CN118291498B - A phosphoethanolamine methyltransferase gene and its preparation method and application - Google Patents

Abstract

The present invention designs a phosphoethanolamine methyltransferase gene, a preparation method and an application, and belongs to the field of gene engineering technology. The nucleotide sequence of the phosphoethanolamine methyltransferase gene is shown in SEQ ID NO.1. The Suaeda salsa phosphoethanolamine methyltransferase gene and the overexpression vector of the present invention can significantly improve the salt tolerance of plants, especially through the determination and analysis of the SOD activity, POD activity and CAT activity of transgenic plants and the detection of the phenotype and biomass of transgenic processing tomato plants, the results show that the processing tomato plants introduced with the plant overexpression vector have significantly enhanced their salt tolerance.

Description

A phosphoethanolamine methyltransferase gene and its preparation method and application

Technical Field

The invention relates to a phosphoethanolamine methyltransferase gene and a preparation method and application thereof, belonging to the technical field of genetic engineering.

Background Art

Plant phosphoethanolamine methyltransferase generates phosphorylcholine through three consecutive methylations, and phosphorylcholine generates choline and phosphatidic acid under the hydrolysis of phosphorylcholine phosphatase. Choline is the substrate for the synthesis of glycine betaine, a small molecule organic osmotic regulating substance. Betaine is a soluble alkaloid that can enhance plant resistance to drought and salt stress. Studies have found that the synthesis rate of choline in higher plants is strictly limited, and phosphoethanolamine methyltransferase is the key rate-limiting enzyme in the synthesis of choline, the substrate of betaine. In addition, phospholipids are not only components of plant cell membranes, but also lipid second messengers in lipid metabolism and processes such as biotic and abiotic stresses. They accumulate rapidly during plant growth and development and under adverse stress conditions, and play an important role in various signal transduction pathways by binding to target proteins and regulating their activity and subcellular localization. Therefore, phosphoethanolamine methyltransferase plays an important role in the regulation of plant resistance to stress.

Summary of the invention

In view of the deficiencies of the prior art, the present invention provides a phosphoethanolamine methyltransferase gene, a preparation method and an application thereof, and specifically relates to a Suaeda salsa phosphoethanolamine methyltransferase gene SdNMT and its application in improving plant salt tolerance. The nucleotide sequence of the gene SdNMT in the present invention is shown in SEQ ID NO.1.

The present invention clones the SdNMT gene and constructs a plant overexpression vector. It is also found that the expression of the phosphoethanolamine methyltransferase gene SdNMT in Suaeda salsa is significantly increased under 800mmo/LNaCl stress. The present invention introduces the constructed plant overexpression vector into the processing tomato genome through Agrobacterium-mediated method to achieve the effect of enhancing the salt tolerance of transgenic plants. The gene fragment inserted in the plant overexpression vector is only the phosphoethanolamine methyltransferase gene SdNMT; preferably, the empty vector is pCAMBI2300.

Furthermore, the primer sequences for amplifying the gene SdNMT are SEQ ID NO.2 and SEQ ID NO.3 respectively.

Furthermore, the method for constructing the plant overexpression vector is as follows: constructing the gene SdNMT into the plant expression vector pCAMBIA2300 by using Kpn Ⅰ and BamH Ⅰ enzymes.

The present invention also includes the application of the transferase gene SdNMT and the constructed plant overexpression vector in plant salt tolerance, and further in creating new salt-tolerant varieties.

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

The Suaeda salsa phosphoethanolamine methyltransferase gene SdNMT and the overexpression vector of the present invention can significantly improve the salt tolerance of plants, especially through the determination and analysis of the SOD activity, POD activity and CAT activity of the transgenic plants and the detection of the phenotype and biomass of the transgenic processing tomato plants, the results show that the processing tomato plants introduced with the plant overexpression vector have significantly enhanced their salt tolerance.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a PCR gel image of gene SdNMT;

FIG2 is a diagram of phosphorylation sites of the N-terminal methyltransferase domain of the gene SdNMT;

FIG3 is a diagram showing phosphorylation sites of the C-terminal methyltransferase domain of the gene SdNMT;

FIG4 is a prokaryotic expression vector map of gene SdNMT;

FIG5 is a diagram showing the restriction enzyme digestion verification of the prokaryotic expression recombinant plasmid of the gene SdNMT;

FIG6 is a diagram showing the expression and identification results of the protein in prokaryotic cells;

Figure 7 is a graph showing the results of induction condition optimization and protein solubility analysis; wherein, lane M: Protein Marker; lane 1: sample after induction with 0.2mM IPTG at 15°C; lane 2: sample after induction with 1.0mM IPTG at 15°C; lane 3: sample after induction with 0.2mM IPTG at 37°C; lane 4: sample after induction with 1.0mM IPTG at 37°C; lane 5: sample without induction; lane 6: sample precipitated after induction with 1.0mM IPTG at 37°C; lane 7: sample supernatant after induction with 1.0mM IPTG at 37°C; lane 8: sample precipitated after induction with 0.2mMIPTG at 37°C; lane 9: sample supernatant after induction with 0.2mM IPTG at 37°C; lane 10: sample precipitated after induction with 1.0mM IPTG at 15°C; lane 11: sample supernatant after induction with 1.0mM IPTG at 15°C; lane 12: sample precipitated after induction with 0.2mM IPTG at 15°C IPTG-induced precipitation sample; Lane 13: 15°C 0.2 mM IPTG-induced supernatant sample;

FIG8 is a graph showing the affinity purification SDS-PAGE results of the protein;

FIG. 9 is a graph showing the results of SDS-PAGE of protein purification samples.

FIG10 is a diagram of a constructed plant overexpression vector;

FIG11 shows the expression of the target gene SdNMT in transgenic plants;

Figure 12 shows the choline content in transgenic tomato plants;

Figure 13 shows the betaine content in transgenic plants;

FIG14 is a diagram showing the SOD activity assay of transgenic plants after salt stress treatment;

FIG15 is a graph showing the POD activity assay analysis of transgenic plants after salt stress treatment;

FIG16 is a diagram showing the CAT activity assay analysis of transgenic plants after salt stress treatment;

FIG17 is a graph showing the growth of transgenic processing tomato plants under salt stress;

FIG. 18 shows the aboveground and underground biomass of transgenic processing tomato plants under salt stress; FIG. 18A shows the aboveground fresh weight, FIG. 18B shows the aboveground dry weight, FIG. 18C shows the underground fresh weight, and FIG. 18D shows the underground dry weight.

DETAILED DESCRIPTION

The technical solution of the present invention is described in detail below through specific implementation methods. It should be understood that the following specific implementation methods are only exemplary, and any changes or modifications, as long as they do not deviate from the technical solution design of the present invention, should be within the scope of protection of the rights of the present invention. The present invention is described in detail below in conjunction with examples.

Example 1: Acquisition of gene SdNMT

1. Biomaterials

Suaeda dendroides, processing tomato (Solanum lycopersicum), plant expression vector pCAMBI2300, Agrobacterium tumefaciens GV3101.

2. Technical methods

2.1 Total RNA extraction and cDNA synthesis from Suaeda salsa leaves

The present invention adopts the method provided by the high-efficiency plant genomic DNA extraction kit (DP350) of Tiangen Biotechnology Company to extract total RNA from Suaeda salsa leaves. The specific steps are as follows:

2.1.1 RNA extraction

(1) Take 0.1 g of fresh leaves and put them into a 2 mL EP tube. Add 400 μl of extraction solution LP1, 6 μl of RNase A (10 mg/ml), and two steel beads (4 mm in diameter). Oscillate at 30 Hz for 2 min until the leaves are homogenized; vortex for 1 min and place at room temperature for 10 min.

(2) Add 130 μl of buffer LP2 and vortex for 1 min to mix thoroughly. ③ Centrifuge at 13,000 g for 5 min and transfer the supernatant into a new EP tube.

(3) Centrifuge at 13,000 g for 5 min and transfer the supernatant into a new EP tube.

(4) Add 1.5 times the volume of buffer LP3 (add anhydrous ethanol before use), immediately shake and mix thoroughly for 15 seconds, add to another adsorption column CB3, centrifuge at 12,000 g for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube.

(5) Add 600 μl of rinse solution PW (add anhydrous ethanol before use) to the adsorption column CB3, centrifuge at 12,000 g for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube. Repeat step 5.

(6) Place the adsorption column CB3 in a collection tube and centrifuge at 12,000 g for 2 min. Discard the waste liquid and place the adsorption column CB3 in a collection tube. Thoroughly dry the residual rinse liquid in the adsorption material.

(7) Transfer the adsorption column CB3 into a new EP tube, add 50-200 μl of RNAase-free dd H ₂ O to the middle of the adsorption membrane, leave it at room temperature for 2-5 minutes, centrifuge it at 12,000 g for 2 minutes, collect the solution in a centrifuge tube, and store it at -20°C for later use.

2.1.2 cDNA synthesis

The cDNA was synthesized using the reverse transcription kit PrimeScriptTM RT reagent kit with gDNAEraser (TaKaRa, Code No. RR047A). The operation method is as follows:

Step 1: Removal of genomic DNA reaction

Prepare the genomic DNA removal reaction solution in Table 1 in a 0.2 ml PCR centrifuge tube.

Table 1 - Genomic DNA removal reaction solution

After mixing, incubate at 42°C for 2 min (or room temperature for 5 min) and store at 4°C.

Continue to prepare the reverse transcription reaction solution in Table 2 in the above PCR tube.

Table 2 - Reverse transcription reaction solution

37℃, 15min; 85℃, 5sec; store at 4℃.

2.2 Cloning of cDNA sequence of gene SdNMT

Take a 0.2mL PCR tube and add the following ingredients in sequence:

Table 3-PCR amplification system

The sequence of the upstream primer SdNMT-F of the gene SdNMT is shown in SEQ ID NO.2;

The sequence of the downstream primer SdNMT-R of gene SdNMT is shown in SEQ ID NO.3.

The PCR reaction procedure was 94℃ denaturation for 10 seconds, 60℃ annealing for 15 seconds, 72℃ extension for 90 seconds, and 35 cycles; 72℃ full extension for 10 minutes. After the PCR reaction was completed, 1.0% agarose gel electrophoresis was performed for detection, and the results are shown in Figure 1. The gel containing the target band size was recovered (according to the "Agarose Gel DNA Purification Kit" of Tiangen Company), connected to T-Vector pMD™19 (Takara) through KpnⅠ and BamHI, and transformed into Escherichia coli competent cells DH5α. On LB plate culture medium, inverted overnight culture at 37℃, single colonies were picked, and sequence determination was performed at Beijing Ruibo Xingke Biotechnology Co., Ltd. The sequence of the gene SdNMT was obtained by splicing and alignment, and its nucleotide sequence is shown in SEQ ID NO.1.

2.2.1 Sequence analysis of the SdNMT gene

The coding sequence of SdNMT is 1485 bp, encoding 494 amino acids, as shown in SEQ ID NO.4. The protein encoded by this gene has a dual methyltransferase domain, that is, both the N-terminus (34-176 aa) and the C-terminus (265-406 aa) have an S-adenosyl-L-methionine binding domain-dependent methyltransferase catalytic activity domain. The online tool http://web.expasy.org/protparam/ predicts that the molecular weight of this protein is 56.56 KDa, the isoelectric point is 5.52, and it contains a large amount of lysine (Lys), leucine (Leu), glutamic acid (Glu), glycine (Gly) and aspartic acid (Asp), which are 8.9%, 8.7%, 8.3%, 7.5% and 7.3% respectively. The average hydrophilicity coefficient (GRAVTY) is -0.416, which is a hydrophilic protein. The online tool NetPhos 3.1 (https://services.healthtech.dtu.dk/service.php?NetPhos-3.1) was used to analyze its phosphorylation sites. As shown in Figure 2, the N-terminal methyltransferase domain of the SdNMT gene has a total of 10 possible phosphorylation sites, including 7 serine phosphorylation sites, 1 threonine phosphorylation site, and 2 tyrosine phosphorylation sites. The C-terminal methyltransferase domain of the SdNMT gene has a total of 15 possible phosphorylation sites, including 5 serine phosphorylation sites, 6 threonine phosphorylation sites, and 4 tyrosine phosphorylation sites, as shown in Figure 3.

2.2.2 Solubility analysis of the protein encoded by the gene SdNMT

The CDS sequence (1485 bp) of the gene SdNMT was codon optimized and then synthesized into the prokaryotic expression vector pET-28a, and the restriction sites NdeI ( CATATG ) and XhoI ( CTCGAG ) were introduced. The expression vector map is shown in Figure 4. The restriction enzyme digestion and sequencing were used for verification, and the result of the restriction enzyme digestion verification is shown in Figure 5.

The amino acid sequence after codon optimization is shown in SEQ ID NO.5.

The verified recombinant plasmid pET28a-SdNMT was transformed into competent cells BL21 (DE3), and the plates were inverted and cultured overnight at 37°C. A single clone was selected in LB medium and cultured at 37°C until the bacterial OD600 was 0.6-0.8. IPTG was added to a final concentration of 0.5mM for induction. After culturing at 37°C for 4h, the cells were centrifuged and the samples were collected. The results of SDS-PAGE showed that the gene SdNMT could be expressed in prokaryotic cells, as shown in Figure 6.

IPTG was further added to the culture after the inoculated seed bacteria were cultured to a bacterial OD600 of 0.6-0.8 to a final concentration of 0.2mM and 1mM, respectively, and cultured at 37℃ and 15℃ 220 rpm for 4h and 16h, respectively, to induce the expression of the fusion protein, and 4 groups of induction optimization conditions (YH1, YH2, YH3, YH4) were set for protein induction expression. The bacterial liquid of each condition was centrifuged to collect the bacterial cells, broken (Tris-NaCl buffer, PH8.0), and the supernatant precipitate was sampled separately. The SDS-PAGE detection results are shown in Figure 7. Under the induction conditions of YH1, YH2, YH3 and YH4, the target protein was expressed in the precipitated bacterial cells and the supernatant, indicating that the target protein was soluble; the optimal induction condition for the expression of the target protein in the precipitated bacterial cells and the supernatant under the induction condition of YH3;

Induction optimization conditions 1 (YH1): 15°C, 0.2 mM IPTG, 16 h;

Induction optimization conditions 2 (YH2): 15°C, 1.0 mM IPTG, 16 h;

Induction optimization condition 3 (YH3): 37°C, 0.2 mM IPTG, 4 h;

Induction optimization condition 4 (YH4): 37°C, 1.0 mM IPTG, 4 h;

The optimal clone strain was expanded and cultured at 37°C for 1 L. When OD600 = 0.6-0.8, the bacteria were collected after induction at 15°C for 16 h. After resuspension, the cells were ultrasonically disrupted and centrifuged. The supernatant was purified by Ni column affinity chromatography. After SDS-PAGE detection, the target protein was detected in both the precipitate and the supernatant after disruption, and the protein content in the eluted sample was high, as shown in Figure 8.

The eluted sample was added to an ultrafiltration tube and concentrated by centrifugation to obtain a 56 kDa target protein, which was consistent with the sequence prediction result, as shown in FIG9 .

2.3 Plant overexpression vector construction

The construction process of the specific expression vector of the phosphoethanolamine methyltransferase gene SdNMT is shown in Figure 10. The gene SdNMT is constructed into the plant expression vector pCAMBIA2300 by Kpn Ⅰ and BamH Ⅰ enzymes. All restriction endonucleases are purchased from Takara Company and operated according to the instructions. The plasmid of the positive clone identified by enzyme digestion, sequencing and other methods is introduced into Escherichia coli DH5α and stored for future use.

2.4 Preparation of transgenic plants

The constructed plant overexpression vector plasmid was introduced into Agrobacterium strain GV3101 by electroporation.

The Suaeda solani phosphoethanolamine methyltransferase gene SdNMT was integrated into the genome of the processing tomato Yaxin-5 (purchased from Xinjiang Huiyuan Company) using the Agrobacterium tumefaciens-mediated method. The culture medium for the genetic transformation of processing tomatoes mediated by Agrobacterium is shown in Table 4. Table 4 - Culture medium for genetic transformation of processing tomatoes mediated by Agrobacterium

MS: Murashige&Skoog purchased from

Ti: Timentia, Timentin

IAA: Indoleacetic acid

6-BA: 6-Benzylaminopurine

Kan: Kanamycin

The expression vector is used as a receptor through Agrobacterium-mediated processing tomato hypocotyls to integrate the target gene into processing tomato sub-heart 87-5.

The transgenic plants obtained in the plant culture room were routinely managed in the experimental field. After maturity, the T1 generation seeds were harvested and a salt tolerance comparison experiment was carried out in the plant culture room.

Experimental Example 1: Identification of transgenic positive plants and detection of target gene expression

Transgenic processing tomato seeds were sown in seedling trays, with one seed per hole. When the seedlings grew to 4 true leaves, the leaf genomic DNA was extracted for PCR detection to screen positive plants. The total RNA of the leaves of positive transgenic processing tomato plants was extracted using the method provided by the high-efficiency plant genomic DNA extraction kit (DP350) of Tiangen Biotechnology Company, and cDNA was synthesized using the reverse transcription kit (PrimeScriptTM RT reagent kit with gDNA Eraser (RR047A)) of Takara Company. The tomato Actin gene was used as an internal reference to detect the expression of the gene SdNMT in transgenic plants, and each sample was amplified three times. The reaction system was 20 μL: containing 10 μL of SYBR Green Master Mix, 0.4 μL of upstream primers and downstream primers, and 2 μL of cDNA. The PCR reaction procedure was as follows: the first step was pre-denaturation at 95℃ for 30 s; the second step was denaturation at 95℃ for 5 s, annealing at 60℃ for 34 s, and 40 cycles; finally, the melting curve was detected, and each sample was repeated three times. Data processing and analysis were performed using excel software. The results are shown in Figure 11. The target gene SdNMT was significantly expressed in all 12 strains. Among them, the expression levels of the gene SdNMT in strains 10 and 21 were significantly higher than those in other strains. They were named OE1 and OE2 for salt tolerance research.

The sequence of the upstream primer SdNMTQ-F for gene SdNMT QPCR is shown in SEQ ID NO.6;

The sequence of the gene SdNMT QPCR downstream primer SdNMTQ-R is shown in SEQ ID NO.7;

The sequence of the upstream primer ActinQ-F for gene Actin QPCR is shown in SEQ ID NO.8;

The sequence of the downstream primer ActinQ-R of gene Actin QPCR is shown in SEQ ID NO.9.

Test Example 2: Salt stress treatment of transgenic processing tomatoes

Transgenic processed tomato seeds were sown in seedling trays, with one seed per hole. When the seedlings grew to 4 true leaves, genomic DNA was extracted for PCR detection to screen positive plants. Positive plants were transplanted into nutrient pots (12×10×8 cm), with one plant per pot. When the transgenic tomato plants grew to 6 to 8 true leaves, 150 mL of 350 mmol/L NaCl solution was irrigated per pot. The second irrigation was carried out on the 7th day. Similarly, 150 mL of 350 mmol/L NaCl solution was irrigated per pot, and the control plants were irrigated with the same volume of tap water. Each treatment was repeated 3 times. The phenotypic changes of the plants were observed, and physiological and biochemical indicators were measured.

The physiological and biochemical indicators were measured as follows:

(1) Determination of choline and betaine contents in transgenic plants after salt stress treatment

The choline content in the leaves of transgenic processing tomato plants was determined according to the enzymatic colorimetric method in GB 5413.20-2022. Take 2g (accurate to 0.001g) of fresh leaves in a 100mL conical flask, add 30mL of 1mol/L hydrochloric acid solution for acid hydrolysis; then place in a 70℃ water bath for hydrolysis for 3h (shake every 30 min), and cool to room temperature. Adjust the pH to 3.5-4.0 with sodium hydroxide solution (500 g/L), transfer to a 50mL volumetric flask, and dilute to the mark with water; filter the hydrolyzate with filter paper, and collect the filtrate for testing. The preparation of the standard curve and the determination of the choline content in the sample were carried out according to the standard instructions. The results of calculating the choline content according to the standard curve are shown in Figure 12. After salt treatment, the choline content in the transgenic processing tomato plants was significantly higher than that in the wild-type processing tomatoes.

The betaine content in transgenic plants was measured using the betaine content assay kit (TCJ-1-G) of Suzhou Keming Biotechnology Co., Ltd. After drying, the fresh leaves were sieved through a 40-mesh sieve, 0.02 g of sample was taken, 0.8 mL of water was added, and the mixture was extracted at 60°C for 30 min with continuous shaking. Then 200 μL of the extract was added, mixed and centrifuged at 10000 g, 25°C for 10 min, and the supernatant was taken for testing. The assay solution was prepared according to the instructions, and the absorbance at 525 nm was measured using an ELISA reader Infinite 200 Pro M Nano (TECAN). The choline content was calculated according to the standard curve. The results are shown in Figure 13. After salt treatment, the choline content in transgenic processing tomato plants was significantly higher than that in wild-type processing tomatoes.

(2) Determination of antioxidant enzyme activity in transgenic plants after salt stress treatment

The superoxide dismutase (SOD) kit (A001-3) of Nanjing Jiancheng Company was used to determine the SOD activity in the leaves of transgenic plants. Plant tissues were taken, and the surface dirt was washed with distilled water, and then dried with absorbent paper. 0.1g was cut into pieces, and an appropriate amount of liquid nitrogen was added. It was quickly ground into powder, and 0.9ml volume of phosphate buffer (phosphate buffer: 0.1mol/L pH7-7.4) was added. The supernatant, i.e., 10% homogenate supernatant, was obtained by vortex mixing for 3 minutes, and the absorbance at 450nm was determined according to the instructions of the kit. The data were processed and analyzed using Excel. The results are shown in Figure 14. Under normal conditions, there was no significant difference between the SOD activity of transgenic plants and the wild type; after salt stress treatment, the SOD activity of transgenic plants increased significantly.

The POD activity in the leaves of transgenic plants was determined using the Nanjing Jiancheng Peroxidase (POD) Kit (A084-1-1). Plant tissues were taken, and the surface dirt was washed with distilled water, and then dried with absorbent paper. 0.1 g of the tissue was cut into pieces, and an appropriate amount of liquid nitrogen was added. The tissue was quickly ground into powder, and 0.9 ml of phosphate buffer (phosphate buffer: 0.1 mol/L pH7-7.4) was added. The tissue was vortexed for 3 minutes, and the supernatant was taken at 3500 rpm or above, and the supernatant was centrifuged for 10 minutes to obtain the 10% homogenate supernatant. The absorbance at 420 nm was determined according to the instructions of the kit, and the data were processed and analyzed using Excel. The results are shown in Figure 15. Under normal conditions, there was no significant difference between the POD activity of transgenic plants and the wild type; after salt stress treatment, the POD activity of transgenic plants increased significantly.

The CAT activity in the leaves of transgenic plants was determined using the catalase kit (A007-1-1) of Nanjing Jiancheng Company. Plant tissues were taken, and the surface dirt was washed with distilled water, and then dried with absorbent paper. 0.1g was cut into pieces, and an appropriate amount of liquid nitrogen was added. It was quickly ground into powder, and 0.9ml volume of phosphate buffer (phosphate buffer: 0.1mol/L pH7-7.4) was added. The supernatant, i.e., 10% homogenate supernatant, was obtained by vortex mixing for 3 minutes, and the absorbance at 405nm was determined according to the instructions of the kit. The data were processed and analyzed using Excel, and the results are shown in Figure 16. Under normal conditions, there was no significant difference between the CAT activity of transgenic plants and the wild type; after salt stress treatment, the CAT activity of transgenic plants increased significantly.

(3) Phenotypic changes of transgenic plants after salt stress treatment

The phenotype of transgenic processing tomato plants treated with salt stress for 20 days is shown in Figure 17. Under normal growth conditions (control), the growth of transgenic processing tomato plants was significantly better than that of wild-type plants. Under 350 mmol/L NaCl treatment, the tips and edges of old leaves of wild-type plants first gradually lost green and turned yellow until they were scorched. As the treatment time increased, the new leaves gradually turned yellow and the plant grew slowly. However, the new leaves of the overexpression strain became darker in color, and some old leaves showed yellowing. The growth of transgenic processing tomato plants was better than that of wild-type plants.

(4) Biomass changes of transgenic plants after 20 days of salt stress treatment

The biomass of the aboveground and underground parts of the transgenic plants treated with salt for 20 days was measured. As shown in Figure 18, the aboveground fresh weight (Figure 18A), aboveground dry weight (Figure 18B), underground fresh weight (Figure 18C), and underground dry weight (Figure 18D) of the transgenic processing tomato plants were significantly higher than those of the wild-type plants.

Claims (8)

1. The phosphoethanolamine methyltransferase gene is characterized in that the nucleotide sequence of the phosphoethanolamine methyltransferase gene is shown as SEQ ID NO. 1.

2. A plant over-expression vector comprising the phosphoethanolamine methyltransferase gene of claim 1.

3. The plant over-expression vector according to claim 2, characterized in that the empty vector of the plant over-expression vector is pCAMBI2300T.

4. A method of constructing a plant overexpression vector according to claim 2 or 3, characterized in that the construction method is as follows:

The phosphoethanolamine methyltransferase gene was constructed on the plant expression vector pCAMBIA2300 by KpnI and BamHI enzymes.

5. Use of the transferase gene according to claim 1 for salt tolerance in tomato.

6. Use of a transferase gene according to claim 1 for the creation of salt tolerant tomatoes.

7. Use of a plant over-expression vector according to claim 2 or 3 in tomato salt tolerance.

8. Use of a plant over-expression vector according to claim 2 or 3 for the creation of salt tolerant tomatoes.