Cloning of tomato MYB transcription factor gene SlMYB94 and application of tomato MYB transcription factor gene SlMYB94 in salt stress resistance

Abstract

The invention discloses clone of a tomato MYB transcription factor gene SlMYB94, a nucleotide sequence of the gene SlMYB94 is shown as SEQ ID No.1, a coded amino acid sequence is shown as SEQ ID No.2, and application of the gene SlMYB94 in salt stress resistance, so that a salt-tolerant transgenic arabidopsis seed material is obtained; through cloning of the tomato MYB transcription factor gene SlMYB94 which is subjected to salt stress induced expression, the salt stress resistance of transgenic arabidopsis thaliana is enhanced in the application of the overexpressed SlMYB94 gene in salt stress resistance, practical significance is achieved for cultivation of a new salt-tolerant tomato variety, a molecular basis is provided for cultivation of the new salt-tolerant tomato variety, and meanwhile, the salt stress resistance of the transgenic arabidopsis thaliana is improved. And gene resources are provided for salt-tolerant molecular breeding and variety improvement of other crops.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to the cloning of a tomato MYB transcription factor gene SlMYB94 and its application in salt stress tolerance. Background Art

[0002] Salt stress is the second largest environmental stress faced by plants worldwide after drought stress, and has a serious impact on agricultural production. Currently, soil salinization occurs mainly in arid, semi-arid and coastal areas worldwide, and due to improper irrigation and environmental deterioration, soil salinization is showing an increasing trend year by year. Therefore, it is of great significance to cultivate new salt-tolerant varieties, improve the utilization rate of saline-alkali land, and thus improve the quality and yield of crops.

[0003] Salt stress seriously affects plant growth and development, which is mainly manifested in the inhibition of plant growth, short plant morphology, and dark green leaf color. Under salt stress, plants will undergo a series of physiological and biochemical changes: changes in cell membrane permeability, membrane lipid peroxidation, reduced activity of membrane-bound enzymes, and metabolic disorders; photosynthesis is inhibited, photosynthetic efficiency decreases, chlorophyll content is significantly reduced, and respiration is enhanced, ultimately leading to a decrease in crop yield.

[0004] Genetic analysis of plant salt tolerance has been reported. It is generally believed that plant salt tolerance is a quantitative trait, genetically controlled by a single or multiple genes. Therefore, cloning salt-tolerant genes and studying their mechanisms of action are very necessary for breeding new salt-tolerant varieties.

[0005] The MYB transcription factor family is one of the largest transcription factor families in plants and is widely involved in plant growth and development, secondary metabolism, and response to adverse stress. In recent years, studies have found that multiple MYB transcription factors play an important role in salt stress tolerance. For example, the SlMYB14 gene has been shown to play an important role in salt tolerance and drought resistance in tomatoes. However, there has been no detailed report on the function of SlMYB94 in salt stress tolerance. Therefore, the present invention proposes a cloning of a tomato MYB transcription factor gene SlMYB94 and its application in salt stress tolerance to solve the problems existing in the prior art. Summary of the Invention

[0006] In response to the above problems, the purpose of the present invention is to propose a cloning method for a tomato MYB transcription factor gene SlMYB94 and its application in salt stress tolerance. The cloning method of the tomato MYB transcription factor gene SlMYB94 realizes the cloning of the tomato MYB transcription factor gene SlMYB94 induced by salt stress. At the same time, overexpression of the SlMYB94 gene enhances the salt stress resistance of transgenic Arabidopsis in the application of salt stress tolerance, which has practical significance for breeding new salt-tolerant tomato varieties and provides a molecular basis for breeding new salt-tolerant tomato varieties.

[0007] To achieve the purpose of the present invention, the present invention is implemented through the following technical solution: a tomato MYB transcription factor gene SlMYB94, the nucleotide sequence of the gene SlMYB94 is shown in SEQ ID No.1, and the encoded amino acid sequence is shown in SEQ ID No.2.

[0008] A recombinant vector comprising the gene S1MYB94.

[0009] When the SlMYB94 gene is constructed into a plant expression vector, an overexpression promoter CaMV 35S is added before its start codon. To facilitate the subsequent screening of transgenic plants, a hygromycin or kanamycin resistance gene or a selective marker GUS gene or luciferase gene is added to the recombinant vector.

[0010] A recombinant bacterium comprising the gene S1MYB94 is Escherichia coli or Agrobacterium.

[0011] A method for cloning a tomato MYB transcription factor gene S1MYB94 comprises the following steps:

[0012] Step 1: Extract total RNA from tomato leaves using an RNA extraction kit;

[0013] Step 2: Use a reverse transcription kit to reverse transcribe and synthesize cDNA using the extracted total RNA;

[0014] Step 3: Using cDNA as a template, specific primers were designed according to the SlMYB94 gene sequence and PCR amplification was performed;

[0015] Step 4: Use a DNA gel recovery kit to recover the PCR product and sequence it.

[0016] A further improvement is that the specific primers in step 3 are SlMYB94-F and SlMYB94-R, and their sequences are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively.

[0017] The further improvement is that the PCR amplification reaction system in the step three is 1.0 μL of cDNA template, 1.0 μL of 10 μM SlMYB94-F primer, 1.0 μL of 10 μM SlMYB94-R primer, 4.0 μL of 2.5 mM dNTP, 5.0 μL of 10×PCR buffer, 0.5 μL of Taq enzyme and 7.5 μL of ddH2O3, and the amplification reaction conditions are first pre-denaturation at 94°C for 5 min, then denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s for a total of 35 cycles, and finally post-extension at 72°C for 10 min, and the amplified product is stored at 4°C.

[0018] The invention relates to an application of a tomato MYB transcription factor gene SlMYB94 in salt stress tolerance, screening Arabidopsis plants overexpressing SlMYB94, and obtaining salt-tolerant transgenic Arabidopsis seed materials.

[0019] The beneficial effects of the present invention are as follows: by cloning the tomato MYB transcription factor gene SlMYB94, which is induced to express under salt stress, the overexpression of the SlMYB94 gene in the application of salt stress resistance enhances the salt stress resistance of transgenic Arabidopsis, which has practical significance for breeding new salt-tolerant tomato varieties, provides a molecular basis for breeding new salt-tolerant tomato varieties, and also provides genetic resources for salt-tolerant molecular breeding and variety improvement of other crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the electrophoresis diagram of the cloning of the tomato gene S1MYB94 in Example 1 of the present invention.

[0021] Figure 2 This is a PCR detection diagram of Escherichia coli monocloned by the pCAMBIA1302-SlMYB94 recombinant vector in Example 2 of the present invention.

[0022] Figure 3 This is a diagram showing the enzyme digestion identification of the pCAMBIA1302-SlMYB94 recombinant vector in Example 3 of the present invention.

[0023] Figure 4 This is a PCR and qPCR identification diagram of Arabidopsis plants overexpressing SlMYB94 in Example 4 of the present invention.

[0024] Figure 5 This is a diagram showing the germination of different strains in Example 5 of the present invention under treatment with different concentrations of NaCl.

[0025] Figure 6 This is a diagram showing the root length of different strains in Example 5 of the present invention under treatment with different concentrations of NaCl. DETAILED DESCRIPTION

[0026] In order to deepen the understanding of the present invention, the present invention will be further described in detail below with reference to the examples. The examples are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0027] Example 1

[0028] according to Figure 1 As shown, this example provides a cloning and enzyme digestion identification of a tomato MYB transcription factor gene SlMYB94.

[0029] 1. Extraction of Total RNA from Tomato Leaves

[0030] Before RNA extraction, soak plastic consumables such as centrifuge tubes and pipette tips in DEPC water for 24 hours, sterilize them at 121°C under high pressure for 20 minutes, and then dry them in a dedicated oven. Wrap the mortar and pestle in tinfoil and dry them at 180°C for 8 hours.

[0031] The specific steps are as follows:

[0032] Step 1: Add 1 mL of lysate to a 1.5 mL centrifuge tube;

[0033] Step 2: Grind the tomato leaves quickly and thoroughly in liquid nitrogen, weigh 0.1g and transfer to the centrifuge tube in step 1, and immediately shake to mix.

[0034] Step 3: Place the sample mixed in step 2 at room temperature for 5 minutes;

[0035] Step 4: Centrifuge the sample from step 3 at 4°C, 12,000 rpm for 5 min, and transfer the supernatant to a new 1.5 mL centrifuge tube.

[0036] Step 5: Add 200 μL of chloroform to the centrifuge tube prepared in step 4, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes;

[0037] Step 6: Centrifuge at 4°C, 12,000 rpm for 10 min. The sample will separate into three layers from bottom to top: a yellow organic phase, an intermediate layer, and a colorless aqueous phase. Transfer the aqueous phase to a new 1.5 mL centrifuge tube.

[0038] Step 7: Add 0.5 times the volume of the aqueous phase of anhydrous ethanol to the centrifuge tube in step 6, pipette to mix, transfer to the CP3 adsorption column, and centrifuge at 4°C, 12,000 rpm for 1 min.

[0039] Step 8: Add 500 μL of protein-removing solution to the CP3 adsorption column, centrifuge at 4°C, 12,000 rpm for 1 min, and discard the waste liquid.

[0040] Step 9: Add 600 μL of rinse solution to the CP3 adsorption column, let it stand at room temperature for 2 minutes, centrifuge at 4°C, 12,000 rpm for 1 minute, discard the waste liquid, and repeat this step once;

[0041] Step 10: Place the CP3 adsorption column into the collection tube and centrifuge at 4°C, 12,000 rpm for 2 minutes.

[0042] Step 11. Place the CP3 adsorption column in a new 1.5 mL centrifuge tube, open the lid and let it stand for 5 minutes, add 30 μL RNase-Free ddH2O, let it stand at room temperature for 2 minutes, and centrifuge at 4°C, 12,000 rpm for 2 minutes; repeat this step once.

[0043] 2. Reverse transcription to synthesize cDNA

[0044] According to the instructions of the Takara reverse transcription kit, the specific steps are as follows:

[0045] The first step was to take 0.2 μL of RNA sample and measure its concentration and quality on a NANODROP 2000 (Thermo Scientific) nucleic acid protein detector to ensure the availability of the sample.

[0046] Step 2: Thaw the reagents and total RNA samples on ice and microcentrifuge before use.

[0047] Step 3: Remove genomic DNA and prepare the reaction solution according to Table 1:

[0048] Table 1 Composition of genomic DNA removal reaction solution

[0049]

[0050] Step 4: Incubate the above mixture at 42°C for 2 minutes, then place on ice for later use;

[0051] Step 5: Reverse transcription reaction. Prepare the reaction solution according to Table 2 below:

[0052] Table 2 Reverse transcription reaction solution composition

[0053]

[0054] The above mixture was first incubated at 37°C for 15 minutes, and then incubated at 85°C for 5 minutes; the cDNA synthesized by reverse transcription was stored at 4°C.

[0055] 3. Cloning of the SlMYB94 gene

[0056] Specific primers SlMYB94-F and SlMYB94-R with Spe I and Pml I restriction sites were designed, and their sequences are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively.

[0057] The PCR reaction system is as shown in Table 3:

[0058] Table 3 PCR reaction system

[0059]

[0060] The PCR reaction program is shown in Table 4 below:

[0061] Table 4 PCR reaction schedule

[0062]

[0063] IV. SlMYB94 gene gel excision and recovery

[0064] First, the PCR reaction product obtained in step 3 was subjected to agarose (1%) gel electrophoresis, and then recovered according to the DNA gel recovery kit of Tiangen Company. The specific steps are as follows:

[0065] Step 1: Cut the target band from the gel in a UV-cutting box, place it in a 1.5 mL centrifuge tube, and add 3 times the volume of PN solution (for example, if the gel mass is 100 mg, add 300 μL PN);

[0066] Step 2: Place the gel in a 60°C water bath until it is completely dissolved. At the same time, add 500 μL of equilibration solution BL to the CP3 adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

[0067] Step 3: Allow the dissolved gel solution to cool to room temperature, then absorb it all into the CP3 adsorption column and let it stand at room temperature for 2 minutes.

[0068] Step 4: Centrifuge at 25°C, 12,000 rpm for 1 min and discard the waste liquid;

[0069] Step 5: Pipette 600 μL of rinse solution PW into the CP3 adsorption column, centrifuge at 25°C, 12,000 rpm for 1 min, discard the waste liquid, and repeat this step once;

[0070] Step 6: Place the CP3 adsorption column into the collection tube and centrifuge at 25°C and 12,000 rpm for 2 minutes.

[0071] Step 7: Place the CP3 adsorption column in a new 1.5 mL centrifuge tube, open the lid and let it stand for 5 minutes, add 50 μL of 60°C preheated ddH2O, let it stand at room temperature for 2 minutes, and centrifuge at 25°C and 12,000 rpm for 2 minutes; store the recovered target gene at -20°C for later use.

[0072] The experimental results are as shown in the attached manual. Figure 1 As shown, agarose (1%) gel electrophoresis showed that the PCR amplification product was a fragment of about 879 bp.

[0073] Example 2

[0074] according to Figure 2 As shown, this example provides PCR detection of Escherichia coli monocloned by the pCAMBIA1302-S1MYB94 recombinant vector.

[0075] 1. Plasmid extraction of plant eukaryotic expression vector pCAMBIA1302

[0076] The specific steps are as follows:

[0077] Step 1: Add 500 μL of equilibrium solution BL to the CP3 adsorption column, centrifuge at 25°C, 12,000 rpm for 1 min, and discard the waste liquid;

[0078] Step 2: Add 1.5 mL of overnight cultured E. coli containing the pCAMBIA1302 plasmid into a 1.5 mL centrifuge tube, centrifuge at 25°C, 12,000 rpm for 1 min, and remove the supernatant.

[0079] Step 3: Add 250 μL of solution P1 to the centrifuge tube in step 2, and use a pipette to suspend and mix the bacterial pellet.

[0080] Step 4: Add 250 μL of solution P2 to the centrifuge tube in step 3, close the tube cap, and gently invert it upside down 6-8 times;

[0081] Step 5. Add 350 μL of solution P3 to the centrifuge tube prepared in step 4, close the tube cap, and quickly invert the tube up and down 6-8 times to mix thoroughly. At this point, a white flocculent precipitate will appear. Centrifuge at 25°C, 12,000 rpm for 10 min.

[0082] Step 6: Transfer the supernatant to a CP3 column and centrifuge at 25°C, 12,000 rpm for 1 min.

[0083] Step 7: Add 500 μL of deproteinized solution PD to the CP3 column and centrifuge at 25°C and 12,000 rpm for 1 min.

[0084] Step 8. Add 600 μL of rinse solution PW to the CP3 column and centrifuge at 25°C and 12,000 rpm for 1 min. Repeat this step once.

[0085] Step 9: Place the CP3 adsorption column back into the collection tube and centrifuge at 25°C, 12,000 rpm for 2 minutes.

[0086] Step 10: Place the CP3 adsorption column in a new centrifuge tube, open the lid, and place it at room temperature for 5 minutes;

[0087] Step 11. Add 50 μL of preheated ddH2O to the CP3 column, place at room temperature for 2 minutes, and centrifuge at 25°C and 12,000 rpm for 2 minutes; store the extracted plasmid at -20°C.

[0088] 2. Enzyme Digestion Reaction

[0089] The target gene and pCAMBIA1302 plasmid DNA recovered from gel excision were simultaneously digested with Spe I and Pml I. The enzyme digestion reaction system is shown in Table 5:

[0090] Table 5 Enzyme digestion reaction system

[0091]

[0092] The reaction mixture was placed in a 37°C water bath and reacted for 30 min.

[0093] 3. Recycling

[0094] The two enzyme digestion products were subjected to agarose gel electrophoresis and then cut and recovered to recover the target bands of SlMYB94 and pCAMBIA1302, respectively.

[0095] 4. Ligation Reaction

[0096] The SlMYB94 gene recovered from the third step was ligated with the pCAMBIA1302 vector. The ligation reaction system is shown in Table 6:

[0097] Table 6 Ligation reaction system

[0098]

[0099] The above mixture was placed in a constant temperature water bath at 16°C for 8 h.

[0100] 5. E. coli Transformation

[0101] Step 1: Thaw the competent E. coli DH5a stored at -80℃ in an ice water bath;

[0102] Step 2: aspirate the ligation product in step 4 into competent E. coli cells and place them in an ice-water bath for 20 minutes.

[0103] Step 3: Heat shock in a 42°C water bath for 90 seconds;

[0104] Step 4: After the heat shock, immediately transfer to an ice water bath for 3 minutes;

[0105] Step 5. On a clean bench, add 900 μL of LB liquid medium without antibiotics to the competent culture, close the tube cap, seal it with sealing film, and culture it at 37°C, 200 rpm, and shake for 1 hour.

[0106] Step 6: Centrifuge at 10,000 rpm for 1 minute to collect the cells, resuspend them in a small amount of supernatant, and evenly spread the cell suspension on LB solid medium containing kanamycin resistance. Incubate the culture in an inverted position at 37°C for 8 hours.

[0107] Step 7: After colonies grow, select monoclonal bacteria, identify positive strains through colony PCR, and sequence them.

[0108] 6. PCR identification of positive strains

[0109] Specific primers 35S and SlMYB94-R were used, and the sequences are shown as SEQ ID No. 5 and SEQ ID No. 4, respectively.

[0110] The PCR reaction system is as shown in Table 7:

[0111] Table 7 PCR reaction system

[0112]

[0113] The PCR reaction program is as follows:

[0114] Table 8 PCR reaction program

[0115]

[0116]

[0117] The experimental results are as shown in the attached manual. Figure 2 As shown, agarose (1%) gel electrophoresis showed that clones 1-9 had a fragment of approximately 982 bp, wherein the negative control using ddH2O as a template had no band.

[0118] Example 3

[0119] according to Figure 3 As shown, this example provides enzyme digestion identification of the pCAMBIA1302-SlMYB94 recombinant vector.

[0120] The positive strains carrying the recombinant plasmid that were correctly sequenced were shaken, and then the plasmids were extracted and identified by enzyme digestion with SpeI and PmlI. The reaction system is as shown in Table 9:

[0121] Table 9 Enzyme digestion identification reaction system

[0122]

[0123] The digested products were subjected to agarose gel electrophoresis to observe whether the recombinant plasmid could be fully digested and separated.

[0124] The experimental results are as shown in the attached manual. Figure 3 As shown, agarose (1%) gel electrophoresis showed that the recombinant plasmid was cut into two fragments by SpeI and PmlI enzymes, the upper fragment was the vector fragment, and the lower fragment was the target gene fragment.

[0125] Example 4

[0126] according to Figure 4 As shown, this example provides PCR and qPCR identification of Arabidopsis plants overexpressing SlMYB94.

[0127] 1. Agrobacterium Transformation

[0128] Step 1: Thaw the competent Agrobacterium Gv3101 stored at -80°C in an ice water bath;

[0129] Step 2: Add 10 μL of pCAMBIA1302-SlMYB94 recombinant plasmid to 100 μL of competent medium and mix gently by pipetting;

[0130] Step 3: Place the competent cells in an ice-water bath for 45 minutes and then immediately place them in liquid nitrogen for 1 minute.

[0131] Step 4: Take out from liquid nitrogen and immediately heat shock in a 37°C water bath for 3 minutes;

[0132] Step 5: Immediately place in ice water bath for 3 minutes;

[0133] Step 6: On a clean bench, add 900 μL of YEP liquid medium without antibiotics to the competent cells and culture on a shaker at 28°C for 3 h.

[0134] Step 7: Centrifuge at 10,000 rpm for 1 min to collect the cells, resuspend them in a small amount of supernatant, and evenly spread the culture medium on YEP solid medium containing kanamycin (50 μg / mL) and rifampicin (100 μg / mL) for resistance. Incubate the culture in an inverted position at 28°C for 2 days.

[0135] Step 8: After colonies grow, select monoclonal bacteria and screen out positive strains through colony PCR identification.

[0136] 2. Agrobacterium-mediated genetic transformation of Arabidopsis thaliana (flower infection method)

[0137] The first step is to plant wild-type Arabidopsis seeds in nutrient soil, wait for the main inflorescence to grow, cut off the top main inflorescence, promote the formation of side inflorescences, and wait until all inflorescences are fully open, ready for infection and transformation;

[0138] Step 2: The positive Agrobacterium strain obtained in the first step was inoculated into YEP liquid medium containing kanamycin (50 μg / mL), cultured at 28°C and 200 rpm for 8 h, and then used.

[0139] Step 3: Centrifuge at 4°C, 6000 rpm for 5 minutes to collect the cells and resuspend them in the prepared infection solution. The infection solution preparation system is as shown in Table 10:

[0140] Table 10 Infection solution configuration system

[0141]

[0142] Step 4: Immerse the Arabidopsis inflorescence in the infection solution and gently shake it for 20 seconds; then, place the seedlings in a dark place for 24 hours, and then continue to culture under normal conditions; after five days, repeat the above steps and infect again; harvest the seeds after one month.

[0143] 3. Screening of transgenic Arabidopsis seeds

[0144] Step 1: Weigh an appropriate amount (30 mg) of transgenic Arabidopsis seeds and place them in a 1.5 mL centrifuge tube;

[0145] Step 2: Add 1 mL of 75% anhydrous ethanol to the centrifuge tube, vigorously invert the centrifuge tube upside down, sterilize for 8 minutes, centrifuge at 12,000 rpm for 10 seconds, and discard the supernatant;

[0146] Step 3: Wash the seeds 4-5 times with sterilized water;

[0147] Step 4: Spread the seeds onto 1 / 2 MS solid medium (containing 30 μg / mL hygromycin), vernalize in a 4°C refrigerator for 2 days, and then culture in a light culture room (22°C, 16 h light / 8 h dark) for 10 days.

[0148] Step 5: Transplant the resistant seedlings into pots to continue growing. After about 2 months, collect the T2 generation seeds produced on the T1 plants.

[0149] 4. Extraction of transgenic Arabidopsis genomic DNA

[0150] The T2 generation seeds collected from the third batch were planted on 1 / 2MS solid culture medium, vernalized in a 4°C refrigerator for 2 days, and then transplanted into pots for further growth. When the plants grew six true leaves, the leaf genomic DNA was extracted.

[0151] Step 1: Preheat the CTAB extraction solution in a 65°C water bath.

[0152] Step 2: Cut an appropriate amount of leaves into a 1.5 mL centrifuge tube, add 300 μL of preheated CTAB extract to the centrifuge tube, and grind quickly with a grinding rod until the leaves are fully ground;

[0153] Step 3: Place in a 65°C water bath for 30 minutes, gently turning it over 2-3 times during this time.

[0154] Step 4: Cool to room temperature, add 200 μL of chloroform / isoamyl alcohol (24:1), mix gently, and centrifuge at 25°C, 12,000 rpm for 10 min;

[0155] Step 5: Transfer the supernatant to a new centrifuge tube, add 300 μL of isopropanol, mix gently, and let it stand at room temperature for 3 minutes;

[0156] Step 6: Centrifuge at 25°C, 12,000 rpm for 10 min;

[0157] Step 7: Discard the supernatant and wash once with 200 μL 75% anhydrous ethanol;

[0158] Step 8: Centrifuge at 25°C, 12,000 rpm for 2 minutes and place at room temperature for 10-20 minutes.

[0159] Step 9. Add 20 μL ddH2O to dissolve genomic DNA.

[0160] 5. PCR Identification of Transgenic Arabidopsis

[0161] Specific primers 35S and SlMYB94-R were used, and the sequences are shown as SEQ ID No. 5 and SEQ ID No. 4, respectively.

[0162] The PCR reaction system is as shown in Table 11:

[0163] Table 11 PCR reaction system

[0164]

[0165]

[0166] The PCR reaction program is as shown in Table 12:

[0167] Table 12 PCR reaction schedule

[0168]

[0169] 6. qPCR Identification of Transgenic Arabidopsis

[0170] Specific primers SlMYB94-qF and SlMYB94-qR were used, and the sequences are shown in SEQ ID No. 6 and SEQ ID No. 7, respectively.

[0171] The PCR reaction system is as shown in Table 13:

[0172] Table 13 PCR reaction system

[0173]

[0174] The PCR reaction program is shown in Table 14 below:

[0175] Table 14 PCR reaction schedule

[0176]

[0177] Melting curve: temperature range 65℃-95℃, temperature gradient 0.5℃, holding time 5s.

[0178] The experimental results are as shown in the attached manual. Figure 4 As shown, agarose (1%) gel electrophoresis showed that a 982 bp fragment appeared in all transgenic lines 1-9, among which the WT line had no band ( Figure 4 qPCR results showed that the relative expression levels of the SlMYB94 gene in overexpression lines 1-9 were several thousand times higher, among which OE1 (7584.82 times), OE4 (4112.84 times) and OE5 (1339.42 times) were selected as the next research materials ( Figure 4 Middle B).

[0179] Example 5

[0180] according to Figure 5 and Figure 6 As shown, this example provides an analysis of salt tolerance of transgenic Arabidopsis thaliana overexpressing SlMYB94.

[0181] Homozygous T3 transgenic Arabidopsis seeds were selected for the experiment. Seeds of overexpressing Arabidopsis plants and wild-type Arabidopsis plants (WT, Columbia-0) were sown on 1 / 2 MS medium containing varying NaCl concentrations (0, 100, 125, and 150 mM). Germination was observed and the germination rate was calculated. To analyze root development after germination, seeds were first sown on 1 / 2 MS medium and vernalized in a 4°C refrigerator for 2 days. After 3 days of growth under normal light conditions, the seedlings were transplanted to 1 / 2 MS medium containing varying NaCl concentrations (0, 100, and 150 mM). Root growth was observed and root length was measured.

[0182] The results showed that: on 1 / 2MS medium containing 0mM NaCl, there was no significant difference in germination rate or root length between overexpression and WT plants. However, on 1 / 2MS medium containing different concentrations of NaCl, the germination rate and root length of overexpression Arabidopsis plants were better than those of WT plants ( Figure 5 A, B and Figure 6 Middle A). Germination rate statistics ( Figure 5 C, D), fresh weight ( Figure 6 B) and chlorophyll content ( Figure 6 The measurement results in D) support the above phenotypic results.

[0183] The specific application of the tomato MYB transcription factor gene SlMYB94 in salt stress tolerance is:

[0184] First, transgenic Arabidopsis thaliana overexpressing the SlMYB94 gene was obtained. Seeds of the overexpressing Arabidopsis thaliana plants and wild-type Arabidopsis thaliana plants (WT, Columbia-0) were planted on 1 / 2 MS medium containing different NaCl concentrations. The germination rate of the seeds was counted, and the root elongation was observed. After sowing, the seed germination rate was measured every day. To analyze the development of the root system after germination, the seeds were first planted on 1 / 2 MS medium and vernalized in a 4°C refrigerator for 2 days. Then, they were transferred to normal light conditions and grown for 3 days. Then, the seedlings were transferred to 1 / 2 MS medium with different NaCl concentrations, and the root elongation was measured. On 1 / 2 MS medium without NaCl, there was no significant difference in germination rate or root length between the overexpressing and WT plants. However, on 1 / 2 MS medium containing NaCl, the germination rate and root length of the overexpressing Arabidopsis plants were better than those of the WT plants, which fully demonstrates that overexpression of the SlMYB94 gene enhances the salt stress resistance of transgenic Arabidopsis thaliana.

[0185] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A tomato MYB transcription factor gene S1MYB94, characterized by: The nucleotide sequence of the gene SlMYB94 is shown in SEQ ID No. 1, and the encoded amino acid sequence is shown in SEQ ID No.

2.

2. A recombinant vector comprising the gene S1MYB94 according to claim 1.

3. A recombinant bacterium comprising the gene S1MYB94 according to claim 1.

4. The clone of a tomato MYB transcription factor gene S1MYB94 according to claim 1, characterized in that: The following steps are involved: Step 1: Extract total RNA from tomato leaves using an RNA extraction kit; Step 2: Use a reverse transcription kit to reverse transcribe and synthesize cDNA using the extracted total RNA; Step 3: Using cDNA as a template, specific primers were designed according to the SlMYB94 gene sequence and PCR amplification was performed; Step 4: Use a DNA gel recovery kit to recover the PCR product and sequence it.

5. The clone of the tomato MYB transcription factor gene S1MYB94 according to claim 4, characterized in that: The specific primers in step 3 are SlMYB94-F and SlMYB94-R, and their sequences are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively.

6. The clone of the tomato MYB transcription factor gene S1MYB94 according to claim 4, characterized in that: The PCR amplification reaction system in the step 3 is 1.0µL of cDNA template, 1.0µL of 10µM SlMYB94-F primer, 1.0µL of 10µM SlMYB94-R primer, 4.0µL of 2.5mM dNTP, 5.0µL of 10×PCR buffer, 0.5µL of Taq enzyme, and 37.5µL. The amplification reaction conditions are first pre-denaturation at 94°C for 5 minutes, then denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds for a total of 35 cycles, and finally post-extension at 72°C for 10 minutes. The amplified product is stored at 4°C.

7. Use of the tomato MYB transcription factor gene SlMYB94 in salt stress tolerance according to claim 1, screening Arabidopsis plants overexpressing SlMYB94 to obtain salt-tolerant transgenic Arabidopsis seed materials.

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