 The (b) row of Figure A shows the pictures taken on the 5th day of flooding treatment. Figure 3 Figure A shows parallel experiments of wild-type tobacco from the first to the third columns from left to right. Figure 3Figure A from left to right 4th to 6th columns are AhMFT1 Parallel trials of genetically modified tobacco, Figure 3 Figure A from left to right 7th to 9th columns are AhMFT5 Parallel trials of genetically modified tobacco, Figure 3 Figure A from left to right 10th to 12th columns are AhMFT6 Parallel trials of genetically modified tobacco; Figure 3 Figure B is AhMFT8 genetically modified tobacco, AhMFT10 genetically modified tobacco, AhMFT14 Phenotypes of transgenic tobacco and wild-type tobacco under waterlogging stress, Figure 3 The (a) row of Figure B is the picture before flooding. Figure 3 The (b) row of Figure B shows the pictures taken on the 5th day of flooding treatment. Figure 3 Figure B shows parallel experiments of wild-type tobacco from the first to the third columns from left to right. Figure 3 Figure B from left to right 4th to 6th columns are AhMFT8 Parallel trials of genetically modified tobacco, Figure 3 From left to right, the 7th to 9th columns of Figure B are AhMFT10 Parallel trials of genetically modified tobacco, Figure 3 From left to right, the 10th to 12th columns of Figure B are AhMFT14 Parallel trials of transgenic tobacco.

[0023] Figure 4 is the survival rate and water loss rate of tobacco after flooding treatment, Figure 4 Figure A shows the survival rate. Figure 4 Figure B shows the water loss rate; WT represents wild-type tobacco, 10-4, 10-6 and 10-8 represent constructed AhMFT1 Three parallel samples of transgenic tobacco lines; ** indicates p <0.01.

[0024] Figure 5 for AhMFT1 Transgenic tobacco has enhanced resistance to waterlogging stress; Figure 5 Row A shows the pictures of the flooding treatment on day 0. Figure 5 Row B shows the pictures of the first day of flooding treatment. Figure 5 Row C shows the pictures taken on the third day of flooding treatment. Figure 5 The D row is the picture of the fifth day after flooding treatment; WT represents the wild-type tobacco as the control group, 10-4, 10-6 and 10-8 are the constructed AhMFT1 Three parallel samples of transgenic tobacco lines; Figure 5 From left to right, the first to third columns are wild-type tobacco. Figure 5 From left to right, the 4th to 6th columns are AhMFT1 Transgenic tobacco 10-4, Figure 5 From left to right, the 7th to 9th columns are AhMFT1 Transgenic tobacco 10-6, Figure 5 From left to right, the 10th to 12th columns are AhMFT1 Transgenic tobacco 10-8.

[0025] Figure 6 for AhMFT1 Subcellular localization of gene-encoded proteins, Figure 6 Figure (a), Figure 6 Figure (b), Figure 6 Figure (c) and Figure 6 Figure (d) is the image of the control without load. Figure 6 Figure (e), Figure 6 Figure (f), Figure 6 The (g) graph and Figure 6 Figure (h) is the image of EGFP-AhMFT1. Figure 6 Figure (a) and Figure 6 Figure (e) shows the image observed through the green fluorescent protein filter. Figure 6 Figure (b) and Figure 6 Figure (f) shows the image observed through the red fluorescent protein filter. Figure 6 Figure (c) and Figure 6 Figure (g) is a bright field image. Figure 6 Figure (d) is Figure 6 Figure (a), Figure 6 Figure (b) and Figure 6 (c) The merged image, Figure 6 The (h) graph is Figure 6 Figure (e), Figure 6 (f) graph and Figure 6 (g) The merged image. DETAILED DESCRIPTION

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

[0027] In the present invention, Agrobacterium EHA105 was purchased from Qingke Biotechnology, and the pCAMBIA1307 expression vector and the vector pCAMBIA1300-35S-EGFP were donated by the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences.

[0028] Peanuts are an important economic crop with high nutritional value and a wide range of uses. However, in regions where plum rains are prevalent, the uneven spatial and temporal distribution of precipitation caused by the monsoon and the terrain's high west and low east make peanuts highly susceptible to flooding during their growing season. Peanuts are sensitive to waterlogging, and flooding can lead to fruit failure, which can even lead to crop failure in severe cases. Therefore, identifying key genes responsible for waterlogging tolerance in peanuts is of great significance for improving plant tolerance and cultivating new waterlogging-tolerant varieties.

[0029] The present invention provides a AhMFT1 The application of genes in improving plant waterlogging tolerance. This paper conducted transcriptome analysis and metabolome analysis on samples of the waterlogging-tolerant peanut variety 'Changhua 18' and the waterlogging-sensitive peanut variety 'Huayu 39', and found a candidate gene related to plant waterlogging tolerance. AhMFT1 , by constructing a AhMFT1 The recombinant expression vector pCAMBIA1307- AhMFT1 Then, the recombinant expression vector was transferred into the plant by Agrobacterium-mediated genetic transformation to obtain transgenic plants. AhMFT1 Genes can improve plant tolerance to waterlogging stress. AhMFT1 Genes can be used to genetically improve plant waterlogging tolerance.

[0030] Example 1: Screening of peanut waterlogging-tolerant MYB transcription factor genes The waterlogged peanut cultivar 'Changhua 18' and the waterlogged peanut cultivar 'Huayu 39', both grown to the flowering stage and showing similar growth, were selected. 'Changhua 18' was labeled CH18, and 'Huayu 39' was labeled HY39. The pots of CH18 and HY39 were placed in a flooding chamber and filled with tap water until the soil reached a depth of 5 cm above the soil surface. Samples were collected at 6 hours, 24 hours, 3 days, and 5 days after the flooding treatment. Three biological replicates were used for each sampling. The plant samples were washed with distilled water until the surface of the plant was free of soil and dirt. The samples were then blotted dry with absorbent paper. The tissues were minced and sent to a biotechnology company for transcriptome and metabolome analysis.

[0031] Through transcriptome analysis and metabolome analysis, 2104 differentially expressed genes (DEGs) that responded to waterlogging stress were screened out from all genes; among these 2104 DEGs, 36 different transcription factor families were found to respond to waterlogging stress, among which the largest was the MYB transcription factor gene family, with 205 members.

[0032] The 205 MYB transcription factor encoding genes screened out were classified, including 90 R2R3-MYB transcription factor encoding genes. Then, a cluster analysis was performed on the 90 R2R3-MYB transcription factor encoding genes during the CH18 and HY39 flooding treatments. It was found that 16 R2R3-MYB genes showed significant differential expression in CH18 and HY39.

[0033] Example 2: Cultivation of transgenic tobacco From the 16 R2R3-MYB genes showing significant differential expression between CH18 and HY39 obtained in Example 1, 6 genes with tissue-specific expression were selected: AhMFT1 、 AhMFT5 、 AhMFT6 、 AhMFT8 、 AhMFT10 、 AhMFT14 By constructing an overexpression gene vector and using Agrobacterium-mediated genetic transformation, the six genes were transferred into tobacco respectively. AhMFT1 genetically modified tobacco, AhMFT5 genetically modified tobacco, AhMFT6 genetically modified tobacco, AhMFT8 genetically modified tobacco, AhMFT10 Genetically modified tobacco and AhMFT14 Genetically modified tobacco. AhMFT1 The construction method of transgenic tobacco is used as an example to illustrate the following: AhMFT1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoding the gene is shown in SEQ ID NO.2.

[0034] SEQ ID NO.1: 。

[0035] SEQ ID NO.2: MEMEYTSSVHMIMRSLSDCGSSVTNGSEEDMEIRKGPWTEEEDSALLNHITTYGLKRSGKSCRLRWLNYLRPNVRRGNITLEEQLLILDLHSRWGNRWSKIAEQLPGRTDNEIKNYWRTRVVKQAKQLKCDV NSKQFRDALRYVWMPRLIEQIQAQGRSTMCVSQAREIVNPVTSVSMASSKSCSSFSGCEQFQASSSVSDSCVSYYSLMGSGSGGGSSEHAEKGATSSSTPFEPENGFGGADLWTDENIWFLQQQLADDDHL.

[0036] S1. Extract RNA and reverse transcribe into cDNA The total RNA of the plant was extracted from the leaves of CH18, and the first-chain cDNA was synthesized using the reverse transcription kit of Takara Company.

[0037] S2. PCR amplification The nucleotide sequence is shown in SEQ ID NO.1 AhMFT1 Design of DNA sequences specific to gene coding regions AhMFT1 A specific primer combination for the gene, comprising an upstream primer having a nucleotide sequence as shown in SEQ ID NO.3 and a downstream primer having a nucleotide sequence as shown in SEQ ID NO.4, was used to amplify the cDNA in step S1 as a template according to the following PCR amplification system and PCR amplification procedure. AhMFT1 Gene, and obtain PCR products.

[0038] SEQ ID NO.3: 5'-CGCGGATCCATGGAGATGGAGTACACAAG-3'; SEQ ID NO. 4: 5'-CCGGAATTCAAGATGATCATCATCAGCAA-3'.

[0039] PCR amplification system: 10 µL Prime STAR Max Premix, 1 µL upstream primer, 1 µL downstream primer, 1 µL cDNA, 8 µL ddH2O.

[0040] PCR amplification program: 95°C for 30 seconds; 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 10 seconds, for 30 cycles.

[0041] S3. Electrophoresis and gel recovery: The PCR products were subjected to agarose gel electrophoresis, and the successfully amplified gene fragments were excised and recovered.

[0042] S4, recombinant expression vector CAMBIA1307- AhMFT1 Construction: Expression vector pCAMBIA1307 map as shown Figure 1 As shown, according to the enzyme digestion system shown below, use restriction endonucleases BamH I and restriction enzymes EcoR I. Double-enzyme digestion was performed on the expression vector pCAMBIA1307 and the recovered gene fragment to obtain the linearized pCAMBIA1307 expression vector and the gene fragment after enzyme digestion, respectively.

[0043] Enzyme digestion system: 5µL 10× QuickCut buffer, 1µL QuickCut BamH I. 1µL QuickCut EcoR I. 5 µL of expression vector pCAMBIA1307 or the gene fragment recovered in step S3, and 38 µL of ddH2O.

[0044] Then, using T4 ligase, the linearized pCAMBIA1307 expression vector and the gene fragment after enzyme digestion were connected according to the following ligation reaction system to obtain the recombinant expression vector CAMBIA1307- AhMFT1 .

[0045] Ligation reaction system: 1µL T4 ligase, 1µL 10× Buffer, 7µL digested gene fragment, and 1µL linearized pCAMBIA1307 expression vector.

[0046] S5, transformation: the recombinant expression vector CAMBIA1307- AhMFT1 The cells were introduced into Agrobacterium EHA105 competent cells and recombinant bacteria were obtained after culture.

[0047] S6. Preparation of infection solution Activation of recombinant bacteria: The recombinant bacteria obtained in step S5 were inoculated into YEB medium containing 50 μg / mL kanamycin and 25 μg / mL rifampicin, and cultured at 28°C and 200 rpm until the bacterial solution OD 600 The value is 0.6, and the activated bacterial solution is obtained.

[0048] Preparation of infection medium: Transfer 1 mL of activated bacterial suspension to 50 mL of YEB medium containing 50 μg / mL kanamycin and 25 μg / mL rifampicin, and culture overnight at 28°C with shaking at 200 rpm. Then centrifuge at 5000 rpm for 5 minutes at room temperature, discard the supernatant, and resuspend the bacterial pellet in acetosyringone at a final concentration of 100 μM to an OD of 0. 600 The value is 1.0 and the mixture is allowed to stand at room temperature for 2 hours.

[0049] S7, tobacco transformation Tobacco pre-culture: Select well-growing, green and thick leaves from Nicotiana benthamiana at the 5-leaf stage, rinse them 5 times with sterile water, dry them, disinfect them with 70v / v% ethanol for 1 min, sterilize them with 25v / v% sodium hypochlorite for 3 min, and rinse them 8 times with sterile water. Gently place the leaves on sterilized filter paper. After the water is completely dried, use a sterilized knife to cut the leaves into small samples with a side length of about 1 cm. Use tweezers to place the samples on the pre-culture MS medium and culture them for 3 days.

[0050] Co-cultivation: Add 200 μL of 100 μg / mL acetosyringone solution to 100 mL of MS. Use this liquid to resuspend the infection solution obtained in step S6 for 30 minutes. Place the tobacco leaves after pre-cultivation in the resuspended infection solution and shake gently for 10 minutes. After the end, place them on sterilized filter paper. After ensuring that the water is completely absorbed, place them in co-cultivation medium and culture in the dark for 3 days.

[0051] S8. Screening culture: After the co-cultivation, take out the leaves, rinse them with sterile water 5 times, and use sterilized filter paper to absorb excess water. Then transfer the leaves to the screening culture medium and repeat the transfer every half a month.

[0052] S9, rooting culture: observe the rooting of leaves every day. When the buds grow to 1 cm long, remove them from the culture medium, cut off the callus tissue at the bottom, and transfer them to the rooting medium for culture. When the buds take root, remove them with tweezers, rinse them with sterile water 5 times, and then transfer them to soil for culture to obtain the overexpressed AhMFT1 T0 generation transgenic tobacco.

[0053] Extract T0 transgenic tobacco DNA, use this DNA as a template, and use the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 AhMFT1 Gene-specific primers were used for PCR amplification, and the PCR products obtained by PCR amplification were subjected to gel electrophoresis. Figure 2 As shown, the stripes indicate the transgenic tobacco positive seedlings that were successfully cultivated.

[0054] Example 3: Identification of waterlogging tolerance in transgenic tobacco The present invention cultivates the AhMFT1 genetically modified tobacco, AhMFT5 genetically modified tobacco, AhMFT6 genetically modified tobacco, AhMFT8 genetically modified tobacco, AhMFT10 Genetically modified tobacco and AhMFT14 Identification of waterlogging tolerance in transgenic tobacco.

[0055] (1) Screening ① Place transgenic tobacco seeds in a sterilized 1.5 mL centrifuge tube, disinfect with 2.5 v / v% sodium hypochlorite for 10 min, then disinfect with 75 v / v% anhydrous ethanol for 2 min, and wash with sterile water five times.

[0056] ② Transfer the disinfected seeds to sterilized filter paper and blow dry the sterile water on the seeds. Then sow the seeds on MS solid medium containing 50 μg / mL hygromycin and culture at 25°C, 16h light / 8h dark conditions.

[0057] (2) Planting The nutrient soil and vermiculite were mixed in a volume ratio of 1:1 to obtain the culture soil. The culture soil was then transferred into the flower pots, and the positive plants screened were transferred into the pots for cultivation. Each pot of plants was numbered and placed in an artificial climate chamber for cultivation at 25°C, 16h light / 8h dark, and a light intensity of 50μmoL / m 2 / s.

[0058] (3) Processing T2 tobacco seeds were dried in a 28°C oven for 5 days to remove moisture, then vernalized in a 4°C refrigerator for 2 days. Vernalized seeds of positive and wild-type strains were placed in 1.5 mL test tubes, sterilized with 75 v / v% anhydrous ethanol for 30 seconds, then disinfected with 10 v / v% H₂O₂ for 5 minutes, and rinsed five times with sterile water. The sterile water on the seeds was then blown dry on sterilized filter paper and sown on MS solid medium.

[0059] Transgenic tobacco lines and wild-type tobacco lines grown on culture medium for 20 days were photographed before flooding. Water was then added to the culture dish to submerge the tobacco lines. Phenotypic changes in the tobacco were observed 5 days after flooding.

[0060] The results are as follows Figure 3 As shown in the figure, compared with the wild-type tobacco plants before treatment, the growth state of the wild-type tobacco plants after waterlogging stress was poor, the root length of the plants did not change significantly, but the leaves turned yellow, wilted, and shrank seriously. AhMFT1 genetically modified tobacco, AhMFT5 genetically modified tobacco, AhMFT6 genetically modified tobacco, AhMFT8 genetically modified tobacco, AhMFT10 Genetically modified tobacco and AhMFT14 The transgenic tobacco plants grew well after waterlogging stress, AhMFT1 The transgenic tobacco leaves showed no shrinkage, the root length of the plants increased significantly, the number of lateral roots increased, and the ability to withstand waterlogging stress was the most outstanding.

[0061] The present invention further verifies AhMFT1 The specific process of the influence of genes on tobacco waterlogging resistance is as follows.

[0062] Will AhMFT1 Transgenic tobacco strains 10-4, 10-6, and 10-8, along with wild-type tobacco strains, were transplanted into pots containing a 1:1 mixture of nutrient soil and vermiculite. After 8 weeks of cultivation, the pots were flooded. Experimental and control groups were set up, with three biological replicates per group. The experimental groups were placed in a flooded chamber, with water added to a depth of 5 cm above the soil. The control group was grown normally under the same conditions. Phenotypic changes in the two groups were observed after 0 h, 1 day, 3 days, and 5 days of flooding, and plant survival and water loss rates were measured.

[0063] The method for determining the water loss rate is as follows: weigh 0.5g of tobacco leaves, record the initial weight, then place the leaves at room temperature, record the weight change every 20 minutes, and finally calculate the water loss rate using the water loss rate formula.

[0064] Water loss rate = (initial weight - measured weight) / initial weight × 100%.

[0065] The survival rate was determined as follows: wild-type control plants and overexpressing AhMFT1 For tobacco seedlings grown in soil, the total number of initial plantings was recorded. After eight weeks of cultivation, waterlogging treatment was performed and the number of surviving plants was recorded five days after waterlogging.

[0066] Survival rate = (number of surviving plants ÷ total number of initial plantings) × 100% like Figure 4 As shown, AhMFT1 The survival rate of transgenic tobacco was significantly higher than that of wild-type tobacco, and the water loss rate was also lower than that of wild-type tobacco, indicating that AhMFT1 Overexpression can improve the plant's resistance to waterlogging stress.

[0067] Figure 5 illustrate, AhMFT1 The growth conditions of the four transgenic tobacco lines after five days of waterlogging treatment were significantly better than those of wild-type tobacco. In summary, AhMFT1 overexpression can improve the plant's resistance to waterlogging stress.

[0068] Example 4: AhMFT1 subcellular localization S1. PCR amplification: According to the nucleotide sequence as shown in SEQ ID NO.1 AhMFT1 The specific DNA sequence of the gene coding region was amplified using the cDNA in Example 2 as a template and EGFP-AhMFT1-F and EGFP-AhMFT1-R as amplification primers according to the following PCR amplification system and PCR amplification procedure. AhMFT1The nucleotide sequence of EGFP-AhMFT1-F is shown in SEQ ID NO.5, and the nucleotide sequence of EGFP-AhMFT1-R is shown in SEQ ID NO.6.

[0069] SEQ ID NO.5: 5'-GCTCTAGAATGGAGATGGAGTACACAAGC-3'; SEQ ID NO. 6: 5'-GCTGCAGAAGATGATCATCATCAGCAAGTT-3'.

[0070] PCR amplification system: 10 µL Prime STAR Max Premix, 1 µL EGFP-AhMFT1-F, 1 µL EGFP-AhMFT1-R, 1 µL cDNA, 8 µL ddH2O.

[0071] PCR amplification program: 95°C for 30 seconds; 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 10 seconds, for 30 cycles.

[0072] S2. Electrophoresis and gel recovery: The PCR products were subjected to agarose gel electrophoresis, and the successfully amplified gene fragments were excised and recovered.

[0073] S3, subcellular localization vector EGFP- AhMFT1 Build: Use restriction enzymes according to the enzyme digestion system shown below. Xba I and restriction enzymes Pst I. Double-enzyme digestion was performed on the vector 1300-35S-EGFP and the recovered gene fragment to obtain the linearized 1300-35S-EGFP vector and the gene fragment after enzyme digestion, respectively.

[0074] Enzyme digestion system: 5µL 10× QuickCut buffer, 1µL QuickCut Xba I. 1µL QuickCut Pst I. 5 µL of vector 1300-35S-EGFP or the gene fragment recovered in step S2, and 38 µL of ddH2O.

[0075] Then, using T4 ligase, the linearized 1300-35S-EGFP vector and the enzyme-digested gene fragment were connected according to the following ligation reaction system to obtain the subcellular localization vector EGFP- AhMFT1 .

[0076] Ligation reaction system: 1µL T4 ligase, 1µL 10× Buffer, 7µL digested gene fragment, 1µL linearized 1300-35S-EGFP vector.

[0077] S4, transformation: The subcellular localization vector EGFP- AhMFT1 The recombinant strain was introduced into EHA105 competent cells of Agrobacterium tumefaciens and cultured to obtain the recombinant bacteria used for localization. The Agrobacterium transformed with only the 1300-35S-EGFP vector was used as a control strain and marked as an empty vector Agrobacterium.

[0078] S5. Tobacco Planting: Wild-type tobacco was planted and cultivated for 5 weeks under the culture conditions of 14 h light / 10 h dark, 25° C., and 70% relative humidity.

[0079] S6. Preparation of Agrobacterium Activation of Agrobacterium: The recombinant bacteria obtained in step S4 for positioning and the empty Agrobacterium were inoculated into YEB medium containing 50 μg / mL kanamycin and 25 μg / mL rifampicin, and cultured at 28°C and 200 rpm until the bacterial solution OD 600 The value was 0.6, and the activated recombinant bacterial solution for positioning and the empty Agrobacterium bacterial solution were obtained respectively.

[0080] Preparation of infection medium: 1 mL of activated recombinant bacteria for positioning and 1 mL of empty Agrobacterium culture were transferred to 50 mL of YEB medium containing 50 μg / mL kanamycin and 25 μg / mL rifampicin, respectively. The culture was shaken at 200 rpm at 28°C overnight. The culture was then centrifuged at 5000 rpm for 5 minutes at room temperature. After discarding the supernatant, the bacterial pellet was resuspended in acetosyringone at a final concentration of 100 μM to an OD value of 0. 600 The value is 1.0 and the mixture is allowed to stand at room temperature for 2 hours.

[0081] Transient infection of tobacco: Using the cultured empty Agrobacterium as a control, mix equal volumes of the empty Agrobacterium and the recombinant bacteria used for positioning to obtain a mixed bacterial solution. Slowly inject the mixed bacterial solution into the tobacco leaves from the back, and mark the injection site with a circle.

[0082] Culture observation: The injected tobacco was cultured in the dark at 21°C for 12 h / d for 2 days, and fluorescence imaging was observed using a laser confocal microscope.

[0083] The results are as follows Figure 6 As shown, by observing the injection of EGFP- AhMFT1 The fluorescence signal of Agrobacterium in tobacco leaves can only be observed in the nuclei of tobacco leaves, indicating that the AhMFT1 protein is localized in the nucleus.

[0084] It should be noted that when numerical ranges are mentioned in the present invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints may be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes preferred embodiments. Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0085] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. AhMFT1 The use of a gene for improving waterlogging tolerance in plants is characterized in that: described AhMFT1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. according to claim 1 AhMFT1 The use of a gene for improving waterlogging tolerance in plants is characterized in that: The application method is as follows: improving the plant by transgenic technology or gene editing technology AhMFT1 The expression level of the gene can be increased to improve the plant's tolerance to waterlogging stress.

3. according to claim 1 AhMFT1 The use of a gene for improving waterlogging tolerance in plants is characterized in that: The plant is tobacco or peanut.

4. A use of AhMFT1 protein in improving plant waterlogging tolerance, characterized in that: The AhMFT1 protein is the one described in claim 1 AhMFT1 Gene encoding; the plant is tobacco or peanut.

5. Use of a recombinant expression vector in improving waterlogging tolerance in plants, characterized in that: The recombinant expression vector contains the AhMFT1 Gene; the plant is tobacco or peanut.

6. Use of a recombinant bacterium in improving waterlogging tolerance in plants, characterized in that: The recombinant bacteria contains the AhMFT1 Gene; the plant is tobacco or peanut.

7. A method for cultivating waterlogging-tolerant transgenic plants, characterized in that: The following steps are involved: The claim 1 AhMFT1 The gene is connected to the expression vector to construct a recombinant expression vector; The recombinant expression vector is introduced into the plant to AhMFT1 The gene is overexpressed in plants to obtain waterlogging-tolerant transgenic plants.

8. The method for cultivating waterlogging-tolerant transgenic plants according to claim 7, wherein: The expression vector is pCAMBIA1307.

Images