BvM14-TAG2 gene and application thereof

Abstract

The invention discloses a BvM14-TAG2 gene and application thereof, and relates to the technical field of biological genes. The CDS sequence of the BvM14-TAG2 gene disclosed by the invention is as shown in SEQ ID NO. 1. The amino acid sequence of the protein coded by the BvM14-TAG2 gene is as shown in SEQ ID No. 2. According to the invention, the BvM14-TAG2 gene is utilized to construct an arabidopsis thaliana plant for heterologous expression of the BvM14-TAG2 gene, stress treatment is carried out, and the seed germination rate and root length of the plant for heterologous expression of the BvM14-TAG2 gene are both superior to those of a wild type; the biomass of a plant for heterologous expression of the BvM14-TAG2 gene is higher than that of a wild plant; the result shows that the BvM14-TAG2 gene can promote the growth and development of the plant under the salt stress, and can improve the adaptive capacity of the BvM14-TAG2 transgenic plant under the salt stress.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological gene technology, and in particular to a BvM14-TAG2 gene and application fields thereof. BACKGROUND

[0002] Extreme soil salinization, temperature and drought and other environmental stresses have seriously affected the production and planting of agricultural crops. Global salinization intensification is a persistent environmental problem in the 21st century.

[0003] Soil salinization is one of the most destructive environmental pressures, which seriously threatens global agricultural production and ecological security. Saline-alkali soil accumulates excessive soluble salts, which are harmful to most plants by limiting their growth and productivity. The area of salinized soil continues to increase, especially irrigated soil. Statistics show that about 33% of global food production relies on irrigated agriculture, however, about 45 million hectares of irrigated farmland is suffering from different degrees of salinization, which directly affects the stability of the global food security system. A recent study shows that about 50% of global arable land will be affected by salinity by 2050. It is estimated that the world population will reach 9.7 billion by 2050, and the increase in food demand driven by the global population explosion has promoted the development of salt-tolerant plants for agricultural saline-alkali land.

[0004] Under salt stress environment, the main physiological obstacles faced by plants are caused by the obstruction of root water uptake. Excessive soluble salts in high-salt soil will significantly reduce the water potential gradient in the rhizosphere region, thereby weakening the plant's ability to obtain water through osmosis. This water absorption obstacle will cause a double stress effect: on the one hand, it causes water deficiency in plant tissues, and on the other hand, it produces osmotic adjustment pressure, which is one of the key factors limiting plant growth and development in salinized habitats. Under salt stress conditions, Na + and Cl - ions are excessively accumulated in plant cells, causing a series of physiological disorders. This destruction of ion homeostasis will interfere with key metabolic processes, leading to direct cellular toxicity, causing imbalances in the absorption of essential nutrients, and the ion toxicity ultimately significantly inhibits the normal growth and development of plants. In addition to directly affecting plant growth, salt stress also leads to the excessive accumulation of reactive oxygen species (ROS) in the plant body. Due to the high reactivity of ROS, it causes oxidative damage to macromolecules such as lipids, nucleic acids, proteins and carbohydrates, leading to severe oxidative stress due to redox imbalance in the plant body.

[0005] 3-methyladenine DNA glycosylase I (TAG) plays a key role in base excision repair pathway, mainly involved in the repair process of DNA single-strand break, and plays an indispensable role in the mechanism of maintaining genome stability under stress induction, but there is no research on BvM14-TAG2 gene and plant salt tolerance. SUMMARY

[0006] The purpose of the present application is to provide BvM14-TAG2 gene and its application in improving plant salt tolerance. The present application firstly clones BvM14-TAG2 gene from sugar beet M14, and the bioinformatics analysis shows that the BvM14-TAG2 protein has a typical Adenine glyco domain, has the function of DNA glycosylase; and it is found that the expression amount of BvM14-TAG2 is significantly increased under 200, 400 mM NaCl stress treatment, indicating that BvM14-TAG2 gene is involved in salt stress response. The heterologous expression of BvM14-TAG2 gene Arabidopsis plants is constructed, and stress treatment is carried out, and the seed germination rate and root length of the heterologous expression of BvM14-TAG2 gene plants are better than those of the wild type; further experiments find that the biomass of the heterologous expression of BvM14-TAG2 gene plants is higher than that of the wild type plants; it is shown that BvM14-TAG2 gene promotes the growth and development of plants under salt stress.

[0007] The CDS sequence of the BvM14-TAG2 gene of the present application is shown as SEQ ID NO. 1.

[0008] The amino acid sequence of the protein encoded by the above-mentioned BvM14-TAG2 gene is shown as SEQ ID No. 2.

[0009] The application of the BvM14-TAG2 gene of the present application in improving plant salt tolerance.

[0010] The CDS region of the BvM14-TAG2 gene of the present application is 960 bp; it encodes 320 amino acids; the relative molecular mass is 36.46 kDa. The conserved domain Adenine glyco segment is 127-305 aa.

[0011] The protein (BvM14-TAG2) encoded by the BvM14-TAG2 gene of the present application has the molecular composition of C 1623 H 2557 N 445 O 481 S 14 The theoretical isoelectric point is 8.90, and the average hydrophobicity is -0.428, indicating that the protein as a whole presents hydrophilic characteristics.

[0012] The application constructs the BvM14-TAG2 gene heterologous expression BvM14-TAG2 gene Arabidopsis plant by using the BvM14-TAG2 gene, carries out stress treatment, and the seed germination rate and root length of the BvM14-TAG2 gene heterologous expression plant are better than those of the wild type; and the biomass of the BvM14-TAG2 gene heterologous expression plant is higher than that of the wild type plant; it is shown that the BvM14-TAG2 gene promotes the growth and development of the plant under salt stress, and can improve the adaptability of the BvM14-TAG2 transgenic plant under salt stress. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 It is a visualization secondary structure prediction diagram of BvM14-TAG2 protein;

[0014] Figure 2 It is a visualization tertiary structure prediction diagram of BvM14-TAG2 protein;

[0015] Figure 3 It is BvM14-TAG2 gene expression analysis in leaves and roots, and in the figure, * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001;

[0016] Figure 4 It is BvM14-TAG2 gene salt stress response expression analysis diagram;

[0017] Figure 5 It is pCAMBIA2300-BvM14-TAG2-eYFP recombinant vector plasmid electrophoresis result diagram, Figure 5 Lanes 1-4 in the figure are pCAMBIA2300-BvM14-TAG2-eYFP recombinant plasmid, and the M lane is Supercoiled DNA Ladder Marker;

[0018] Figure 6 It is the pCAMBIA2300-BvM14-TAG2-eYFP subcellular localization contrast observation result diagram under the confocal microscope;

[0019] Figure 7 It is the screening result of BvM14-TAG2 gene heterologous expression Arabidopsis plant T0 generation seed in 1 / 2 MS solid culture medium;

[0020] Figure 8 It is the RT-PCR product electrophoresis result diagram of BvM14-TAG2 gene heterologous expression Arabidopsis plant cDNA, and in the figure, WT is wild type Arabidopsis plant; OE#1-OE#3 is BvM14-TAG2 gene heterologous expression plant;

[0021] Figure 9Real-time PCR of Arabidopsis plants heterologously expressing BvM14-TAG2 gene;

[0022] Figure 10 Figure of germination phenotype experiment results of Arabidopsis plants heterologously expressing BvM14-TAG2 gene under 0, 150 mM NaCl treatment;

[0023] Figure 11 Figure of root length determination of Arabidopsis plants heterologously expressing BvM14-TAG2 gene under 0, 150 mM NaCl treatment;

[0024] Figure 12 Figure of phenotype observation of wild type plants and Arabidopsis plants heterologously expressing BvM14-TAG2 under 0, 150 mM NaCl treatment;

[0025] Figure 13 Data of dry and fresh weight determination of Arabidopsis plants heterologously expressing BvM14-TAG2 under 0, 150 mM NaCl treatment;

[0026] Figure 14 Na + , K + standard solution curve figure in Example 7;

[0027] Figure 15 Results of ion content determination of Arabidopsis plants heterologously expressing BvM14-TAG2 and wild type plants;

[0028] Figure 16 Results of POD, SOD, CAT, APX enzyme activity determination of Arabidopsis plants heterologously expressing BvM14-TAG2 and wild type plants;

[0029] Figure 17 Pro content determination experiment of Arabidopsis plants heterologously expressing BvM14-TAG2 and wild type plants;

[0030] Figure 18 Results of MDA content determination of Arabidopsis plants heterologously expressing BvM14-TAG2 and wild type plants;

[0031] Figure 19 Results of O2 - content determination of Arabidopsis plants heterologously expressing BvM14-TAG2 and wild type plants;

[0032] Figure 20 Results of H2O2 content determination of Arabidopsis plants heterologously expressing BvM14-TAG2 and wild type plants. DETAILED DESCRIPTION

[0033] With reference to the accompanying drawings, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the protection scope of the present application.

[0034] Specific implementation manner one: the CDS sequence of the BvM14-TAG2 gene in the embodiment is shown as SEQ ID NO. 1.

[0035] In the embodiment, total RNA of the beet M14 strain is extracted, and then full-length gene cDNA is obtained by reverse transcription using the total RNA as a template; then, specific primers BvM14-TAG2-F and BvM14-TAG2-R are used as primers, and KOD high-fidelity enzyme is used for PCR amplification. The sequence of the primer BvM14-TAG2-F is 5'-ATAATGTCACGAGCAAGTGCAAAG-3'; and the sequence of the primer BvM14-TAG2-R is 5'-CTTAACCATGCCTCCAAGGATT-3'.

[0036] The PCR reaction system is shown in Table 1.

[0037] Table 1

[0038] System components Volume (μL) Mg 2+ ]]> 1.5 Beta vulgaris M14 cDNA 1 KOD enzyme 0.5 BvM14-TAG2-F 1 BvM14-TAG2-R 1 dNTP Mixture 2.5 10×KOD Buffer 2.5 ddH2O up to 25

[0039] The PCR reaction program is as follows: 94℃ 2 min; 98℃ 10 s, 55℃ 30 s, 68℃ 2 min, 30 cycles; and 4℃ storage. After the PCR ends, 1% agarose gel electrophoresis detection is performed.

[0040] The PCR product (BvM14-TAG2) obtained above is purified and recovered, and then the recovered BvM14-TAG2 gene fragment is tailed and connected with a T vector. After 16℃ connection for 6-12 h, Escherichia coli DH5α is transformed; then a single colony is picked in LB liquid medium containing a corresponding antibiotic and is expanded; after the bacterial liquid is turbid, bacterial liquid PCR verification is performed, and plasmid extraction is performed for sequencing according to the TaKaRa Mini BEST Plasmid Purification Kit Ver. 4.0 instruction manual.

[0041] The reaction system of the BvM14-TAG2 gene fragment tailing is shown in Table 2.

[0042] Table 2

[0043] System components Volume (μL) Gel recovery PCR product (BvM14-TAG2) 20 10x PCR Buffer (Mg 2+ plus) 2.5 dNTPs 2 rTaq DNA polymerase 0.25

[0044] The connection T vector reaction system is shown in Table 3:

[0045] Table 3

[0046] System components Volume (μL) Tailed gel recovery product 4 pMD TM 18-T]] 1 Solution I 5

[0047] The recombinant plasmid was extracted and sequenced using an alkaline lysis method.

[0048] Sequencing results: The full-length cDNA of BvM14-TAG2 gene is 1189 bp; the CDS region is 960 bp; a total of 320 amino acids are encoded; the relative molecular weight is 36.46 kDa. The CDS region has an "ATG" start codon and a "TAA" stop codon, and the conserved domain Adenine glyco segment is 127-305 aa.

[0049] Specific embodiment two: the amino acid sequence of the protein encoded by the BvM14-TAG2 gene of the present embodiment is shown in SEQ ID No. 2.

[0050] The physicochemical properties of the protein of the present embodiment were characterized by the Expasy Protparam tool. The results showed that the molecular composition of the protein was C 1623 H 2557 N 445 O 481 S 14 , the theoretical isoelectric point is 8.90, and the average hydrophobicity is -0.428, indicating that the protein as a whole presents hydrophilic characteristics.

[0051] The secondary structure of BvM14-TAG2 protein was predicted and analyzed using the NPS online platform, as shown in Figure 1 The secondary structure of BvM14-TAG2 protein is mainly composed of α-helix (41.38%), β-sheet (5.02%) and random coil (53.61%). Among them, α-helix and random coil constitute the main structural elements of the protein, while β-sheet accounts for a relatively small proportion; this structural feature suggests that BvM14-TAG2 protein may have specific spatial conformation and functional properties.

[0052] The tertiary structure of BvM14-TAG2 protein was predicted using the SWISS MODEL database, and the results are shown in Figure 2The C-terminal of BvM14-TAG2 protein has a highly conserved helix structure according to the tertiary structure prediction result, and the SMART domain analysis shows that it contains a conserved domain Adenine glyco at 127-305 aa, indicating that the protein is a typical 3-methyladenine DNA glycosylase I protein. The protein may recognize and bind to damaged DNA through the Adenine glyco conserved domain to play the function of DNA glycosylase.

[0053] Specific embodiment three: application of BvM14-TAG2 gene in improving plant salt tolerance.

[0054] Example 1

[0055] Reverse transcription of sugar beet RNA:

[0056] Extract the RNA of sugar beet M14 leaves and roots treated with 200, 400 mM NaCl, and adjust the concentration to below 500 ng / μL. Perform reverse transcription, and the system is as follows:

[0057] Table 4 Real-time PCR reverse transcription system

[0058] System components Volume (μL) 5× Prime Script RT Master Mix 2 Total RNA 1 ddH2O up to 10

[0059] The steps of Real-time PCR are as follows:

[0060] (1) Use primer 3 plus website (https: / / www.primer3plus.com / ) and NCBI-primerblast to design fluorescent quantitative primers;

[0061] (2) Obtain cDNA by reverse transcription as a template, and add the reaction system (Table 5) to an eight-tube tube;

[0062] (3) Set the fluorescent quantitative program as shown in Table 6;

[0063] (4) Analyze the data results, use Excel to calculate and integrate the data, and use 2 -ΔΔCT method to calculate the relative expression amount of genes.

[0064] Table 5 Real-time PCR reaction system

[0065] System components Volume (μL) 2× SGExcel FastSYBR Mixture 15 Upstream primer F (10 μM) 0.6 Downstream primer R (10 μM) 0.6 cDNA template 2 100× ROX Reference Dye 0.3 RNase-Free ddH2O up to 30

[0066] Table 6 Real-time PCR reaction program

[0067]

[0068] Real-time PCR technology was used to detect the expression level of BvM14-TAG2 gene in sugar beet root and leaf tissues, 18S rRNA was used as an internal reference gene for standardization, and the relative expression amount was calculated by 2 -ΔΔCT The test results are shown in Figure 3 The expression of BvM14-TAG2 gene in leaf tissue was significantly higher than that in root tissue, and the expression amount was about 2.87 times that of root tissue. This result shows that BvM14-TAG2 gene is expressed in sugar beet leaves and roots, but may play a more important biological function in leaves.

[0069] Real-time PCR method was used to analyze the response of BvM14-TAG2 gene under 200 mM and 400 mM NaCl treatment at different time points, the treatment time was 0 h, 0.5 h, 1 h, 3 h, 6 h, 9 h, 12 h, 24 h, 48 h, and the results are shown in Figure 4 As can be seen from Figure 4 Under 200 mM NaCl salt stress, the expression amount of BvM14-TAG2 gene in leaves gradually increased with the treatment time, and reached the highest at 12 h, about 6.89 times of 0 h; BvM14-TAG2 gene in roots was significantly up-regulated at 3 h and 6 h, and reached the highest at 9 h, about 13.06 times of 0 h. Under 400 mM salt stress, the expression amount of BvM14-TAG2 gene in leaves and roots showed a trend of gradually increasing first and then decreasing. The expression amount in leaves reached the peak at 6 h of salt stress; while in roots, the expression amount reached the peak at 3 h of salt stress.

[0070] Analysis shows that the expression amount of BvM14-TAG2 in sugar beet M14 roots is shorter than that in leaves under salt stress; the up-regulation of gene expression in leaves and roots of sugar beet M14 line under 400 mM NaCl treatment is faster than that under 200 mM NaCl, and the time to reach the highest expression is earlier. It shows that sugar beet M14 line responds to salt stress faster with the increase of salt concentration. Under two kinds of concentration treatment, the roots respond to salt stress faster than the leaves.

[0071] Example 2

[0072] Construction of eukaryotic expression vector of BvM14-TAG2 gene

[0073] 1. Construction of pCAMBIA1300S-3×FLAG-BvM14-TAG2 vector

[0074] (1) Design specific primers FLAG-TAG2-F and FLAG-TAG2-R with restriction endonuclease BamH I and Kpn I enzyme cutting sites, use T vector with target gene BvM14-TAG2 as template, and use KOD high fidelity enzyme for PCR amplification. The PCR amplification reaction system is consistent with Table 1, and the product is recovered and purified after the reaction.

[0075] (2) Perform double enzyme cutting of vector pCAMBIA1300S-3xFLAG with BamH I and Kpn I (reaction temperature 37℃, reaction time 3 h), and the system is as shown in Table 7:

[0076] Table 7 Enzyme cutting reaction system of pCAMBIA1300S-3xFLAG plasmid

[0077] System components Volume (μL) pCAMBIA1300S-3×FLAG vector ≤1 μg BamH I 1 Kpn I 1 10×K Buffer 2 ddH2O up to 20

[0078] After enzyme cutting, the enzyme cutting product is detected by electrophoresis and recovered and purified.

[0079] (3) According to the homologous recombination enzyme ligation system in Table 8 below, the target gene BvM14-TAG2 is connected with the linearized pCAMBIA1300S-3xFLAG vector using homologous recombination enzyme, and the connection is performed at 37℃ for 30 min.

[0080] Table 8 Homologous recombination enzyme ligation system

[0081] System components Volume (μL) Linearized pCAMBIA1300S-3×FLAG 0.03 pmol Target gene BvM14-TAG2 0.06 pmol 5×CE Buffer 4 Exnase II 2 ddH2O up to 20

[0082] 2, Construction of pCAMBIA2300-BvM14-TAG2-eYFP vector

[0083] (1) According to the enzyme cutting sites on the MCS of pCAMBIA2300-eYFP vector, design specific primers eYFP-TAG2-F and eYFP-TAG2-R using Primer 5 software, the sequences are 5'-CGACTCTAGAGGATCATGTCACGAGCAAGTGCAAAG-3', 5'-CCATACTAGTGGATCACCATGCCTCCAAGGATTTTCAG-3', and the T vector with the target gene BvM14-TAG2 is amplified using KOD high fidelity enzyme, and the PCR program is the same as Table 1.

[0084] (2) Perform single enzyme cutting of vector pCAMBIA2300-eYFP with BamH I (reaction temperature 30℃, reaction time 3 h), and the system is as shown in Table 9:

[0085] Table 9 Single enzyme cutting reaction system of vector

[0086] System components Volume (μL) pCAMBIA2300-eYFP 10 10×K Buffer 2 BamH I 1 ddH2O 7

[0087] After the completion of the enzyme digestion, agarose gel electrophoresis was performed for detection.

[0088] (3) The obtained pCAMBIA2300-BvM14-TAG2-eYFP target gene fragment was purified and recovered together with the single-digested pCAMBIA2300-eYFP linearized vector. The recovered product was subjected to 50°C metal bath connection for 15 min using Infusion enzyme, and the system is shown in Table 10:

[0089] Table 10 Infusion reaction system

[0090] System components Volume (μL) 5×Infusion HD Ligase 2 Insert fragment 2 Linearized vector 1 ddH2O 5

[0091] Connection and transformation of gel recovery product

[0092] (1) 50 μL of E. coli DH5α competent cells (which need to be thawed in advance on ice) were uniformly mixed with 10 μL of pCAMBIA1300S-3xFLAG-BvM14-TAG2 and pCAMBIA2300-BvM14-TAG2-eYFP connection liquid, and then E. coli was transformed.

[0093] (2) Agrobacterium GV3101 preserved at -80°C was thawed on ice, and then 1 μL of plasmid containing pCAMBIA1300S-3xFLAG-BvM14-TAG2 and pCAMBIA2300-BvM14-TAG2-eYFP recombinant vector (plasmid volume <10 μL, plasmid concentration ≤1 μg) was added;

[0094] (3) After mixing, ice for 5 min, liquid nitrogen for 5 min, 28°C water bath for 5 min, and ice bath for 5 min;

[0095] (4) 1 mL of LB medium without antibiotics was added to the transformation tube, and the mixture was incubated for 2 h;

[0096] (5) After centrifugation at 6000 rpm for 2 min, about 100 μL of liquid medium was reserved, the precipitate was resuspended, and then plated on LB medium containing antibiotics and incubated in an incubator for 24-48 h;

[0097] (6) Colony PCR was performed to verify whether the transformation was successful;

[0098] (7) The bacterial liquid containing the target band was expanded to 50 mL of LB liquid medium containing kanamycin and rifampicin antibiotics;

[0099] (8) The bacterial liquid was preserved and used for subsequent experiments.

[0100] The BvM14-TAG2 gene was inserted into the pCAMBIA2300-eYFP vector by molecular cloning technology, and a fusion expression vector pCAMBIA2300-BvM14-TAG2-eYFP was successfully constructed. The recombinant plasmid was transformed into DH5a E. coli competent cells, and the plasmid was extracted after amplification and verified by electrophoresis. The agarose gel electrophoresis results showed that (Figure 1) Figure 5 ), a specific band was observed at the expected molecular weight position, confirming that the recombinant plasmid was correctly constructed.

[0101] Example 3

[0102] Subcellular localization experiment

[0103] (1) Recombinant positive vector colonies (Agrobacterium containing pCAMBIA2300-eYFP vector as control) were transferred to 50 mL LB liquid medium for expansion;

[0104] (2) 8 mL of bacterial solution was added to a 10 mL centrifuge tube, and the bacterial body was collected by centrifugation at 8000 rpm for 10 min at room temperature. The operation was repeated to collect the bacterial body;

[0105] (3) Discard the supernatant, add 1 mL of tobacco transformation solution, resuspend the bacterial body, and centrifuge again;

[0106] (4) Discard the supernatant, add transformation solution at a ratio of about 2:1 of bacterial solution: transformation solution, resuspend the bacterial body, and use a spectrophotometer to detect OD 600 , adjust the OD value to 0.8-1.0, and stand in the dark at room temperature for 2 h;

[0107] (5) Use a syringe to inject the bacterial solution into tobacco leaves, filter paper to absorb surface moisture, and place in the dark for 24 h after injection and then move to light;

[0108] (6) After 24 h of light recovery, inject DAPI nuclear staining solution at the same position, and use a confocal microscope to observe the subcellular localization after 2 h.

[0109] In this example, Agrobacterium carrying pCAMBIA2300-eYFP empty vector and pCAMBIA2300-BvM14-TAG2-eYFP recombinant plasmid were injected into Nicotiana benthamiana leaves for transient expression. After 48 h of transformation, DAPI staining solution was injected, incubated in the dark for 2 h, and then the fluorescence signal was observed using a laser confocal microscope. The results are shown in Figure 2 Figure 6As shown, the fluorescence signal of BvM14-TAG2-eYFP fusion protein was co-localized with the nuclear marker DAPI, indicating that the protein was located in the nucleus, which was consistent with the prediction of bioinformatics analysis, confirming the hypothesis that BvM14-TAG2 protein played a DNA glycosylase function in the nucleus.

[0110] Example 4

[0111] Obtaining and identification of BvM14-TAG2 gene heterologous expression plant

[0112] 1. Using inflorescence dip method to transform Arabidopsis

[0113] (1) The Agrobacterium monoclonal with target gene prepared in Example 2 was inoculated into liquid medium and incubated at 28°C constant temperature shaker until the bacterial solution was turbid; then centrifuged at 4°C, 8000 rpm for 10 min to collect the bacterial body;

[0114] (2) Add 5 mL Agrobacterium transformation buffer to each centrifuge tube, gently blow to resuspend the bacterial body; centrifuge again at 4°C, 8000 rpm for 5 min, and discard the supernatant;

[0115] (3) Resuspend the precipitated bacterial body with an appropriate amount of transformation buffer, and use a spectrophotometer to measure the OD value, adjust the bacterial solution concentration to 0.8-1.0, and prepare for plant transformation; 600

[0116] (4) Select well-developed Arabidopsis inflorescences for transformation treatment; immerse the inflorescences in the prepared bacterial solution for 1 min, remove the excess liquid on the surface with sterile filter paper, and mark the treated inflorescences with a label rope;

[0117] (5) The transformed plants were cultured in the dark for 24 h and then transferred to light conditions for growth; repeat the transformation operation every 24 h, and each Arabidopsis plant can be treated 2-3 times;

[0118] (6) Regularly water the plants, collect and dry the seeds when they are mature, and perform vernalization treatment at 4°C;

[0119] (7) Seed disinfection: 75% ethanol treatment for 1 min, 5 min, and 10 min gradient disinfection, then 1 min of anhydrous ethanol treatment, and then evenly sowing on MS medium containing 30 mg / L hygromycin; after 12-14 d of culture, select seedlings with strong root system and good growth, transfer them to MS medium containing only cefotaxime for further culture for about 10 d, then transplant them to nutrient soil, cover with plastic wrap to keep the soil moist, and regularly water the plants;

[0120] ​(8) When the plants grow to have three pairs of true leaves, collect leaf tissues to extract genomic DNA, and perform molecular identification of the heterologous expression gene plants.

[0121] 2. Identification of the heterologous expression BvM14-TAG2 gene Arabidopsis plants

[0122] (1) The extraction of plant genomic DNA was performed by urea method, and the specific experimental procedure was performed according to the method described in Biological Basic Experiment;

[0123] (2) The wild type Arabidopsis was used as a control, and the genomic DNA of the heterologous expression BvM14-TAG2 gene Arabidopsis was used as a template for PCR verification of positive heterologous expression plants;

[0124] (3) After reverse transcription, RT-PCR and Real-time PCR were performed for verification, and positive plants were confirmed.

[0125] In this example, the flower dip method was used to infect wild type Arabidopsis (WT) with Agrobacterium tumefaciens GV3101 strain carrying pCAMBIA1300S-BvM14-TAG2-3xFLAG recombinant plasmid, and T1 generation transformed seeds were successfully harvested. The seeds were evenly sown on 1 / 2 MS selection medium containing 30 mg / L hygromycin, and resistant seedlings were selected after 7 days of culture. As shown in FIG. 1, seedlings with typical hygromycin resistance phenotype (main root elongation was significantly, and true leaf unfolded well) were selected, transplanted into sterile substrate for further culture, and used to obtain homozygous Arabidopsis lines. Figure 7

[0126] The plants were further verified, and the genomic DNA of the positive plants was extracted for genomic PCR and RT-PCR verification. In the genomic DNA level verification results, some contained bands while the wild type plants did not, proving that the BvM14-TAG2 gene was successfully inserted. The RNA was extracted, and after reverse transcription, the DNA concentration was adjusted to be consistent, and RT-PCR was performed. The verification results showed that the transcription level of BvM14-TAG2 gene in each line was different, and from the 10 lines with BvM14-TAG2 gene inserted, the lines with clear and obvious bands were named OE#1, OE#2, and OE#3 (as shown in FIG. 2). Figure 8

[0127] After screening and identification, T3 generation OE#1, OE#2, and OE#3 were all homozygous lines, and Real-time PCR was used to detect the expression amount of BvM14-TAG2 gene, and the results are shown in FIG. 3. The above results show that the heterologous expression of BvM14-TAG2 gene in Arabidopsis plants is successfully established. Figure 9

[0128] Example 5 ​​​

[0129] Phenotype observation of heterologous expression gene plant and determination of physiological and biochemical indexes

[0130] In this example, wild type Arabidopsis (WT) was used as control, and salt-related indexes of BvM14-TAG2 gene-expressed plants (OE#1, OE#2, OE#3) were determined.

[0131] 1. In order to clarify the effect of salt stress on the germination rate of Arabidopsis seeds with heterologous expression of BvM14-TAG2 gene, the germination rates of wild type Arabidopsis and Arabidopsis with heterologous expression of BvM14-TAG2 gene under 0 mM salt treatment and 150 mM salt stress were counted in this example.

[0132] According to the statistical results of Figure 10 , the germination rate of wild type Arabidopsis seeds under salt stress condition was significantly reduced to 31%, while the average germination rate of Arabidopsis seeds with heterologous expression of BvM14-TAG2 gene maintained at the level of 79%, showing obvious salt tolerance advantage. This comparative analysis shows that salt stress environment can significantly inhibit the germination process of plants, but the heterologous expression of BvM14-TAG2 gene can effectively alleviate the inhibitory effect of salt stress on seed germination, so that Arabidopsis seeds with heterologous expression of BvM14-TAG2 gene maintain high germination ability.

[0133] 2. Growth of root length of wild type plants (WT) and Arabidopsis plants with heterologous expression of BvM14-TAG2 gene (OE#1, OE#2, OE#3) under salt stress

[0134] The seeds of wild type and Arabidopsis plants with heterologous expression of BvM14-TAG2 gene were cultured in 1 / 2 MS solid medium after sterilization, and then transferred to normal culture after 48 h of 4°C dark culture. After 7 days, they were transferred to 1 / 2 MS solid medium with 0, 150 mM NaCl, 3 biological replicates were set for each group, and the root length phenotype was observed and photographed after vertical culture for 7 days.

[0135] The plant root length phenotype results are shown in Figure 11 a, under 0 mM salt treatment, wild type Arabidopsis and Arabidopsis seedlings with heterologous expression of BvM14-TAG2 gene grew normally, but the root length of Arabidopsis seedlings with heterologous expression of BvM14-TAG2 gene was longer; while under 150 mM NaCl, wild type Arabidopsis showed yellowing and wilting of seedlings, and the center turned red, but Arabidopsis seedlings with heterologous expression of BvM14-TAG2 gene grew well, with slightly curled leaves and longer root system.

[0136] The plant root length statistical results are shown in Figure 11As shown in b, a statistical comparison of root development between wild-type Arabidopsis thaliana and Arabidopsis thaliana plants expressing the BvM14-TAG2 gene revealed that, under normal growth conditions, the root length of the Arabidopsis thaliana line expressing the BvM14-TAG2 gene was significantly better than that of the wild-type plant. When subjected to 150 mM NaCl stress, although root growth of all plants was inhibited, the Arabidopsis thaliana line expressing the BvM14-TAG2 gene still maintained a relatively long root system.

[0137] Experimental results show that the expression of the BvM14-TAG2 gene can effectively alleviate the inhibitory effect of salt stress on root growth, and enhance the plant's ability to absorb nutrients and its resistance to stress by promoting root development.

[0138] Example 6

[0139] Determination of plant dry weight and fresh weight

[0140] Seeds of wild-type Arabidopsis thaliana and Arabidopsis thaliana heterologously expressing the BvM14-TAG2 gene were sown in a 1:1 mixture of vermiculite and potting soil and cultured in an artificial climate chamber. Culture conditions were set at a daytime temperature of 22℃, a photoperiod of 8 h light / 16 h dark, and culture for 4 weeks. The stress treatment group was irrigated with 150 mM NaCl solution, while the control group received an equal volume of distilled water. Five days later, plants from both groups were collected, and their fresh weight and dry weight were measured to assess the effect of salt stress on plant growth.

[0141] Wild-type Arabidopsis thaliana (WT) and heterologous BvM14-TAG2 gene-expressing plants (OE#1, OE#2, OE#3) grown for 4 weeks were subjected to salt stress treatment with two concentration gradients of 0 mM and 150 mM NaCl. Salt treatment was performed every 48 hours for a total of three treatments. Phenotypic changes were observed 5 days after treatment. Figure 12 As shown, both lines developed normally under 0 mM salt stress, with well-developed rosette leaves and bright green leaf color. However, the OE line exhibited more vigorous growth than the WT line. When treated with 150 mM NaCl, all plants showed significant growth inhibition symptoms, including leaf wilting and yellowing. Notably, the WT plants showed more severe salt damage symptoms, exhibiting more pronounced leaf yellowing, curling, and growth retardation, while the OE line showed relatively stronger tolerance to salt stress.

[0142] according to Figure 13As shown, under normal growth conditions (0 mM NaCl), the dry weight and fresh weight of leaves of Arabidopsis plants heterologously expressing the BvM14-TAG2 gene were higher than those of wild-type plants. However, under 150 mM NaCl stress treatment, the dry weight and fresh weight of leaves of Arabidopsis plants heterologously expressing the BvM14-TAG2 gene were significantly greater than those of wild-type plants. The dry weight and fresh weight of roots were consistent with the trend in leaves. The data show that expression of the BvM14-TAG2 gene can effectively alleviate the inhibition of plant growth by salt stress, confirming that the gene plays an important role in improving plant salt tolerance.

[0143] Example 7

[0144] To determine the Na + and K + content of leaves, 0.5 g of Arabidopsis true leaves (wild-type plants and Arabidopsis plants heterologously expressing the BvM14-TAG2 gene) were taken, killed at 110°C for 30 min, dried at 70°C to constant weight, ground into powder, and filtered through a 0.5 mm sieve. The ground sample was soaked with water, digested in concentrated H2SO4 after 10 min, and left to stand at room temperature overnight. After heating to brown, 30% H2O2 was added to ensure complete decomposition of H2O2 to transparency of the solution. After cooling, the Na + and K + content of the sample was determined using a flame photometer. The standard solution was prepared as shown in Tables 11 and 12, and a standard curve was plotted according to the theoretical point concentration and absorbance value, as shown in Figure 14 , and the ion content was calculated according to the standard curve.

[0145] Table 11 Na + standard solution preparation table

[0146] Sample 1 2 3 4 5 6 7 Sodium standard solution (100.0 mg / L) 0.00 0.10 0.30 0.50 0.70 1.00 1.30 CsCl solution (10.0 g / L) 0.30 0.30 0.30 0.30 0.30 0.30 0.30 65% HNO3 solution 0.10 0.10 0.10 0.10 0.10 0.10 0.10 Theoretical concentration of each point (mg / L) 0.00 0.20 0.60 1.00 1.50 2.00 2.50 ddH2O 49.60 49.50 49.30 49.10 48.85 48.60 48.30 Absorbance A 589 ]]> 0.002 0.075 0.252 0.343 0.513 0.74 0.923

[0147] Table 12 K + standard solution preparation table

[0148] Sample 1 2 3 4 5 6 Sodium standard solution (100.0 mg / L) 0.00 0.05 0.10 0.15 0.20 0.25 CsCl solution (10.0 g / L) 0.30 0.30 0.30 0.30 0.30 0.30 65% HNO3 solution 0.10 0.10 0.10 0.10 0.10 0.10 Theoretical concentration of each point (mg / L) 0 1.00 2.00 3.00 4.00 5.00 ddH2O 4.60 4.55 4.50 4.45 4.40 4.35 Absorbance A 766 ]] 0.007 0.375 0.572 0.813 1.252 1.426

[0149] The Na + and K + content of wild-type plants and Arabidopsis plants heterologously expressing the BvM14-TAG2 gene in the control group and the salt treatment group was determined. As shown in Figure 15 , the experimental results show that under salt stress conditions, plants heterologously expressing the BvM14-TAG2 gene can better maintain ion balance in cells and effectively alleviate the ion toxicity effect caused by salt stress.

[0150] Under 0 mM salt treatment stress conditions, the Na+ K + No significant differences were observed in ion concentration. However, when treated with 150 mM NaCl, Arabidopsis plants heterologously expressing the BvM14-TAG2 gene exhibited a significant ability to regulate ion homeostasis, with their NaCl concentrations decreasing. + Accumulation was significantly lower than that of wild-type plants, while maintaining a higher K content. + This level, thus maintaining a more favorable K level. + / Na + Proportion.

[0151] Under salt stress, Arabidopsis plants heterologously expressing the BvM14-TAG2 gene exhibited superior ion regulation capabilities compared to wild-type plants. Expression of this gene can enhance Na+ ion regulation. + Improve discharge efficiency while reducing K + The loss of [K] effectively maintains a high level of intracellular K. + / Na + The ratio significantly improved the plant's ability to adapt to high-salt environments.

[0152] Example 8

[0153] Weigh 0.1 g of fresh leaves from wild-type Arabidopsis thaliana and Arabidopsis thaliana plants heterologously expressing the BvM14-TAG2 gene, add 1 mL of protein extraction buffer, and grind thoroughly under ice bath conditions until the tissue is completely ruptured. Centrifuge the homogenate at 12000 rpm for 15 min at 4℃, collect the supernatant as the test sample, and place it on ice for later use. The obtained data will be used for subsequent calculation and analysis of physiological and biochemical indicators. All operations were performed under low temperature conditions to ensure the stability of the protein samples.

[0154] The POD enzyme activity, SOD enzyme activity, CAT enzyme activity, APX enzyme activity and MDA content were determined using kits from Suzhou Greens Biotechnology Co., Ltd. (G0109W, G0101W, G0107W, G0106W and G0203W). The specific procedures were performed in accordance with the relevant kit instructions.

[0155] Enzyme activity assay results as follows Figure 16 As shown, changes in these enzyme activities can reflect the degree of physiological response of plants to salt stress. Under normal growth conditions (0 mM NaCl), the activities of POD, SOD, and APX in Arabidopsis plants heterologously expressing the BvM14-TAG2 gene were comparable to those in wild-type plants, while the CAT enzyme activity was significantly higher in wild-type plants. However, when subjected to 150 mM NaCl stress, the activities of antioxidant enzymes in wild-type plants were significantly lower than those in Arabidopsis plants heterologously expressing the BvM14-TAG2 gene. These results indicate that expression of the BvM14-TAG2 gene can enhance the antioxidant defense capacity of plants under salt stress conditions.

[0156] Under high salt stress, BvM14-TAG2 gene can remove the active oxygen free radicals accumulated in the plant body by activating the activities of POD, SOD, APX and CAT, which helps to maintain the balance of active oxygen metabolism in cells and significantly reduces the damage caused by oxidative stress to plant cells.

[0157] Example 9

[0158] Take 0.1 g of fresh leaves of wild-type Arabidopsis and BvM14-TAG2 gene-expressed Arabidopsis plants, add 1 mL of protein extraction buffer, and grind thoroughly in an ice bath until the tissue is completely broken. Centrifuge the homogenate at 4°C, 12000 rpm for 15 min, collect the supernatant as the sample to be tested, and store on ice. The data obtained are used for subsequent calculation and analysis of physiological and biochemical indicators, and all operations are carried out in a low-temperature environment.

[0159] MDA is a key end product of membrane lipid peroxidation, and the concentration of MDA is positively correlated with the degree of cell membrane damage. This embodiment determines the MDA content of BvM14-TAG2 gene-expressed Arabidopsis plants and wild-type plants, and the results are shown in Figure 18

[0160] Under 0 mM salt stress, the MDA content of wild-type and BvM14-TAG2 gene-expressed Arabidopsis plants is basically the same. However, when treated with 150 mM NaCl, the MDA accumulation of BvM14-TAG2 gene-expressed lines is significantly less than that of wild-type plants. This indicates that under salt stress, BvM14-TAG2 gene can effectively inhibit membrane lipid peroxidation and reduce the production of MDA, thereby enhancing the stability of plant cell membrane.

[0161] Example 10

[0162] Proline (Pro) content determination under salt stress

[0163] Take 0.1 g of fresh leaf tissue of wild-type Arabidopsis and BvM14-TAG2 gene-expressed Arabidopsis plants, add 1 mL of pre-cooled extraction buffer, and grind thoroughly in an ice bath until homogenized. Transfer the homogenate to a 1.5 mL centrifuge tube and heat in a 90°C water bath for 10 min. Then centrifuge at 12000 rpm for 10 min at room temperature, collect the supernatant, and store it at room temperature after cooling to room temperature. The composition and volume ratio of the specific reaction system are shown in Table 13.

[0164] Table 13 Proline mixture sample

[0165] Reagent name Test tube (μL) Blank tube (μL) Sample 150 - Distilled water - 150 Glacial acetic acid 150 150 Reagent one 300 300 ​

[0166] Heat in 95℃ water bath for 30 min, cool to room temperature, measure absorbance value at 520 nm. △A=A 测定 -A 空白 ;

[0167] Proline content: calculated according to the standard curve equation;

[0168] y=0.1625x-0.0064; x is the mass of standard (μg), y is △A.

[0169] This example determines the Pro level of Arabidopsis plants heterologously expressing BvM14-TAG2 gene and wild type plants under different treatment conditions, as shown in Table 6. Figure 17 As shown in Table 6, under non-stress conditions, the Pro level of Arabidopsis plants heterologously expressing BvM14-TAG2 gene is basically consistent with that of wild type Arabidopsis. However, under 150 mM NaCl treatment, plants heterologously expressing BvM14-TAG2 gene show more significant Pro accumulation. It is indicated that the gene can enhance the osmotic regulation function of cells by regulating the Pro biosynthesis pathway, thereby improving the adaptability of plants to high salt environment.

[0170] The results show that under salt stress, BvM14-TAG2 gene can increase the accumulation amount of proline in Arabidopsis plants, and maintain the osmotic balance of cells by regulating osmotic pressure.

[0171] Example 11

[0172] Determination of superoxide anion (O2-·) content under salt stress

[0173] Take 0.1 g of fresh plant leaf sample, add 1 mL of extraction solution, grind on ice to homogenate, centrifuge at 12000 rpm, 4℃ for 10 min, take the supernatant and measure on ice, the reaction system is as shown in Table 14;

[0174] Mix well, centrifuge at 25℃, 8000 rpm for 5 min, carefully pipette 200 μL of the upper aqueous phase into a 96-well plate, measure the absorbance value at A 530 , calculate △A 标准 =A 标准管 -A 空白管 , △A 测定 =A 测定管 -A 空白管 ;

[0175] Superoxide anion (O2 - ·) content: calculated according to the instructions of biochemical kit D799772-0100 of GenScript.

[0176] Table 14 Samples of mixed solutions containing superoxide anions

[0177] In this embodiment, O2 levels were measured in wild-type plants and BvM14-TAG2 heterologous expression plants under 0 and 150 mM NaCl. - • Content. For example... Figure 19 As shown, the BvM14-TAG2 gene can significantly reduce plant O2 under salt stress. - ·content.

[0178] Determination of hydrogen peroxide (H2O2) content under salt stress conditions

[0179] Take 0.1 g of fresh leaves from wild-type Arabidopsis thaliana and Arabidopsis thaliana plants expressing the BvM14-TAG2 gene heterologously, grind them with 1 mL of extract, homogenize them in an ice bath, centrifuge at 8000 g for 10 min at 4℃, collect the supernatant, place it on ice for testing, and the reaction system is shown in Table 15.

[0180] Table 15 Samples of hydrogen peroxide mixture

[0181] After adding reagent four to dissolve the precipitate, let it stand at room temperature for 5 min, then transfer 200 μL to a 96-well plate to determine A. 415 Calculate ΔA based on the absorbance value at the specified location. 测定 =A 测定管 -A 空白管 , △A 标准 =A 标准管 -A 空白管 ;

[0182] Hydrogen peroxide (H2O2) content: calculated according to the instructions of Sangon Biotech Biochemical Reagent Kit D799774-0100.

[0183] In this embodiment, the H2O2 content of wild-type plants and BvM14-TAG2 heterologous expression plants was measured under 0 and 150 mM NaCl conditions. Figure 20 As shown, the BvM14-TAG2 gene can significantly reduce the H2O2 content of plants under salt stress.

[0184] The experimental results of this embodiment show that the BvM14-TAG2 gene can effectively improve the plant's ability to scavenge ROS and effectively reduce ROS oxidative damage caused by salt stress.

Claims

1. A BvM14-TAG2 gene characterized by: The CDS sequence of the gene is shown as SEQ ID NO.

1.

2. The protein encoded by the BvM14-TAG2 gene according to claim 1, characterized by, The amino acid sequence of the protein is shown as SEQ ID No.

2.

3. Use of the BvM14-TAG2 gene of claim 1 in improving salt tolerance of plants.

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