Use of ahmft1 gene in improving plant waterlogging tolerance

Abstract

This invention belongs to the field of plant genetic engineering technology, specifically relating to a... AhMFT1 Application of genes in improving plant waterlogging tolerance AhMFT1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO.2. This invention first constructs a protein containing... AhMFT1 The recombinant gene expression vector pCAMBIA1307- AhMFT1 Then, using Agrobacterium-mediated genetic transformation to... AhMFT1 Genes were transferred into plants to obtain transgenic plants. Flooding treatment experiments revealed that overexpression... AhMFT1 Genes can enhance a plant's tolerance to waterlogging stress, utilizing... AhMFT1 Genes can be used to genetically modify the waterlogging tolerance of plants.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and particularly relates to AhMFT1 Application of a gene in improving plant waterlogging tolerance. BACKGROUND

[0002] Peanut is an important economic crop with high nutritional value and wide application. In the areas where plum rain prevails, due to the uneven temporal and spatial distribution of precipitation caused by monsoon and the west-high and east-low terrain, the peanut growth period is easily affected by flood disasters. Peanut is a subterranean fruiting plant, and the development of pods requires coordination of water and air in the soil. Studies have shown that the water requirement of peanut is at the lower level among crops, and waterlogging or excessive soil moisture can affect peanut development. After waterlogging, the peanut roots are in a soaking state for a long time, the root system is oxygen-deficient, the respiration is strengthened, the consumption is increased, and part of the metabolic pathways are changed to cause physiological and biochemical reactions, affecting the development of peanut pods, resulting in peanut empty fruit, and even causing peanut to be absolutely lost in severe cases.

[0003] Therefore, cultivating peanut varieties with waterlogging tolerance can ensure high yield and stable yield of peanuts in waterlogging areas, and is also the most economical, safe and effective method. However, using conventional breeding methods is time-consuming, labor-intensive, and difficult to improve target traits, and with the continuous deepening of the research on functional genomics and molecular assisted design breeding technology, it is an effective way to combine the key genes for waterlogging response in peanuts with conventional breeding methods to efficiently, accurately and quickly cultivate new waterlogging-tolerant varieties.

[0004] Therefore, it is of great significance to mine the key genes for waterlogging response in peanuts for improving the waterlogging tolerance of plants and cultivating new waterlogging-tolerant plant varieties. SUMMARY

[0005] In order to solve the above problems, the application provides a kind of AhMFT1 Application of a gene in improving plant waterlogging tolerance. Through the exploration of the function of the gene, it is found that overexpression of the gene can improve the tolerance of plants to low temperature stress. AhMFT1 AhMFT1

[0006] To achieve the above purpose, the specific technical solutions of the application are as follows:

[0007] The first aspect of the application provides a kind of AhMFT1 Application of a gene in improving plant waterlogging tolerance, the nucleotide sequence of the gene is shown as SEQ ID NO. 1. AhMFT1

[0008] Further, the application method is as follows: improving the gene in the plant by transgenic technology or gene editing technology AhMFT1 ​​​The expression amount of the gene can improve the tolerance of the plant to waterlogging stress.

[0009] Further, the plant is tobacco or peanut.

[0010] The second aspect of the present application provides an application of the AhMFT1 protein in improving the waterlogging tolerance of the plant, wherein the AhMFT1 protein is encoded by the gene described above. AhMFT1 The gene.

[0011] Further, the plant is tobacco or peanut.

[0012] The third aspect of the present application provides an application of the recombinant expression vector in improving the waterlogging tolerance of the plant, wherein the recombinant expression vector contains the gene described above. AhMFT1 The gene.

[0013] Further, the plant is tobacco or peanut.

[0014] The fourth aspect of the present application provides an application of the recombinant bacteria in improving the waterlogging tolerance of the plant, wherein the recombinant bacteria contains the gene described above. AhMFT1 The gene.

[0015] Further, the plant is tobacco or peanut.

[0016] The fifth aspect of the present application provides a method for cultivating a waterlogging-tolerant transgenic plant, comprising the following steps:

[0017] The gene described above is connected to an expression vector to construct a recombinant expression vector; AhMFT1 The recombinant expression vector is introduced into the plant by the Agrobacterium transformation method, so that the gene is overexpressed in the plant, and a waterlogging-tolerant transgenic plant is obtained.

[0018] The recombinant expression vector is introduced into the plant by the Agrobacterium transformation method, so that the gene is overexpressed in the plant, and a waterlogging-tolerant transgenic plant is obtained. AhMFT1 The gene is overexpressed in the plant, and a waterlogging-tolerant transgenic plant is obtained.

[0019] Further, the expression vector is pCAMBIA1307.

[0020] Further, the Agrobacterium is EHA105.

[0021] Compared with the prior art, the present application has the beneficial effects that:

[0022] The present application first proposes and verifies that the gene can regulate the waterlogging tolerance of the plant. AhMFT1 Through the transcriptome analysis and metabolome analysis on the samples of the waterlogging-tolerant peanut variety 'Changhua 18' and the waterlogging-sensitive peanut variety 'Huayu 39', a candidate gene AhMFT1 related to the waterlogging tolerance of the plant is found, and a recombinant expression vector pCAMBIA1307- AhMFT1 containing the gene is constructed. AhMFT1Then the recombinant expression vector is introduced into plants by Agrobacterium-mediated genetic transformation to obtain transgenic plants. AhMFT1 The overexpression of the gene can improve the tolerance of plants to waterlogging stress. Figure 1 The gene can be used for genetic improvement of waterlogging tolerance of plants. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0024] BsaB For the expression vector pCAMBIA1307 map, * indicates that the I enzyme cutting site on the expression vector pCAMBIA1307 is blocked by Dam methylation. Figure 2 I enzyme cutting site is blocked by Dam methylation.

[0025] Figure 2 For electrophoretograms of positive seedlings of T0 generation of transgenic tobacco of 6 candidate genes; Figure 2 A of the figure, Figure 2 B of the figure, Figure 2 C of the figure, Figure 2 D of the figure, Figure 2 E of the figure, AhMFT1 F of the figure are samples of AhMFT5 transgenic tobacco, AhMFT6 transgenic tobacco, AhMFT8 transgenic tobacco, AhMFT10 transgenic tobacco, AhMFT14 transgenic tobacco, Figure 2 transgenic tobacco. Figure 3 WT in the electrophoretograms is wild type tobacco, and Arabic numerals are different samples of transgenic tobacco.

[0026] Figure 3 For phenotypic results of transgenic tobacco and wild type tobacco of 6 candidate genes under waterlogging stress; AhMFT1 A of the figure is AhMFT5 transgenic tobacco, AhMFT6 transgenic tobacco, Figure 3 transgenic tobacco and wild type tobacco under waterlogging stress, Figure 3 (a) row of A of the figure is a picture before waterlogging, Figure 3 (b) row of A of the figure is a picture on the 5th day of waterlogging treatment, Figure 3 the first column to the third column from left to right of A of the figure are parallel tests of wild type tobacco, AhMFT1Figure 1A is from left to right column 4 to column 6 Figure 3 Parallel test of transgenic tobacco, AhMFT5 Figure 1A is from left to right column 7 to column 9 Figure 3 Parallel test of transgenic tobacco, AhMFT6 Figure 1A is from left to right column 10 to column 12 Figure 3 Parallel test of transgenic tobacco; AhMFT8 Figure 1B is AhMFT10 Transgenic tobacco, AhMFT14 Transgenic tobacco, Figure 3 Phenotype of transgenic tobacco and wild type tobacco under waterlogging stress, Figure 3 Row (a) of Figure 1B is picture before waterlogging, Figure 3 Row (b) of Figure 1B is picture on the 5th day of waterlogging treatment, Figure 3 Figure 1B is from left to right column 1 to column 3 parallel test of wild type tobacco, AhMFT8 Figure 1B is from left to right column 4 to column 6 Figure 3 Parallel test of transgenic tobacco, AhMFT10 Figure 1B is from left to right column 7 to column 9 Figure 3 Parallel test of transgenic tobacco, AhMFT14 Figure 1B is from left to right column 10 to column 12 Figure 4 Parallel test of transgenic tobacco.

[0027] Figure 4 Survival rate and water loss rate of tobacco after waterlogging treatment, Figure 4 Figure 1A is survival rate, AhMFT1 Figure 1B is water loss rate; WT represents wild type tobacco, 10-4, 10-6 and 10-8 are three parallel samples of transgenic tobacco lines constructed; Figure 5 Figure 1A is survival rate, p <0.01.

[0028] AhMFT1 Figure 1A is survival rate, Figure 5 Transgenic tobacco enhances resistance to waterlogging stress; Figure 5 Row A of Figure 1A is picture on the 0th day of waterlogging treatment, Figure 5 Row B of Figure 1A is picture on the 1st day of waterlogging treatment, Figure 5 Row C of Figure 1A is picture on the 3rd day of waterlogging treatment, AhMFT1 Row D of Figure 1A is picture on the 5th day of waterlogging treatment; WT represents wild type tobacco as control group, 10-4, 10-6 and 10-8 are three parallel samples of transgenic tobacco lines constructed; Figure 5 Figure 1A is survival rate, Figure 5 From left to right column 1 to column 3 is wild type tobacco, AhMFT1 From left to right column 4 to column 6 is Figure 5 Transgenic tobacco 10-4,AhMFT1 From left to right, columns 7-9 are Figure 5 Transgenic tobacco 10-6, AhMFT1 From left to right, columns 10-12 are Figure 6 Transgenic tobacco 10-8.

[0029] AhMFT1 are Figure 6 Subcellular localization of the protein encoded by the gene, Figure 6 Fig. (a) of Figure 6 Fig. (b) of Figure 6 Fig. (c) of Figure 6 Fig. (d) of is an image of the control empty vector, Figure 6 Fig. (e) of Figure 6 Fig. (f) of Figure 6 Fig. (g) of Figure 6 Fig. (h) of is an image of EGFP-AhMFT1, Figure 6 Fig. (a) of and Figure 6 Fig. (e) of are images observed through a green fluorescent protein filter, Figure 6 Fig. (b) of and Figure 6 Fig. (f) of are images observed through a red fluorescent protein filter, Figure 6 Fig. (c) of and Figure 6 Fig. (g) of are bright field images, Figure 6 Fig. (d) of Figure 6 Fig. (a) of Figure 6 Fig. (b) of and Figure 6 Fig. (c) of are images merged, AhMFT1 Fig. (h) of AhMFT1 Fig. (e) of AhMFT1 Fig. (f) of and AhMFT1 Fig. (g) of are images merged. DETAILED DESCRIPTION

[0030] The specific embodiments of the present application are described in detail below, but the scope of protection of the present application is not limited to the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present application. The experimental methods described in the embodiments of the present application are all conventional methods, and the materials and reagents used in the following examples are commercially available unless otherwise specified.

[0031] Agrobacterium EHA105 in the present application was purchased from Genesee Scientific, and pCAMBIA1307 expression vector and vector pCAMBIA1300-35S-EGFP were donated by the Institute of Oil Crops, Chinese Academy of Agricultural Sciences.

[0032] Peanut is an important economic crop with high nutritional value and wide application. However, in the plum rain area, due to the uneven temporal and spatial distribution of precipitation caused by monsoon and the west-high and east-low topography, the peanut growing period is easily affected by flood disasters. Peanut is sensitive to flooding, and after being flooded, it is easy to have unripe fruits, and in severe cases, it will lead to absolute yield. Therefore, it is of great significance to improve the flooding tolerance of plants and breed new varieties of plants with flooding tolerance by excavating the key genes of peanut with flooding tolerance.

[0033] The application provides a kind of AhMFT1 The application provides a kind of AhMFT1 The application provides a kind of AhMFT1 The application provides a kind of AhMFT5 The application provides a kind of AhMFT6 The application provides a kind of AhMFT8 The application provides a kind of

[0034] Example 1: Screening of peanut flooding-tolerant MYB transcription factor gene

[0035] The flooding-tolerant peanut variety 'Changhua 18' and the flooding-sensitive peanut variety 'Huayu 39' growing to the flowering stage and having the same growth vigor were selected, and 'Changhua 18' was marked as CH18, and 'Huayu 39' was marked as HY39. The pots planted with CH18 and HY39 were placed in a flooding box and injected with tap water, and the water was added to 5 cm above the soil surface. At the 6th hour, 24th hour, 3rd day and 5th day of the flooding treatment time, three biological replicates were taken each time, the plant samples were washed with distilled water until the surface was free of soil and dirt, and then the water was absorbed with absorbent paper. Each tissue was cut into small pieces and sent to a biological company for transcriptome and metabolome analysis.

[0036] Through transcriptome analysis and metabolome analysis, 2104 differentially expressed genes DEGs responsive to flooding stress were screened from all genes; among the 2104 DEGs, 36 different transcription factor families were found to respond to flooding stress, of which the most was the MYB transcription factor gene family, with 205 genes.

[0037] The 205 MYB transcription factor encoding genes screened were classified, of which 90 were R2R3-MYB transcription factor encoding genes. The 90 R2R3-MYB transcription factor encoding genes in CH18 and HY39 during the flooding treatment were subjected to cluster analysis, and it was found that 16 R2R3-MYB genes were significantly differentially expressed in CH18 and HY39.

[0038] Example 2: Cultivation of transgenic tobacco

[0039] Of the 16 R2R3-MYB genes obtained from Example 1 that were significantly differentially expressed in CH18 and HY39, 6 genes that were tissue-specifically expressed were selected: AhMFT10 、 AhMFT14 、 AhMFT1 、 AhMFT5 、 AhMFT6 、 AhMFT8 . By constructing overexpression gene vectors, the 6 genes were respectively introduced into tobacco by Agrobacterium-mediated genetic transformation to obtain AhMFT10 transgenic tobacco, AhMFT14 transgenic tobacco, AhMFT1 transgenic tobacco, AhMFT1 transgenic tobacco, AhMFT1 transgenic tobacco, and AhMFT1 transgenic tobacco. The construction method of the AhMFT1 transgenic tobacco is specifically described as follows:

[0040] AhMFT1 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO. 2.

[0041] SEQ ID NO. 1:

[0042] ATGGAGATGGAGTACACAAGCAGTGTTCACATGATCATGAGAAGTTTATCGGATTGTGGCTCTTCAGTAACAAATGGGAGCGAGGAAGATATGGAAATCAGAAAAGGTCCATGGACAGAGGAAGAGGATTCTGCTCTGTTGAACCATATCACCACCTATGGTCTAAAACGAAGCGGGAAAAGCTGCAGATTAAGATGGTTAAATTACTTGCGTCCGAATGTTAGACGTGGGAACATCACCCTCGAAGAACAACTCTTGATTCTTGACCTCCATTCGCGATGGGGCAATAGGTGGTCGAAAATAGCAGAACAATTGCCTGGAAGAACAGACAACGAGATCAAGAACTACTGGAGAACGAGAGTGGTGAAGCAGGCGAAGCAACTAAAATGCGACGTCAACAGCAAACAGTTCAGAGACGCGTTGCGCTACGTATGGATGCCGCGCCTTATTGAGCAGATTCAAGCCCAAGGAAGATCCACCATGTGCGTCTCCCAAGCTCGCGAGATAGTTAATCCTGTCACTTCAGTTTCGATGGCTTCGTCTAAATCTTGCTCTTCATTCTCAGGATGTGAACAATTCCAAGCCTCTTCTTCGGTCTCTGATTCTTGTGTGTCTTATTACAGTTTAATGGGTAGTGGAAGTGGTGGGGGTTCTTCAGAGCATGCGGAAAAGGGAGCCACGTCATCATCCACGCCATTTGAACCGGAAAATGGTTTCGGGGGTGCTGATTTGTGGACTGATGAAAACATATGGTTCTTGCAGCAGCAACTTGCTGATGATGATCATCTTTGA.

[0043] SEQ ID NO. 2:

[0044] MEMEYTSSVHMIMRSLSDCGSSVTNGSEEDMEIRKGPWTEEEDSALLNHITTYGLKRSGKSCRLRWLNYLRPNVRRGNITLEEQLLILDLHSRWGNRWSKIAEQLPGRTDNEIKNYWRTRVVKQAKQLKCDVNSKQFRDALRYVWMPRLIEQIQAQGRSTMCVSQAREIVNPVTSVSMASSKSCSSFSGCEQFQASSSVSDSCVSYYSLMGSGSGGGSSEHAEKGATSSSTPFEPENGFGGADLWTDENIWFLQQQLADDDHL.

[0045] S1, extracting RNA and reverse transcribing into cDNA

[0046] The total RNA of the plant was extracted from the plant leaf of CH18, and the synthesis of the first strand of cDNA was completed using the reverse transcription kit of Takara Company after the qualified total RNA of the plant was extracted.

[0047] S2, PCR amplification

[0048] The nucleotide sequence is shown as SEQ ID NO. 1 Figure 1 The DNA sequence specific to the coding region of the gene was designed BamH The primer combination specific to the gene comprises the upstream primer with the nucleotide sequence shown as SEQ ID NO. 3 and the downstream primer with the nucleotide sequence shown as SEQ ID NO. 4, and the cDNA in the step S1 is used as the template to amplify according to the PCR amplification system and the PCR amplification procedure as follows EcoR The gene, and the PCR product is obtained.

[0049] SEQ ID NO. 3: 5'-CGCGGATCCATGGAGATGGAGTACACAAG-3';

[0050] SEQ ID NO. 4: 5'-CCGGAATTCAAGATGATCATCATCAGCAA-3'.

[0051] PCR amplification system: 10 μL of Prime STAR Max Premix, 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of cDNA, 8 μL of ddH2O.

[0052] PCR amplification procedure: 95℃ 30S; 98℃ 10S, 55℃ 15S, 72℃ 10S, 30 cycles.

[0053] S3, electrophoresis, gel recovery: PCR products were subjected to agarose gel electrophoresis, and the successfully amplified gene fragments were cut and recovered.

[0054] S4, recombinant expression vector CAMBIA1307- BamH Construction: The expression vector pCAMBIA1307 map is shown as follows, and the linearized pCAMBIA1307 expression vector and the digested gene fragments were obtained by double digestion of the expression vector pCAMBIA1307 and the recovered gene fragments using restriction endonuclease I and restriction endonuclease I. EcoR AhMFT1 I and restriction endonuclease I. AhMFT1 I and restriction endonuclease I.

[0055] Enzyme digestion system: 5 μL of 10×QuickCut buffer, 1 μL of QuickCut AhMFT1 I, 1 μL of QuickCut AhMFT1 I, 5 μL of expression vector pCAMBIA1307 or S3 step recovered gene fragments, 38 μL of ddH2O.

[0056] The linearized pCAMBIA1307 expression vector and the digested gene fragments were connected using T4 ligase according to the following ligation reaction system, and the recombinant expression vector CAMBIA1307- Figure 2 was obtained.

[0057] Ligation reaction system: 1 μL of T4 ligase, 1 μL of 10×Buffer, 7 μL of digested gene fragments, 1 μL of linearized pCAMBIA1307 expression vector.

[0058] S5, transformation: the recombinant expression vector CAMBIA1307- AhMFT1 constructed in S4 step was introduced into Agrobacterium EHA105 competent cells, and after culture, the recombinant bacteria were obtained.

[0059] S6, preparation of infection solution

[0060] Activation of recombinant bacteria: the recombinant bacteria obtained in S5 step were inoculated in YEB medium containing 50 μg / mL kanamycin and 25 μg / mL rifampicin, and cultured at 28°C with 200 rpm shaking until the OD 600 value of the bacterial solution was 0.6, and the activated bacterial solution was obtained.

[0061] ​Preparation of infection solution: 1 mL of activated bacterial suspension was transferred to 50 mL of YEB medium containing 50 μg / mL kanamycin and 25 μg / mL rifampin, and cultured overnight at 28°C with shaking at 200 rpm. Then, the suspension was centrifuged at 5000 rpm for 5 min at room temperature, the supernatant was discarded, and the bacterial cells were resuspended in acetylsyleugenol solution to a final concentration of 100 μM until the bacterial suspension reached the OD value. 600 The value is 1.0, and it is left to stand at room temperature for 2 hours.

[0062] S7, Tobacco Conversion

[0063] Tobacco pre-culture: Select well-grown, green and thick leaves from 5-leaf stage Nicotiana benthamiana, rinse 5 times with sterile water, air dry, disinfect with 70 v / v% ethanol for 1 min, sterilize with 25 v / v% sodium hypochlorite for 3 min, rinse 8 times with sterile water, gently place the leaves on sterilized filter paper, and air dry completely. 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 pre-culture MS medium and incubate for 3 days.

[0064] Co-culture: Add 200 μL of 100 μg / mL acetylsyl syringone solution to 100 mL MS, resuspend the infection solution obtained in step S6 in this solution for 30 min, place the tobacco leaves after pre-culture into the resuspended infection solution and shake gently for 10 min, then place them on sterilized filter paper, and after confirming that the moisture has been completely absorbed, place them into the co-culture medium and incubate in the dark for 3 days.

[0065] S8. Screening Culture: After co-culture, remove the leaves, rinse them 5 times with sterile water, and then use sterile filter paper to absorb the excess water. Then transfer the leaves to the screening culture medium and repeat the transfer every half month.

[0066] S9. Rooting Culture: Observe the rooting of leaves daily. When the buds reach 1 cm in length, remove them from the culture medium, cut off the callus tissue at the base, and transfer them to rooting medium. After the buds have rooted, remove them with tweezers, rinse them 5 times with sterile water, and then transfer them to soil for further culture to obtain overexpressed samples. AhMFT5 T0 generation genetically modified tobacco.

[0067] DNA was extracted from T0 generation transgenic tobacco plants. Using this DNA as a template, nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4 were analyzed. AhMFT6 Gene-specific primers were used for PCR amplification. The PCR products obtained from the PCR amplification were then subjected to gel electrophoresis, and the results are as follows: AhMFT8 As shown, the striped sections indicate successfully cultivated transgenic tobacco seedlings that are positive.

[0068] Example 3: Phenotypic Identification of Flood Tolerance in Transgenic Tobacco

[0069] The transgenic tobacco of embodiment 2 was subjected to waterlogging tolerance identification. AhMFT10 The transgenic tobacco, AhMFT14 The transgenic tobacco, Figure 3 The transgenic tobacco, AhMFT1 The transgenic tobacco, AhMFT5 The transgenic tobacco and AhMFT6 The transgenic tobacco were subjected to waterlogging tolerance identification.

[0070] (1) Screening

[0071] ① The transgenic tobacco seeds were placed in a sterilized 1.5 mL centrifuge tube, disinfected with 2.5 v / v% sodium hypochlorite for 10 min, then disinfected with 75 v / v% anhydrous ethanol for 2 min, and washed with sterile water for 5 times.

[0072] ② The disinfected seeds were transferred to a sterilized filter paper and the sterile water on the seeds was blown dry, and then the seeds were sowed on MS solid medium containing 50 μg / mL hygromycin, and cultured at 25°C under the condition of 16 h light / 8 h dark.

[0073] (2) Planting

[0074] The nutrient soil was mixed with vermiculite at a volume ratio of 1:1 to obtain culture soil, and then the culture soil was moved into a flowerpot, and the screened positive plants were moved into the pot for culture. Each plant in the pot was numbered and placed in an artificial climate chamber for culture under the condition of 25°C, 16 h light / 8 h dark, and illumination of 50 μmoL / m 2 / s.

[0075] (3) Treatment

[0076] The T2 generation tobacco seeds were placed in a 28°C oven for 5 d to dry the water, and then were vernalized in a 4°C refrigerator for 2 d. The vernalized positive strains and wild type strain seeds were respectively placed in 1.5 mL test tubes, disinfected with 75 v / v% anhydrous ethanol for 30 s, then disinfected with 10 v / v% H2O2 for 5 min, washed with sterile water for 5 times, and then the sterile water on the seeds was blown dry on a sterilized filter paper. The seeds were sowed on MS solid medium.

[0077] The transgenic tobacco strains and wild type tobacco strains grown on the culture medium for 20 d were photographed before waterlogging treatment, then water was added to the culture dish to submerge the tobacco strains, and the tobacco phenotype changes were observed after 5 d of waterlogging.

[0078] The results are shown in Table 1. AhMFT8 Compared with before treatment, the wild type tobacco plants after waterlogging stress had a poor growth state, and the root length had no obvious change, but the leaves were yellow, wilted, and severely shriveled. Compared with the wild type tobacco plants, AhMFT10 the transgenic tobacco, AhMFT14Transgenic tobacco, AhMFT1 Transgenic tobacco, AhMFT1 Transgenic tobacco, AhMFT1 Transgenic tobacco and AhMFT1 Transgenic tobacco grows well after waterlogging stress, wherein Figure 4 The transgenic tobacco leaf has no wrinkling phenomenon, the root length of the strain has obvious growth, the number of lateral roots increases, and the waterlogging stress resistance is most prominent.

[0079] The present application further verifies AhMFT1 The effect of the gene on the waterlogging resistance of tobacco, and the specific process is as follows.

[0080] The AhMFT1 Transgenic tobacco strain samples 10-4, 10-6, 10-8 and wild type tobacco strains are transplanted into pots for culture, the soil in the pot is mixed with nutrient soil and vermiculite at a mass ratio of 1:1, and after 8 weeks of culture, waterlogging treatment is carried out, and experimental groups and control groups are set up, each group has 3 biological repeats. The experimental group is placed in a waterlogging box for treatment, and water is added to the position 5cm above the soil, and the control group grows normally, and the other conditions are the same, and after 0h, 1d, 3d and 5d of waterlogging, the phenotype changes of the two groups of tobacco are observed, and the survival rate and the water loss rate of the plants are measured.

[0081] The determination method of the water loss rate is as follows: 0.5g of tobacco leaves is weighed, the initial weight is recorded, then the leaves are placed at room temperature, and the weight change is recorded every 20min, and finally the water loss rate is calculated by the water loss rate formula.

[0082] The water loss rate = (initial weight value-measured weight value) / initial weight value * 100%.

[0083] The determination method of the survival rate is as follows: wild type control plants and overexpression Figure 5 Tobacco soil culture seedlings are cultured in a greenhouse, the initial total number of planting is recorded, and after 8 weeks of culture, waterlogging treatment is carried out, and after 5 days of waterlogging, the number of surviving plants is recorded.

[0084] The survival rate = (the number of surviving plants ÷ the initial total number of planting) * 100%

[0085] As AhMFT1 shown, AhMFT1 The survival rate of the transgenic tobacco is significantly higher than that of the wild type tobacco, and the water loss rate is also lower than that of the wild type tobacco, indicating AhMFT1 Overexpression can improve the resistance of plants to waterlogging stress.

[0086] AhMFT1 It is shown that Xba The growth of the four transgenic tobacco strains after 5 days of waterlogging treatment is obviously better than that of the wild type tobacco, and in summary, overexpression of AhMFT1 can improve the resistance of plants to waterlogging stress.

[0087] Example 4: Subcellular localization of AhMFT1

[0088] S1, PCR amplification: according to the nucleotide sequence shown in SEQ ID NO. 1 Pst The DNA sequence specific to the coding region of the gene was used as a template, and EGFP-AhMFT1-F and EGFP-AhMFT1-R were used as amplification primers, and the PCR amplification system and PCR amplification program were as follows: Xba The gene was amplified to obtain a PCR product. The 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.

[0089] SEQ ID NO. 5: 5'-GCTCTAGAATGGAGATGGAGTACACAAGC-3';

[0090] SEQ ID NO. 6: 5'-GCTGCAGAAGATGATCATCATCAGCAAGTT-3'.

[0091] PCR amplification system: 10 μL of Prime STAR Max Premix, 1 μL of EGFP-AhMFT1-F, 1 μL of EGFP-AhMFT1-R, 1 μL of cDNA, 8 μL of ddH2O.

[0092] PCR amplification program: 95°C for 30 s; 98°C for 10 s, 55°C for 15 s, 72°C for 10 s, 30 cycles.

[0093] S2, electrophoresis, gel recovery: the PCR product was subjected to agarose gel electrophoresis, and the successfully amplified gene fragment was recovered by cutting the gel.

[0094] S3, subcellular localization vector EGFP- Pst Construction:

[0095] According to the following enzyme digestion system, the linearized 1300-35S-EGFP vector and the digested gene fragment were obtained by using restriction endonuclease AhMFT1 I and restriction endonuclease AhMFT1 I on the vector 1300-35S-EGFP and the recovered gene fragment.

[0096] Enzyme digestion system: 5 μL of 10×QuickCut buffer, 1 μL of QuickCut Figure 6 I, 1 μL of QuickCut AhMFT1I, 5 μL of vector 1300-35S-EGFP or the gene fragment recovered in step S2, 38 μL of ddH2O.

[0097] Then, the linearized 1300-35S-EGFP vector and the digested gene fragment were connected using T4 ligase in a connection reaction system as follows to obtain a subcellular localization vector EGFP- ​ .

[0098] Connection reaction system: 1 μL of T4 ligase, 1 μL of 10x Buffer, 7 μL of the digested gene fragment, 1 μL of the linearized 1300-35S-EGFP vector.

[0099] S4, transformation: the subcellular localization vector EGFP- ​ obtained in step S3 was introduced into the competent cells of Agrobacterium EHA105, and after cultivation, the recombinant bacteria for localization were obtained. The Agrobacterium into which only the 1300-35S-EGFP vector was transferred was used as a control strain, and was marked as empty Agrobacterium.

[0100] S5, planting of tobacco: wild-type tobacco was planted and cultivated for 5 weeks under the conditions of 14 h light / 10 h darkness, 25°C, and 70% relative humidity.

[0101] S6, preparation of Agrobacterium

[0102] Activation of Agrobacterium: the recombinant bacteria for localization and the empty Agrobacterium obtained in step S4 were inoculated into YEB medium containing 50 μg / mL kanamycin and 25 μg / mL rifampicin, and cultivated at 28°C with 200 rpm shaking until the OD 600 value of the bacterial liquid was 0.6, and the activated recombinant bacteria for localization and the empty Agrobacterium were obtained.

[0103] Preparation of infection liquid: 1 mL of the activated recombinant bacteria for localization and the empty Agrobacterium were transferred into 50 mL of YEB medium containing 50 μg / mL kanamycin and 25 μg / mL rifampicin, and cultivated at 28°C with 200 rpm shaking overnight. Then, the bacterial pellet was resuspended to an OD 600 value of 1.0 by centrifugation at 5000 rpm for 5 min at room temperature, and the supernatant was discarded. The bacterial pellet was resuspended to an OD

[0104] Transient infection of tobacco: the empty Agrobacterium and the recombinant bacteria for localization were mixed in equal amounts, and the mixed bacterial liquid was slowly injected into the tobacco leaves from the back of the leaves, and the injected parts were marked by circling.

[0105] Culture observation: the injected tobacco is cultured in dark condition for 12h / d, 21℃, continuously cultured for 2d, and finally observed by laser confocal microscope for fluorescence imaging.

[0106] The results are shown in ​ As shown in the results, by observing the fluorescence signal of tobacco leaf injected with Agrobacterium containing EGFP- ​ , only fluorescence signal can be observed in the nucleus of tobacco leaf cell, which indicates that AhMFT1 protein is located in the nucleus.

[0107] It should be noted that when the present application involves numerical ranges, both endpoints of each numerical range and any number between the two endpoints can be selected. Since the same steps and examples are used, the preferred embodiments of the present application are described to prevent redundancy. Although the preferred embodiments of the present application have been described, those skilled in the art can make additional changes and modifications to these embodiments once they understand the basic inventive concept. Therefore, the appended claims are intended to include the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0108] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application also intends to include these modifications and variations.

Claims

1. AhMFT1 The use of a gene to improve the waterlogging tolerance of a plant, characterized in that The AhMFT1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the improvement of the plant's [specific genetic information] through transgenic technology or gene editing technology is described. AhMFT1 The expression level of genes is adjusted to improve the plant's tolerance to waterlogging stress; the plant is tobacco or peanut.

2. Use of an AhMFT1 protein for increasing the waterlogging tolerance of a plant, characterized in that, The AhMFT1 protein is as described in claim 1. AhMFT1 Genetic encoding; the plant in question is either tobacco or peanut.

3. Use of a recombinant expression vector for increasing the waterlogging tolerance of a plant, characterized in that The recombinant expression vector contains the gene of claim 1 AhMFT1 ; the plant is tobacco or peanut.

4. Use of a recombinant bacterium for improving the waterlogging tolerance of a plant, characterized in that, The recombinant bacteria contain the gene of claim 1 AhMFT1 ; the plant is tobacco or peanut.

5. A method for cultivating waterlogging-tolerant transgenic plants, characterized in that, comprising the steps of: The method of claim 1, wherein the gene is linked to an expression vector to construct a recombinant expression vector. AhMFT1 The method of claim 1, wherein the gene is linked to an expression vector to construct a recombinant expression vector. The recombinant expression vector is introduced into a plant, so that AhMFT1 The gene is overexpressed in the plant to obtain a transgenic plant resistant to waterlogging; the plant is tobacco or peanut.

6. The method of breeding a transgenic plant tolerant to waterlogging as claimed in claim 5, wherein, The expression vector is pCAMBIA1307.

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