CN116463374A - Application of GhSTP18 Gene in Regulation of Plant Salt Tolerance - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of a GhSTP18 gene in regulation and control of salt tolerance of plants. The nucleotide sequence of the GhSTP18 gene is shown as SEQ ID NO.1, and the function of the GhSTP18 gene is deleted by using a biotechnology means, so that the salt tolerance of plants is improved. According to the invention, through the expression analysis and the function research of the GhSTP18 gene in cotton, the specific transport galactose is found, the expression of the GhSTP18 gene in cotton is inhibited, the sensitivity of the cotton to salt stress is reduced, the GhSTP18 is overexpressed in Arabidopsis thaliana, the sensitivity of plants to salt stress is increased, the gene is proved to negatively regulate the salt stress tolerance of the plants, and an effective way is provided for improving the salt stress tolerance of the plants.

Description

Application of GhSTP18 Gene in Regulation of Plant Salt Tolerance

technical field

The invention belongs to the field of biotechnology, and in particular relates to the application of GhSTP18 gene in regulating plant salt tolerance.

Background technique

Cotton is not only a major economic crop, but also an important raw material for the textile industry. Cotton cultivation is mainly distributed in the southern half of Asia, southern America, Latin America and Africa. Cotton production in southern Asia accounts for more than 50% of the world's total cotton production, including China, India, Pakistan, Central Asia, Transcaucasus and parts of West Asia. Xinjiang Province is the main cotton producing area in China, and its cotton fiber production is limited by external environments such as cold, drought, and saline-alkali soil. Sugars are important substrates for carbon and energy metabolism and play important roles in plant responses to abiotic stresses. Sugars are synthesized in the leaves and then transported by phloem sap to storage tissues. Sugar transport is mediated by sucrose transporters (SUTs), sugar eventual export transporters (SWEETs) and monosaccharide transporters (MSTs). MST plays an important role in the transmembrane transport of various monosaccharides, including seven subfamilies, STP, PLT, XTPH, INT, AZT, pGlcT and ERD. As a subfamily of the MST superfamily, monosaccharide transporters (STP) have been shown to have important effects on plant growth and development and biotic stress responses, but less research has been done on abiotic stress, especially salt stress.

In view of this, the present invention is proposed.

Contents of the invention

One of the objectives of the present invention is to provide the application of the GhSTP18 gene in regulating the salt tolerance of plants, and use biotechnology means to make the function of the GhSTP18 gene ineffective, thereby improving the salt tolerance of the plant; the nucleotide sequence of the GhSTP18 gene is shown in SEQ ID NO.1.

Further, by transferring the biological material targeted to suppress or knock out the GhSTP18 gene into the plant, the function of the GhSTP18 gene is lost, thereby improving the salt tolerance of the plant.

Furthermore, the biological material includes gene expression cassettes, expression vectors or host cells for realizing GhSTP18 gene mutation, gene knockout, gene interference or gene silencing.

Further, the nucleotide sequence of the open reading frame of the GhSTP18 gene is shown in SEQ ID NO.2.

Furthermore, the amino acid sequence of the protein encoded by the GhSTP18 gene is shown in SEQ ID NO.3.

Furthermore, the protein specifically transports galactose.

Further, the plants include cotton.

Further, the GhSTP18 gene is overexpressed by using biotechnology, thereby reducing the salt tolerance of the plant.

Furthermore, the GhSTP18 gene is overexpressed by transferring the GhSTP18 gene or the biological material containing the GhSTP18 gene into plants.

Furthermore, the plants include cotton and Arabidopsis.

The present invention has following beneficial effect:

The present invention confirms from the real-time fluorescence quantitative results that the expression of the GhSTP18 gene in cotton is obviously induced by salt stress. The GhSTP18 gene is located on the cell membrane, specifically transports galactose, inhibits the expression of the GhSTP18 gene in cotton, and enhances the salt tolerance of the plant; overexpressing the GhSTP18 gene in Arabidopsis increases the sensitivity of the plant to salt stress; Favorable genetic resources of cotton germplasm.

Description of drawings

In Figure 1, A is the expression level of GhSTP18 gene in different tissues or organs of cotton, B is the expression level of GhSTP18 under salt treatment, C is the expression level of GhSTP18 under drought treatment, D is the expression level of GhSTP18 under low temperature treatment, and E is the expression level of GhSTP18 under high temperature treatment.

Fig. 2 shows the results of subcellular localization of GhSTP18 in tobacco (Nicotiana benthamiana) leaves.

Figure 3 shows the results of the study on the monosaccharide transport properties of GhSTP18 using the hexose-deficient yeast mutant strain EBY.VW4000.

In Fig. 4, A is the phenotype of positive control (pTRV1:CLA1), blank control (pTRV1:00) and GhSTP18 silenced plants. B is the expression level of GhSTP18 gene in blank control and GhSTP18 silenced plants. C is the chlorophyll content of blank control and GhSTP18 silenced plants. D is the activity of catalase (CAT) in blank control and GhSTP18 silenced plants. E is the content of malondialdehyde (MDA) in blank control and GhSTP18 gene silenced plants.

In Fig. 5, A is the phenotype of wild-type Arabidopsis and overexpressed Arabidopsis without salt treatment after 15 days of normal growth; B is the phenotype of wild-type Arabidopsis and overexpressed Arabidopsis plants after being treated with 400mM NaCl solution for 15 days; C is the study of the expression level of GhSTP18 gene in wild-type Arabidopsis and overexpressed Arabidopsis plants by using semi-quantitative PCR technology.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

Example 1: Expression analysis of GhSTP18

1. Experimental materials and processing

The cotton material selected in this experiment is Upland Cotton TM-1, and the cotton plants used are planted in the greenhouse, the specific environment is 16 hours of light, 8 hours of darkness, the temperature is 25-28°C, and the relative humidity is 80%. The rest of the management measures are managed according to the normal laboratory.

Cotton plants at the stage of two leaves and one heart were treated with 37°C, 4°C, 400mM NaCl solution, and 20% polyethylene glycol (PEG) 600 to simulate high temperature, low temperature, high salt, drought, and environment respectively.

2. Experimental method

2.1 Acquisition of cDNA

Collect various parts of Upland cotton TM-1, including roots, stems, leaves, anthers, petals, and ovules or fibers at different stages, and collect young leaves at different stages such as 0h, 1h, 3h, 6h, 12h, and 24h under the treatment of 37°C, 4°C, 400mM NaCl solution, and 20% polyethylene glycol (PEG) 600.

The RNAprep Pure Plant Plus kit of Tiangen Biochemical Technology (Beijing) Co., Ltd. was used to extract the RNA of each tissue or organ of Upland cotton; the kit TransCript All-in-One First-Strand cDNA Synthesis SuperMix for qpcr (One-Step gDNA Removal) of Tiangen Biochemical Technology (Beijing) Co., Ltd. was used to reverse transcribe the obtained RNA to obtain the cDNA of Upland cotton TM-1.

The reaction system is shown in Table 1. The reaction conditions are mixing, incubating at 42°C for 15 minutes, and paying attention to adjusting the initial temperature to 60°C; heating at 85°C for 5 seconds; and forever at 8°C. After finishing, store in -20°C refrigerator.

Table 1 Reverse transcription reaction system

2.2 Real-time fluorescence quantitative PCR (qRT-PCR)

Using Histone 3 primers as internal reference, the upstream SEQ ID NO.4 is TCAAGACTGATTT GCGTTTCCA, and the downstream SEQ ID NO.5 is GCGCAAAGGTTGGTGTCTTC; each sample has 3 technical replicates.

Use the TransStart Top Green qPCR SuperMix kit to design primers for the GhSTP18 gene (the nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.3). The upstream primer SEQ ID NO.6 is GTTCGAGGTACCCCCAATGTCG, and the downstream primer SEQ ID NO.7 is TGGGATCCCCAAAGCCCCTAAT. qRT-PCR detects the GhSTP18 gene expression volume.

The qRT-PCR reaction system is shown in Table 2. After adding the samples, carefully cover the plate with a membrane, and be careful not to touch the membrane with your hands; after covering the membrane, perform centrifugation, put it in a fluorescence quantitation instrument (ABI7500) for reaction, and collect data.

Table 2 qRT-PCR reaction system

3. Analysis of results

According to the analysis of the obtained data, the results are shown in Figure 1. GhSTP18 is expressed in all tissues, and the expression level is higher in roots and petals. The expression of GhSTP18 gene shows a significant upward trend from 0h to 12h of salt treatment. Drought, high temperature and low temperature stress can induce GhSTP18 gene expression to varying degrees.

Example 2: Subcellular localization of the GhSTP18 gene

1. Experimental materials, reagents or carriers used

The material used in the experiment was Nicotiana benthamiana, which was planted in a greenhouse and managed normally.

Large intestine competence, yeast extract YEAST EXTRACT; tryptone, TRYPTONE; NaCl, 5*pClone007versatile simple vector Mix (T load), AMP antibiotics, 3G Taq Master Mix for PAGE (Red Dye) enzyme provided by Nanjing Novizan Biotechnology Co., Ltd., plasmid extraction kit EasyPure PlasmidMiniPrep Kit, endonuclease SpeI1, SaI1 And corresponding Buffer, GV3101 Agrobacterium, Super Promoter-GFP vector.

2. Experimental method

2.1 Acquisition of open reading frame sequence of GhSTP18

(1) Toyobo/Toyobo KOD-Plus-Neo high-fidelity enzyme is used. The template is the leaf cDNA of Upland Cotton TM-1 obtained above, and the upstream primer SEQ ID NO.8: TGCTTTCAGAGATT GTCCACCA; the downstream primer SEQ ID NO.9: ATTACGTGGTTGGCTTTTTGAT is used for cloning. The reaction system and conditions are shown in Table 3 and Table 4.

Table 3 Amplification reaction system

Table 4 Amplification reaction conditions

(2) Electrophoresis: use a large-pore plate to prepare the gel, mix with loading buffer before electrophoresis detection, and cut the gel to recover after the target band is found.

(3) Glue recovery steps:

Use the EasyPure Quick Gel Extraction Kit, the specific steps are as follows:

a. Cut out the target fragment band in the agarose gel, put it into a clean centrifuge tube, and weigh it.

b. Add GSB solution 3 times the weight and volume of the glue (100mg glue weight can be equal to 100μl), and put it in a water bath at 55°C for about 10 minutes to ensure that the glue block is completely melted, and pay attention to ensure that the glue is completely melted.

c. The gel solution to be melted will reach room temperature (at high temperature, the ability of the spin column to bind DNA is weak), add it to the spin column, let it stand for 1 min, centrifuge at 12000 rpm for 1 min, and discard the effluent.

d. Add 650 μl of WB solution, centrifuge at 12,000 rpm for 1 min, and discard the effluent.

e. Centrifuge at 12000 rpm for 1-2min to completely remove residual WB.

f. Put the spin column in a clean centrifuge tube, open the cover and let it stand for 1 min to evaporate the residual ethanol. Add 30 μl deionized water to the center of the column and let it stand at room temperature for 1 min.

Centrifuge at g.12000 rpm for 1 min to elute the DNA.

h. Detect the DNA concentration, mark it, and store it at -20°C, and finally obtain the open reading frame sequence SEQ ID NO.2 containing the GhSTP18 gene.

2.2 Ligation reaction

Pipette 1 μl of 5*pClone007 versatile simple vector Mix (T load) and 4 μl of the target gene DNA obtained from the above clone into a centrifuge tube, and gently pipette and mix evenly. Place it at room temperature (22-30°C) for 1-5 minutes (generally 5 minutes) to fully react.

2.3 Conversion reaction

(1) Prepare culture medium

LB liquid medium preparation method: make up a total of 400ml and add reagents: YEAST EXTRACT 2g; TRYPTONE 4g; NaCl 4g;

Preparation of LB solid medium: 6g of agar can be directly added to the original sample of the liquid medium, sterilized, cooled, and poured into the plate. If you want to add AMP resistance, kanamycin, rifampicin, after the above medium is sterilized, cool down, absorb 200μl AMP resistance, 400μl kanamycin solution or rifampicin solution, shake well, and then perform corresponding operations;

(2) Take 100 μl of the competent large intestine and melt it on ice, add 5 μl of the product after the above ligation reaction, blow gently to stir and mix, and let stand on ice for 5 minutes.

(3) Heat shock in a water bath at 42°C for 45 seconds, then quickly transfer to an ice bath, and let stand for 2 minutes.

(4) Add 500 μl of non-resistant LB medium solution to the centrifuge tube without resuscitation, shake up and down to mix well, draw 100 μl as needed and evenly spread it on the LB plate containing AMP antibiotics, and invert the incubator at 37°C overnight (12-24h).

Note: This is a rapid transformation step, the resistance marker gene carried by T is AMP, and AMP can be transformed quickly without recovery in the middle.

2.4 Picking a single colony for cloning

(1) Observing the single colony that grows and confirming that it can be picked.

(2) In the ultra-clean workbench, according to the ratio of 1ml LB solution: 1μl AMP, add the corresponding volume of AMP antibiotics to LB, and shake gently.

(3) Add about 300 μl of the above-mentioned LB solution to the centrifuge tube, then pick a single colony, and put it into the centrifuge tube together with the tip of the pipette.

(4) Shake the bacteria in a shaker at 37°C for more than 2 hours for detection.

(5) Detection system: 20 μl system, including 10 μl of 3G Taq Master Mix for PAGE (Red Dye), 0.8 μl of upstream primer (SEQ ID NO.10: GTAAAACGACGGCCAGT), 0.8 μl of downstream primer (SEQ ID NO.9), 1 μl of colonies, and 7.4 μl of sterile water;

The PCR reaction conditions were according to the instructions of the enzyme 3G Taq Master Mix for PAGE (Red Dye) used.

(6) After PCR detection, select the target bands with brighter bands to send samples for detection, and according to the results of sample sequencing feedback, select the ones with better quality for shaking bacteria.

(7) Bacteria shaking step: add 30ml of LB culture solution of 30μl AMP antibiotics, inhale all the above-mentioned selected bacteria solution from the centrifuge tube, shake the bacteria on a shaker at 37°C, and extract the plasmid of the target fragment from the bacteria after 6 hours.

2.5 Extraction of target fragment plasmid

Use the EasyPure Plasmid MiniPrep Kit, add RNase to RB before use; add different volumes of absolute ethanol to the WB solution according to the instructions. According to the volume of bacterial solution, add different reagents in the following steps according to Table 5 below.

Table 5 Reagents used for target fragment plasmid extraction

The specific operation is as follows:

(1) The bacterial solution was centrifuged at 12000 rpm for 1 min, and the supernatant was removed.

(2) According to the above table, add the above colorless solution RB (containing RNase A), oscillate to suspend the bacterial sediment, and no small bacterial clumps should be left.

(3) According to the above table, add the blue solution LB, gently turn up and down and mix 4 to 6 times, so that the cells are fully lysed to form a clear blue solution. The color changes from translucent to clear blue, which means complete lysis.

(4) According to the above table, add the yellow solution NB and mix gently for 5-6 times (the color changes from blue to yellow completely, indicating uniform mixing and complete neutralization), until a firm yellow agglutination is formed, and stand at room temperature for 2 minutes.

(5) Centrifuge at 12000 rpm for 5 minutes, carefully absorb the supernatant and add it to the spin column. Centrifuge at 12000 rpm for 1 min, and discard the effluent. If the volume of the supernatant is greater than 800 μl, it can be added to the spin column several times, centrifuged as above, and the effluent is discarded.

(6) Add 650 μl of solution WB, centrifuge at 12,000 rpm for 1 min, and discard the effluent.

(7) 1-2min at 12000 rpm to completely remove residual WB.

(8) Put the spin column in a clean centrifuge tube, add 30 μl of deionized water to the center of the column, and let stand at room temperature for 1 min. (Note that the deionized water is preheated at 60°C-70°C in advance, the effect is better.)

(9) Centrifuge at 10,000 rpm for 1 min to elute the DNA and store it at -20°C after concentration detection to prepare the DNA template for cloning the ligation carrier.

2.6 Design adapter primers and cloning fragments connected to the vector

(1) In order to connect the GFP vector, primers need to be designed according to the sequence of the GFP vector, and the DNA fragment of the target gene with the linker should be re-cloned. The restriction site of this test is Sa1I and SpeI. Based on this site, the primers are designed in the software SnapGene. The upstream primer SEQ ID NO.11 is GGGGCCCGGGGT CGACATGGCTGGTGGAGGGTTT, and the downstream primer SEQ ID NO.12 is TACCGGATC CACTAGTCGT GGTTGGCTTTTTGATATCCTTCT.

(2) Cloning system and reaction system are the same as the above steps.

(3) Electrophoresis, gel recovery, measure its concentration, follow the above steps, and put it in -20°C for use in connection with GFP.

2.6 Digestion and ligation reactions:

The enzyme digestion reaction was carried out according to the system shown in Table 6, and the reaction condition was 37° C. for 3 h. Electrophoresis of digested products, gel recovery, and detection of concentration, in preparation for ligation of cloned fragments. The connection system is shown in Table 7, and the connection reaction conditions are 37°C, 30min.

Table 6 enzyme digestion system

Table 7 connection system

2.7 Transformation reaction

(1) Take 100 μl of the competent large intestine and melt it on ice, add 10 μl of the product after the above ligation reaction, blow gently to stir and mix, and let stand on ice for 30 minutes.

(2) Heat shock in a water bath at 42°C for 45 seconds, then quickly transfer to an ice bath, and let stand for 2 minutes.

(3) Add 500 μl of non-resistant LB medium solution to the centrifuge tube, shake it up and down to mix well, and put it in a shaker at 37°C for 1-2 hours to recover.

(4) As needed, 100 μl was pipetted and spread evenly on the LB plate containing kanamycin, and the 37°C incubator was inverted overnight (12-24h).

2.8 Pick a single colony and detect:

(1) Observing the single colony that grows and confirming that it can be picked.

(2) In the ultra-clean workbench, according to the ratio of 1ml: 1μl, add the corresponding volume of kanabioxin to LB, and shake gently.

(3) Add about 300 μl of the above-mentioned LB solution to the centrifuge tube, then pick a single colony, and put it into the centrifuge tube together with the tip of the pipette.

(4) Shake the bacteria in a shaker at 37°C for more than 2 hours for detection.

(5) Detection, as described above.

(6) After PCR detection, select the brighter target bands to send samples for detection. According to the results of sample sequencing feedback, pick the ones with better quality for shaking, and save the corresponding positive plasmids.

(7) Bacterial shaking step: add 30 μl of LB culture solution of kanabioxin to 30 ml, suck all the above-mentioned selected bacterial solution from the centrifuge tube, and shake the bacteria on a shaker at 37°C.

(8) After 6 hours, take out the bacterial solution, pipette 500 μl of glycerol and 500 μl of the corresponding bacterial solution in a clean workbench, put them in a clean centrifuge tube, mix them upside down, and put them in -80°C to preserve the bacteria.

2.9 Detection of GV3101-transformed Agrobacterium

(1) Take the GV3101 Agrobacterium (100 μl size) stored at -80°C, put it at room temperature or in the palm of your hand for a while, and mix it with ice and water. Add 5 μl of the positive plasmid obtained in step 2.8 and add it to it. Gently tap the bottom of the tube to mix well. Place on ice for 5 minutes, liquid nitrogen for 5 minutes, 37°C water bath for 5 minutes, and ice bath for 5 minutes.

(2) Add 700 μl of LB liquid medium without antibiotics, shake and culture on a shaker at 28° C. for 2-3 hours.

(3) Pipette 50 μl for coating, and the plate is a double-antibody medium (RK medium) containing kanamycin and rifampicin antibiotics.

(4) Finally, place it upside down in an incubator at 28°C for 2-3 days.

(5) After a single colony grows, pick a single colony and place it in 300 μl RK liquid medium, shake the bacteria at 28° C. for 2-3 hours, and perform detection.

(6) After the electrophoresis test, select positive bacteria and carry out bacteria preservation, and the bacteria preservation steps are the same as above.

2.10 Buffer preparation

Before injecting cotton seedlings, a corresponding buffer solution needs to be prepared to activate the activity of the bacteria.

The buffer solution configuration steps are as follows: 500ml required volume

Add 1.066g MES to 50ml ultrapure water, dissolve, and adjust the pH to 5.6;

Add 1.0165g MgCI ₂ .6H ₂ O to 50ml ultrapure water and dissolve;

Mix the above solutions to a volume of 500ml

Add AS solution at a ratio of 1ml:1μl, for example, 500ml requires 500μl.

Note that the buffer prepared above should be placed in the dark.

2.11 Injection of seedlings:

Resuspend the shaken bacterial solution with the above buffer solution, and adjust the OD value to about 1.5. Pay special attention to the wavelength of 600nm when adjusting the OD value.

Inject the seedlings directly after 2 to 3 hours in the dark.

Tobacco is injected and placed in the dark for 12 hours after injection;

Tobacco is transiently expressed and needs to be observed under a confocal laser microscope within 48 hours after injection.

3. Result analysis:

As shown in Figure 2, the empty GFP is expressed on the nucleus and the cell membrane, while the GFP linked to the GhSTP18 gene is only expressed on the cell membrane, indicating that the GhSTP18 gene is localized on the cell membrane.

Example 3: Study on monosaccharide transport properties of GhSTP18 gene

1. Main reagents or materials

Hexose transporter full knockout strain (EBY.VW4000), maltose, glucose, fructose, mannose, galactose, xylose, yeast extract, peptone, agar powder, pDR196 vector, 5*pClone007versatile simple vectorMix (T load).

2. Experimental steps

2.1 Construction of expression vector

(1) Cloning fragment primer design with pDR196 vector adapter

Primers were designed according to the enzyme cleavage site of the carrier, the vector enzyme cleavage site was EcoRI, SalI, and the final designed linker primer was the upstream primer SEQ ID NO.13: CGGGCTGCAGGAATTCATGGC TGGTGGAGGGTTT; the downstream primer SEQ ID NO.14: CCCCCTCGAGGTCGACTACG TGGTTGGCTTTTTGATATCCTTCT;

(2) The steps of cloning, electrophoresis, gel recovery, enzyme digestion, ligation and transformation of the open reading frame sequence of GhSTP18 are the same as in Example 2.

2.2 Yeast complementation experiment

In this study, yeast genetic transformation was performed using Coolaber's Super Yeast Competent Preparation and Transformation Kit. Its operation is based on the LiAc/SS carrier DNA/PEG method. Use this kit to transform empty pDR196 and GhSTP18-PDR196 into EBY.VW4000. The specific conversion method is as follows:

(1) Preparation of culture medium

YPDA medium preparation 100mL ingredients: yeast extract 1g, peptone 2g, maltose 2g, adenine sulfate 0.003g; if solid medium is required, add 2g agar;

Note that maltose may react chemically with other ingredients at high temperature, so maltose needs to be sterilized on a single plant basis, that is, the best specific steps are: 1g Yeast Extract (yeast extract), 2g Peptone (peptone), 0.003 adenine sulfate in 90ml water, add 2g agar powder to make a plate, autoclave at 121 degrees for 15-20min; add 10ml 2g maltose solution after sterilization, and mix with the above.

(2) Preparation of Competent Cells: Super Yeast Competent Preparation and Transformation Kit (Plus) was used

a. Activated strains. Streak the strain of EBY.VW4000 stored at -80°C on the solid YPDA medium with maltose as the carbon source, and culture it at 30°C for 2-4 days;

b. Pick a single clone (colony), draw a short line of 3-5mm on the YPDA medium plate, and culture at 30°C for 2-4 days.

c. When the diameter of a single yeast colony grows to 2mm, inoculate the yeast cells into 3mL liquid YPDA medium and inoculate at 30°C overnight (cultivate at 200r/min for 1 day).

d. The next day, transfer to a Erlenmeyer flask containing 50ml liquid YPDA medium to continue culturing (200r/min). When the OD600 value reaches 0.4-0.5, centrifuge at 3000rpm for 5min and discard the supernatant. (Preserve the yeast liquid within 1 week at 4°C, use 3mL to inoculate 50mL YPDA liquid medium for overnight culture).

e. Use 10ml of Y1 solution (competent preparation solution) to resuspend the pellet and centrifuge at 3000rpm for 5min, discard the supernatant.

f. Add 1 mL of Y2 solution (competent cryopreservation solution) to resuspend, and aliquot 50 μl into 1.5 mL sterile cryopreservation tubes to obtain yeast competent cells, which can be directly used for transformation.

g. The prepared competent cells need to be frozen slowly, and then placed in a -80°C refrigerator for long-term storage. Put the competent cells into a programmed cooling box, or wrap them in a foam box with multiple layers of paper, and put them at -80°C overnight, then take out the competent cells and put them in a -80°C refrigerator, which can be stored for one year. Thaw at room temperature before use for transformation.

(3) Preparation of transformation master mix

The total volume of the premix is 360 μl: Y3 solution (competent transformation solution) 350 μl, plasmid 5-10 μl, if less than 360 μl, make up the volume with sterile water.

(4) Yeast plasmid transformation

a. Pipette 360 μl of the premix solution and add it to 50 μl of competent cells, and repeatedly pipette with a pipette tip to completely suspend the yeast cells at the bottom of the centrifuge tube in the premix solution.

b. Place in a 30°C water bath for heat shock for 60 minutes, and mix well every 10 minutes. For some strains, extending the incubation time can improve the transformation efficiency, but do not exceed 3 hours.

c. Centrifuge at 12000rpm for 15s, discard the supernatant.

d. Add 400 μl sterile water to resuspend the bacteria. After diluting 10 times, pipette 200 μl and spread it on URA-deficient SD medium, use maltose as carbon source, and culture at 30°C for 2-4 days;

e. Pick a single clone, and use the bacterial liquid PCR method to detect, the detection steps are as described above, and determine whether the target vector is transferred into yeast.

(5) Growth analysis of transgenic yeast

URA-deficient solid SD medium (with maltose as carbon source) used in the experiment;

URA-deficient solid SD medium (with maltose as carbon source) 1L preparation method: Weigh 8g of Ura MinusMedia, add 950mL of water for autoclaving; at the same time, add 20g of maltose to 50mL of water for autoclaving to obtain 40% sterile maltose; after sterilization, mix the above to obtain URA-deficient solid SD liquid medium with maltose as carbon source.

The transgenic yeasts GhSTP18-PDR196-EBY.VW4000 and pDR196-EBY.VW4000 were cultured on URA-deficient solid SD medium (with maltose as carbon source), and single clones were picked and cultured in URA-deficient liquid SD medium (with maltose as carbon source) for 2 days. Centrifuge, remove the supernatant, wash the precipitate 3 times with sterile water, and adjust the OD600 to 0.2 with sterile water. Then it was diluted serially to 1 times, 5 times, 25 times and 125 times respectively. Then the above bacterial liquids were respectively transferred to SD solid medium with different sugars (maltose, glucose, fructose, xylose, galactose, sorbitol, arabinose) as carbon sources (2%), cultured at 30°C for 2-3 days, and photographed and recorded.

3. Analysis of results

The results are shown in Figure 3. Both pDR196 and pDR196-GhSTP18 can grow on the medium with maltose as the carbon source, indicating the success of the experimental system. pDR196-GhSTP18 only grows on the medium containing galactose as the sole carbon source, indicating that GhSTP18 specifically transports galactose.

Example 4: Application research of GhSTP18 gene in regulating cotton tolerance to salt stress

1. Experimental steps

1.1 Synthesis of target sequence ligation vector

In order to further clarify the effect of GhSTP18 gene on drought resistance of cotton, this study silenced the GhSTP18 gene in cotton by virus-mediated gene silencing technology, and used the cotton variety Zhongmian Institute 24 to conduct VIGS experiments. A specific expression fragment of the selected GhSTP18 gene (sequence: SEQ ID NO.15) was inserted into the pTRV (pYL156) vector using double restriction sites EcoRI and BamHI, and sent to Beijing Qingke Biotechnology Co., Ltd. to construct the pTRV:GhSTP18 vector.

1.2 Transform LBA4404 Agrobacterium

After the synthesized gene plasmid and bacterial liquid are returned, the plasmid is directly transformed into Agrobacterium LBA4404, and the steps of transforming Agrobacterium are as follows:

(1) Centrifuge the dry gene powder synthesized by the company and add 40 μl sterile water, shake and mix well;

(2) Take the competent LBA4404 Agrobacterium stored at -80°C in the greenhouse or the palm of your hand for a while, and insert it into the ice when it is partially melted and in the state of ice-water mixture;

(3) Add 5 μl of plasmid DNA to the competent Agrobacterium, gently tap the bottom of the tube to mix evenly, place on ice for 5 minutes, liquid nitrogen for 5 minutes, 37°C water bath for 5 minutes, and ice bath for 5 minutes;

(4) Add 700 μl of L liquid medium without antibiotics, shake and culture at 28° C. for 2-3 hours;

(5) Draw 50 μ l for coating, and the plate is a double-antibody medium (ie, RK solid medium) containing kana and rifampicin;

(6) Place the plate upside down in a 28°C incubator for 2-3 days;

(7) After the long-lived colonies in the plate, select monoclonal colonies in 300 μl RK liquid medium, shake the bacteria at 28°C for about 3 hours, and perform detection;

(8) Detect, select positive bacterium and carry out bacterium preservation, or transfer to 30ml RK nutrient solution and carry out shaking bacterium to turbid yellow in order to prepare for injection cotton seedling plant;

1.3 Configuration of buffer

Before injecting cotton seedlings, a corresponding buffer solution needs to be prepared to activate the activity of the bacteria.

The buffer solution configuration steps are as follows: 500ml required volume

Add 1.066g MES to 50ml ultrapure water, dissolve, and adjust the pH to 5.6;

Add 1.0165g MgCI ₂ .6H20 to 50ml ultrapure water to dissolve;

Mix the above solutions to a volume of 500ml

Add AS solution, add according to the ratio of 1ml:2μl, for example, 500ml needs 1000μl.

(AS solution configuration: configure 1ml, need 19.62mg powder, dissolve with DMSO solution)

Note that the buffer prepared above should be placed in the dark.

1.4 Injection of seedlings

When shaking the bacteria, in addition to the bacteria solution containing the pTRV:GhSTP18 carrier, positive bacteria (PDS), negative bacteria (VA), and active bacteria VB are required.

Resuspend the shaken bacterial solution, add the above buffer solution to mix, and use a spectrophotometer to adjust the OD value to about 1.5 (use the buffer solution to zero before adjusting the OD value). Pay special attention to adjusting the OD value when the wavelength is 600nm; directly inject the seedlings after 2 to 3 hours in the dark.

When injecting, each bacterial solution needs to be added with an equal proportion of VB bacterial solution, and then injected after mixing; the seedlings should be plants whose cotyledons have just been flattened;

After the injection, the plants were treated in the dark for more than 12 hours, and then taken out for normal culture. Note that the temperature where the Agrobacterium is active should not be too high.

1.5 Detection of expression level

Detection of expression level: After the yellowing phenotype appeared in the plants injected with positive bacteria (PDS), take injection-negative bacteria (VA) and cotton tender leaves containing pTRV:GhSTP18 bacterial solution for RNA extraction and reverse transcription to obtain corresponding cDNA.

The steps of real-time fluorescent quantitative PCR (qRT-PCR) were as above.

1.6 Salt stress treatment and physiological index detection

After the albino phenotype appeared in the positive plants, a salt solution with a concentration of 400 mM was prepared, and the TRV: 00 plants and TRV: GhSTP18 were irrigated for 7 consecutive days.

For the detection of catalase (CAT) activity, the catalase (CAT) activity assay kit provided by Beijing Suo Lai Bao Technology Co., Ltd. was used; for the detection of malondialdehyde (MDA) content, the malondialdehyde (MDA) content detection kit provided by Beijing Suo Lai Bao Technology Co., Ltd. was used.

The leaves of the control plants and the plants at the same position as the plants subjected to salt stress were selected to measure the content of chlorophyll a and b, and the determination process was as follows.

(1) Measure 225mL of ethanol with a graduated cylinder, pour 225mL of acetone into a 500mL blue-necked bottle, and add sterile water to make up to 500mL to prepare the extract.

(2) Use an analytical balance to weigh 0.2 g of leaves and put them into a 5 mL centrifuge tube, and add 5 mL of the prepared extract. Extract at 4°C in the dark for 12 hours until the leaves are completely decolorized.

(3) Use the extract as a blank control to calibrate the spectrophotometer, then measure and record the absorbance at wavelengths of 645nm and 663nm.

(4) Calculate the values of chlorophyll a and b respectively according to the modified Arnon method, and calculate chlorophyll a/b.

Chlorophyll a=(12.72OD663-2.59OD645)×v/w×1,000

Chlorophyll b=(22.88OD645-4.67OD663)×v/w×1,000

Chlorophyll a/b = Chlorophyll a/Chlorophyll b

2. Result analysis

The results are shown in Figure 4. The positive control (pTRV1:CLA1) plants showed albino phenotype (Figure 4A), and the expression of GhSTP18 was significantly inhibited (Figure 4B). The leaves of blank control plants showed obvious yellowing, while the leaves of GhSTP18 gene silenced plants showed no obvious yellowing ( FIG. 4A ). The chlorophyll content of silenced plants is higher than that of blank control plants (Figure 4C); catalase is an effective scavenging substance for reactive oxygen species (ROS), which can prevent cell damage and is considered to be an important indicator of stress resistance. The catalase (CAT) activity of the silenced plants was significantly higher than that of the control plants (Figure 4D), and the content of malondialdehyde (MDA) in the silenced plants was significantly lower than that of the control plants (Figure 4E). Combining the above results, it can be found that the GhSTP18 gene plays a negative correlation role in the regulation of cotton salt stress tolerance.

Embodiment 5: the research of GhSTP18 gene in Arabidopsis

1. Experimental steps

1.1 Construction of overexpression vector

The vector uses Super Promoter-GFP vector, and the steps are as above.

1.2 Suspension preparation, OD value determination, infection

Suspension preparation with a volume of 100mL: 0.217g of 1/2MS, 5g of sucrose, dissolved in constant volume, adjusted the pH to 5.8, and then added 20μl of Silwet-L77.

Centrifuge the bacterial solution at 3500r/min for 10 minutes, discard the supernatant, add the suspension, shake to dissolve, adjust the OD value of 600nm with a spectrophotometer to 0.7-1.0, and directly immerse the Arabidopsis in the suspension for 60 seconds (before infecting, subtract the flowering stamens, leaving only the unflowered, dewy stamens.)

After infestation, place in the dark (protect from light) for 24 hours before removing.

1.3 Screening of overexpressed transgenic Arabidopsis

Collect the infected Arabidopsis seeds to obtain the T0 generation seeds, and pay attention to adding a desiccant to the collection tube.

After drying at room temperature for 7 to 15 days, vernalize at low temperature for 7 days (or directly sow on a medium containing corresponding resistance and then vernalize at low temperature).

Preliminary preparation: sterilization of filter paper, pipette tips, ultrapure water, etc., preparation of medium.

1/2MS medium preparation: 100ml: 1/2MS, 0.22g; Agar, 0.8g; sucrose, 3g; after preparation, adjust the pH value to 5.6 to 5.8 before sterilization. After sterilization, wait for the solution to cool down to room temperature, add 60 μl of hygromycin with a concentration of 50 mg/μl to the pre-sterilized ultra-clean workbench, shake well, and then pour the plate. Note that the plate can be slightly thicker.

Sterilization of Arabidopsis seeds:

(1) Vortex the seeds in 2-3% sodium hypochlorite for 5 minutes;

(2) Centrifuge, pour off the sodium hypochlorite, add 75% alcohol, and vortex for 2 minutes;

(3) Centrifuge, pour off 75% alcohol, add absolute ethanol, shake;

(4) Sprinkle the seeds on the filter paper together with absolute ethanol, and after the alcohol volatilizes, sprinkle the seeds on the filter paper directly into the culture medium.

Finally, the medium plate containing Arabidopsis seeds was placed at 4°C, vernalized at low temperature for 2 days, and then placed under normal conditions for growth.

After the plants grow out, the seedlings are transplanted and tested to determine whether they are overexpressed transgenic plants, and the detection steps are as described above.

1.4 Salt treatment

After the transgenic Arabidopsis plants overexpressing GhSTP18 grew for 15 days, they were irrigated with a salt solution of 400 mM concentration for 15 days, and the phenotypes were observed.

2. Result analysis

The results are shown in Figure 5. After 15 days of normal growth, it was found that there was no significant difference between the phenotypes of wild-type Arabidopsis and overexpressed Arabidopsis without salt treatment (Figure A); after 400 mM concentration treatment for 15 days, the growth of transgenic GhSTP18 overexpressed Arabidopsis plants was significantly inhibited; the expression of GhSTP18 gene in wild-type Arabidopsis and overexpressed Arabidopsis plants was analyzed by semi-quantitative PCR, and it was found that the expression of GhSTP18 was significantly expressed in overexpressed Arabidopsis plants Higher than the wild type; the above analysis shows that the overexpression of the GhSTP18 gene in Arabidopsis reduces its salt tolerance.

While preferred embodiments of the invention have been described, additional changes and modifications to these embodiments can be made by those skilled in the art once the basic inventive concept is appreciated. Therefore, it is intended that the appended claims be construed to cover the preferred embodiment as well as all changes and modifications which fall within the scope of the invention.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims (10)

1. The application of the GhSTP18 gene in regulating the salt tolerance of plants, characterized in that the function of the GhSTP18 gene is lost by means of biotechnology, thereby improving the salt tolerance of the plant; the nucleotide sequence of the GhSTP18 gene is shown in SEQ ID NO.1. 2. The application according to claim 1, characterized in that, by transferring the biological material targeted to inhibit or knock out the GhSTP18 gene into the plant, the function of the GhSTP18 gene is lost, thereby improving the salt tolerance of the plant. 3. The application according to claim 2, wherein the biological material comprises a gene expression cassette, an expression vector or a host cell for realizing GhSTP18 gene mutation, gene knockout, gene interference or gene silencing. 4. The application according to claim 1, characterized in that the nucleotide sequence of the open reading frame of the GhSTP18 gene is shown in SEQ ID NO.2. 5. The application according to claim 4, characterized in that the amino acid sequence of the protein encoded by the GhSTP18 gene is shown in SEQ ID NO.3. 6. The use according to claim 5, wherein the protein specifically transports galactose. 7. The use of claim 1, wherein the plant comprises cotton. 8. The application according to claim 1, characterized in that the GhSTP18 gene is overexpressed by using biotechnology, thereby reducing the salt tolerance of plants. 9. The application according to claim 8, wherein the GhSTP18 gene is overexpressed by transferring the GhSTP18 gene or the biological material containing the GhSTP18 gene into plants. 10. The application according to claim 9, wherein said plants comprise cotton and Arabidopsis.