CN107012147A - A kind of arid and/or high salt evoked promoter SlWRKY8P and its application from tomato - Google Patents

Abstract

The invention discloses a tomato-derived drought and/or high-salt inducible promoter S1WRKY8P and its application, belonging to the fields of biotechnology and plant genetic engineering. The nucleotide sequence of the promoter S1WRKY8P is (a) or (b) ) or (c); wherein (a) has the nucleotide sequence shown in SEQ ID NO:1; (b) has more than 75% identity with the nucleotide sequence shown in SEQ ID NO:1, and A DNA molecule with a promoter function; (c) is a DNA molecule that hybridizes to the nucleotide sequence described in (a) or (b) under high stringency conditions and has a promoter function. The SlWRKY8P of the present invention can be used as an element for constructing a plant expression vector, which is connected before the target gene, so that the expression of the target gene is induced by drought and/or high salt. Therefore, the promoter S1WRKY8P provided by the present invention is of great significance for improving the tolerance of transgenic plants to drought and/or high salt by promoting the high expression of the target gene under drought and/or high salt stress.

Description

A drought and/or high salt inducible promoter SlWRKY8P derived from tomato and its application

technical field

The present invention relates to the fields of biotechnology and plant genetic engineering, in particular to a tomato-derived drought and/or high-salt inducible promoter SlWRKY8P and applications thereof, which can drive target genes under drought and/or high-salt stress conditions expressed in plants.

Background technique

my country is one of the three major tomato growing regions in the world, and exports account for 30% of the world's trade volume, while my country's tomato planting areas are mainly distributed in Xinjiang, Inner Mongolia and Gansu (Ma Zhen, 2013). In recent years, due to the deteriorating ecological environment and frequent occurrence of extreme weather in such areas, such as adversity stress such as drought, high temperature, land salinization and desertification, the planting and production of tomatoes have been seriously affected, which in turn has caused major losses to tomato production (Li Jingfu , 2011). Therefore, cultivating and promoting stress-resistant varieties is an effective way to ensure stable and high yield of tomato. Using the stress-resistant genes of plants themselves, new stress-resistant varieties can be bred through conventional breeding and molecular marker-assisted breeding methods. However, the use of resistance genes between species has certain limitations. For example, the stress-resistant genes of rice cannot Applied in tomato. The emergence of transgenic engineering technology overcomes the above-mentioned limitations and opens up a new way for crop stress-resistant breeding.

Plant gene regulation is mainly carried out at the transcriptional level, which is coordinated by a variety of cis-acting elements and trans-acting factors. Plant gene promoters are important cis-acting elements. It is a DNA sequence located in the upstream region of the 5' end of the structural gene to regulate gene transcription. It can activate RNA polymerase to accurately combine with template DNA to ensure accurate and efficient transcription initiation. It is the center of transcription regulation. According to the gene Promoters can be divided into two categories: constitutive promoters and specific promoters. Constitutive promoters can be transcribed in all cells at any time; specific promoters can be divided into tissue-specific promoters and inducible promoters. Inducible promoters usually do not initiate transcription or have very low transcriptional activity, but in a certain Under the stimulation of some specific stress signals, the transcriptional activity can be significantly increased. In transgenic plants, persistent overexpression of transformed foreign genes by constitutive promoters can stunt plant growth and reduce yield (Kasuga et al, 1999; Karaba et al, 2007), because overexpression of foreign genes tends to Competes for energy needed by plants under normal growth conditions and hinders protein or RNA synthesis (Rai et al., 2009). Therefore, it is best to use stress-induced plant promoters to cultivate new varieties of stress-resistant crops, so that foreign genes can be expressed in large quantities only under stress.

At present, there are still few inducible promoters that can be applied to the research of tomato transgenic stress resistance. The discovery, cloning, and analysis of homeopathic elements of new related stress-inducible promoters are still the focus of future research. The successful application of promoters in transgenic plants to regulate the expression of stress-resistant genes is the research direction of plant stress-resistant genetic engineering. Current studies have shown that stress-related genes in the WRKY family often respond to a variety of stresses, mostly because the promoters of these genes contain a variety of stress-related cis-acting elements (Eulgem et al., 2007). Our research in tomato found that there are 81 SlWRKY genes in tomato, and through gene chip and real-time fluorescent quantitative PCR analysis, we identified 27 SlWRKY genes were affected by 6 different biotic stresses (pathogenic bacteria) and abiotic stresses respectively (drought, high salinity) induced expression (Huang et al., 2012). Therefore, through the study of the above stress-related WRKY family gene promoters, a variety of stress-related key DNA fragments and cis-acting elements can be obtained, which is of great significance for the construction of effective stress-inducible promoters.

Therefore, after long-term research, the present inventors have discovered a drought and/or high-salt-induced promoter SlWRKY8P derived from tomato, which has a good application prospect in plant genetic engineering.

references

1. Ma Zhen. Research on Xinjiang Tomato Industry Development. Jilin University, 2013

2. Li Jingfu. Tomato Breeding in China. China Agricultural Press, 2011

3. Kasuga M, Liu Q, Miura S, et al. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. [J]. Nature Biotechnology, 1999,17(3):287.

4. Karaba A, Dixit S, Greco R, et al. Improvement of water use efficiency in rice by expression of HARDY, an Arabidopsis drought and salt tolerance gene. [J]. Proceedings of the National Academy of Sciences of the United States of America, 2007 , 104(39):15270.

5. Rai M, He C, Wu R. Comparative functional analysis of three abiotic stress-inducible promoters in transgenic rice[J].Transgenic Research,2009,18(5):787-799.

6. Eulgem T, Somssich I E. Networks of WRKY transcription factors indefense signaling. Current Opinion in Plant Biology, 2007, 10(4): 366-371

7. Huang S, Gao Y, Liu J, et al. Genome-wide analysis of WRKYtranscription factors in Solanum lycopersicum. Molecular Genetics and Genomics, 2012, 287(6): 495-513

Contents of the invention

The object of the present invention is to provide a tomato drought and/or high-salt-induced promoter S1WRKY8P and its application. The SlWRKY8P of the present invention is of great significance to improving the tolerance of transgenic plants to drought and/or high salt by promoting the high expression of the target gene under drought and/or high salt stress.

In order to achieve the above object, the present invention adopts the following technical solutions:

A kind of inducible promoter S1WRKY8P, its nucleotide sequence comprises following (a) or (b) or (c) sequence:

(a) has the nucleotide sequence shown in SEQ ID NO: 1; or

(b) A DNA molecule having more than 75% identity with the nucleotide sequence defined in a) and having a promoter function; or

(c) A DNA molecule that hybridizes to the nucleotide sequence defined in (a) or (b) under high stringency conditions and has a promoter function.

An expression cassette comprising said DNA molecule.

A recombinant vector comprising the expression cassette, the recombinant vector is preferably pBI121K-SlWRKY8P. The recombination vector is a binary vector or a joint vector.

A recombinant microorganism comprising the recombinant vector.

A transgenic cell line comprising the recombinant vector, the recombinant microorganism is preferably Escherichia coli or Agrobacterium tumefaciens EHA105.

A primer pair, the primer pair is used to amplify the above-mentioned inducible promoter, and the primer pair is:

Forward primer: 5'-GGTACCAAATTCATTTAGCGTTGCAT-3';

Reverse primer: 5'-GGATCCCTCACACAGCCATATTATAGTAAC-3'.

An application of the inducible promoter S1WRKY8P expressed in plants.

An application of the inducible promoter S1WRKY8P to promote the expression of a target gene in plants under drought and/or high salt induction.

The plant is a dicotyledonous plant.

The dicotyledonous plant is tomato, Arabidopsis or tobacco; the target gene is preferably a drought-tolerant, salt-tolerant and stress-resistant gene.

The present invention also discloses a PCR method for extracting and amplifying the tomato inducible promoter S1WRKY8P, comprising the following steps:

1) extracting the genomic DNA of tomato AC ⁺ ;

2) using the genomic DNA of tomato AC ⁺ as a template, using primers, and using the high-fidelity enzyme PrimeSTAR HS to amplify the SlWRKY8P promoter;

Among them, the total volume of amplification is 50 μL, including 1 μL of forward primer, 1 μL of reverse primer, 1 μL of DNA template, 25 μL of PrimeSTARHS, and 22 μL of ddH ₂ O. The amplification program is: 98°C for 10 seconds, 55°C for 5 seconds, 72°C for 5 seconds, 30 cycles;

The primers are:

Forward primer: 5'-GGTACCAAATTCATTTAGCGTTGCAT-3';

Reverse primer: 5'-GGATCCCTCACACAGCCATATTATAGTAAC-3'.

To achieve the above object, the present invention provides a tomato drought and/or high-salt-induced promoter S1WRKY8P and its application. The promoter in the present invention, named S1WRKY8P, is derived from tomato and is as follows a), b) or c) DNA molecule: a) the nucleotide sequence is the DNA molecule of SEQ ID No.1 in the sequence table; b) has 75% or more identity with the nucleotide sequence defined in a) and has a promoter function c) a DNA molecule that hybridizes to the nucleotide sequence defined in a) or b) under high stringency conditions and has a promoter function. Among them, SEQ ID No.1 consists of 1097 nucleotides, and the specific sequence is shown in SEQ ID No.1 in the sequence table.

The high stringency conditions are 2 × SSC (sodium citrate), 0.1% SDS (sodium dodecyl sulfate) solution, hybridization at 68 ° C and washing membrane 2 times, each 5min; then use 0.5 × In SSC and 0.1% SDS solution, hybridize at 68°C and wash the membrane twice, 15min each time.

Those skilled in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the promoter nucleotide sequence of the present invention. Those artificially modified nucleotides with 75% or higher identity with the isolated promoter nucleotide sequence of the present invention, as long as the promoter activity for expressing the target gene is maintained, are all derived from the nuclear Nucleotide sequence and is equivalent to the sequence of the present invention. The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes nucleotide sequences that are 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher identical to the promoter nucleotide sequence of the present invention. Identity can be assessed visually or with computer software. Using computer software, identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

Meanwhile, based on the inducible promoter of the present invention, expression cassettes, recombinant vectors, recombinant microorganisms or transgenic cell lines containing S1WRKY8P also belong to the protection scope of the present invention.

In the expression cassette, the downstream of the promoter SlWRKY8P is connected with a structural gene, or a regulatory gene, or an antisense gene of a structural gene or a regulatory gene, or the coding DNA of a small RNA capable of interfering with endogenous gene expression, for driving the structural gene , or a regulatory gene, or a structural gene or an antisense gene of a regulatory gene, or the expression of natural small RNA or artificially synthesized small RNA.

The expression cassette may be composed of the promoter S1WRKY8P, the gene of interest to be expressed by the promoter S1WRKY8P, and a transcription termination sequence; the promoter is functionally connected to the gene of interest, and the gene of interest is connected to the gene of interest Transcription termination sequence ligation. In one embodiment of the present invention, the target gene is specifically a β-glucuronidase (GUS) gene.

The recombinant vector can be a recombinant vector containing the above expression cassettes, including but not limited to plasmids and viruses. The recombinant vector can also be a recombinant plant expression vector. The recombinant plant expression vector contains the above-mentioned expression cassette and can transfer the expression cassette into the plant host cell, tissue or organ and its progeny and can or at least facilitate the integration of the expression cassette into the genome of the host, which includes but not Limited to binary vectors, co-conjugate vectors. The host cells, tissues or organs and their progeny refer to all plant cells or plant tissues or plant organs or whole plants (including seeds) that are matured through tissue differentiation or asexual embryo regeneration from these cells, tissues or organs. In one embodiment of the present invention, the recombinant vector is specifically a recombinant vector obtained by replacing the 35S promoter of pBI121K (pBI121K is transformed from pBI121 vector, refer to Liu Jikai et al., 2017) with the promoter. The target gene is specifically a β-glucuronidase gene (β-glucuronidase, GUS).

The recombinant expression vector can transform plant organs or tissues or cells by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium-mediated or gene gun, to obtain the transgene Plant cells or tissues or organs and the complete plants differentiated and regenerated therefrom and their clones or their progeny. In the embodiments of the present invention, the Agrobacterium-mediated method is specifically used.

The application of the above-mentioned SlWRKY8P to promote the expression of the target gene in plants also belongs to the protection scope of the present invention.

In the above application, the plant is a dicotyledonous plant, such as tomato, Arabidopsis, rice and/or tobacco.

The primer pair for amplifying S1WRKY8P also belongs to the protection scope of the present invention.

The applicant has actually verified the present invention. The experimental results prove that the expression of the target gene initiated by the drought and/or high-salt-induced promoter SlWRKY8P of the present application under the environment of 300mM mannitol (Mannitol) for 24 hours is about 2.6 times respectively under normal growth conditions, and SlWRKY8P The expression level of the target gene activated under the environment of 200mM NaCl treatment for 24 hours is about 2.7 times that of normal growth. Experimental results show that the SlWRKY8P of the present invention is a drought and/or high-salt-induced promoter and has a good effect.

In summary, the SlWRKY8P of the present invention can be used as an element for constructing a plant expression vector, and it is connected in front of the target gene, so that the expression of the target gene is induced by drought and/or high salt. Therefore, the SlWRKY8P of the present invention is of great significance to improving the tolerance of transgenic plants to drought and/or high salt by promoting the high expression of the target gene under drought and/or high salt stress.

Description of drawings

Fig. 1 is the electrophoresis diagram of PCR amplification of SlWRKY8P promoter, wherein M is DL2000DNA Marker; lane 1 is the PCR amplification band of SlWRKY8P promoter.

Figure 2 is a schematic diagram of constructing the SlWRKY8P promoter in the pBI121K vector plasmid, wherein Figure A is a schematic diagram of pBI121K; Figure B is a schematic diagram of pBI121K-SlWRKY8P, which shows the use of the SlWRKY8P promoter to drive the expression of the GUS gene located downstream of it.

Figure 3 shows the staining of GUS in the seedlings of tomato seedlings transfected with pBI121K-SlWRKY8P treated with 300mM Mannitol and 200mM NaCl for 24 hours respectively, where Figure A shows the GUS staining of normal culture for 24 hours; Figure B shows the staining of 300mM Mannitol for 24 hours Figure C is the GUS staining situation of 200mM NaCl treatment for 24 hours.

Fig. 4 is a quantitative statistical analysis of GUS activity in tomato seedlings transfected with pBI121K-SlWRKY8P treated with 300mM Mannitol and 200mM NaCl for 24 hours respectively.

detailed description

All features disclosed in this specification, or steps in all methods or processes disclosed, may be combined in any manner, except for mutually exclusive features and/or steps.

Any feature disclosed in this specification, unless specifically stated, can be replaced by other alternative features that are equivalent or have similar purposes. That is, unless expressly stated otherwise, each feature is one example only of a series of equivalent or similar features.

The present invention will be described below with reference to specific examples. Those skilled in the art can understand that these examples are only used to illustrate the discovery of the present invention, and they do not limit the scope of the present invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1

(1) Acquisition of drought and/or high salt-induced promoter SlWRKY8P

1. Plant material

Tomato wild-type seeds are AC ⁺ , donated by the laboratory of Professor Liu Yongsheng of Sichuan University.

2. Carrier

The cloning vector pEASY-Blunt Cloning Kit was purchased from Beijing Quanshijin Company.

3. Reagents and medicines

The high-fidelity enzyme PrimeSTAR HS was purchased from TAKARA Company, and the agarose gel recovery kit was purchased from Omega Bio-Tek Company. The primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd., and the sequencing was completed by Huada Gene Technology Co., Ltd.

Buffers, reagents, and bacterial culture medium formulations can be found in "Molecular Cloning Experiment Guide" (third edition, author: [US] J. Sambrook, translated by Huang Peitang, publisher: Science Press, ISBN: 7030103386).

4. Cloning of tomato SlWRKY8P promoter

According to the whole genome sequence of tomato provided by SGN (http://solgenomics.wur.nl/), the amplification primers were designed according to the upstream sequence of tomato SlWRKY8 gene, the forward primer contained the KpnI recognition sequence, and the reverse primer contained the BamHI recognition sequence :

Forward: 5'-GGTACCAAATTCATTTAGCGTTGCAT-3';

Reverse: 5'-GGATCCCTCACACAGCCATATTATAGTAAC-3'.

The above primers were used to amplify the SlWRKY8P promoter using the high-fidelity enzyme PrimeSTAR HS using the genomic DNA of tomato AC ⁺ as a template. The total volume of amplification is 50 μL, including 1 μL of forward primer, 1 μL of reverse primer, 1 μL of DNA template, 25 μL of PrimeSTAR HS, and 22 μL of ddH ₂ O. The amplification program was: 98°C for 10 seconds, 55°C for 5 seconds, 72°C for 5 seconds, 30 cycles. After agarose gel electrophoresis, a fragment similar in size to the target promoter was recovered from the gel. Ligated cloning vector pEASY-Blunt. The ligation product was transformed into Escherichia coli and incubated at 37°C for 16-18 hours. A single colony of Escherichia coli was picked, inoculated into LB liquid medium containing ampicillin, and placed in a shaker at 37° C. at 150 rpm for 16-18 hours for expansion. The expanded Escherichia coli was sent to Huada Gene Technology Co., Ltd. for sequencing analysis, and the clones with correct sequencing were used to construct expression vectors.

5. Glue recycling

The Binding Buffer (XP2), SPW Buffer and Elution Buffer used are all from the agarose gel recovery kit of OmegaBio-Tek Company.

(1) Cut out the agarose gel containing the target DNA under ultraviolet light and place it in a 1.5mL centrifuge tube.

(2) Add the same volume of Binding Buffer (XP2) as the cut gel piece into the centrifuge tube. After mixing evenly, place it at 55-60°C for about 7 minutes, and shake it constantly (every 2-3 minutes) until the gel block is completely melted.

(3) Place the DNA adsorption tube in a 2mL centrifuge tube (provided in the kit), add the completely melted gel solution in the previous step into the DNA adsorption tube, centrifuge at 8000-10000×g for 1 minute at room temperature, and discard the filtrate.

(4) Put the DNA adsorption tube back into the 2mL centrifuge tube, add 300 μL of Binding Buffer (XP2), centrifuge at 10000×g for 1 minute at room temperature, and discard the filtrate.

(5) Put the DNA adsorption tube back into the 2mL centrifuge tube, add 700 μL of SPW Buffer, let it stand for 2-3 minutes, centrifuge at 10000×g for 1 minute at room temperature, and discard the filtrate.

(6) Put the DNA adsorption tube back into the 2mL centrifuge tube and centrifuge at 10000×g for 1 minute at room temperature.

(7) Place the DNA adsorption tube in a 1.5mL centrifuge tube, add 30-50 μL of ElutionBuffer to the center of the membrane of the adsorption tube, and let stand at room temperature for 1 minute. Centrifuge at 10,000×g for 1 minute at room temperature to elute DNA fragments. After sequencing, the nucleotide sequence of the obtained fragment recovered from the gel is shown in the sequence SEQ ID No.1.

6. Connection with cloning vector

Add sequentially to a 200 μL centrifuge tube: 4 μL of gel-recovered DNA fragments, 1 μL of pEASY-Blunt Cloning Vector, mix gently, and react at room temperature for 10 minutes. After the reaction, place the centrifuge tube on ice for E. coli transformation.

7. Transformation of Escherichia coli

(1) Add the ligation product to 50 μL of Trans1-T1 competent cells (provided by pEASY-Blunt Cloning kit), flick to mix well, and ice-bath for 20-30 minutes.

(2) Heat shock at 42°C for 30 seconds, and immediately place on ice for 2 minutes.

(3) Add 250 μL of SOC/LB equilibrated to room temperature, and incubate at 37°C for 1 hour at 200 rpm.

Centrifuge the bacterial solution at 4000 rpm for 1 minute, discard part of the supernatant, keep 100-150 μL, flick the suspended bacterial cells, take all the bacterial solution to spread on the plate, and incubate overnight at 37°C.

(2) Construction of recombinant expression vector pBI121K-SlWRKY8P

1. Strains and vectors

Escherichia coli DH5ɑ, Agrobacterium tumefaciens EHA105, and expression vector pBI121K were all preserved by the Department of Biotechnology, School of Life Science and Engineering, Southwest University of Science and Technology.

2. Reagents and medicines

Restriction enzymes were purchased from Thermo Company, Taq enzyme was purchased from Tiangen Company, T4 DNA ligase was purchased from Promega Company, agarose gel recovery kit and small batch plasmid extraction kit were purchased from Omega Bio-Tek Company. Sequencing was performed by Huada Gene Technology Co., Ltd.

Buffers, reagents, bacterial culture medium formulations, Escherichia coli competent preparation refer to "Molecular Cloning Experiment Guide" (third edition, author: [US] J. Sambrook, translated by Huang Peitang, publisher: Science Press, ISBN :7030103386).

3. Plasmid extraction

The correctly sequenced pEASY-Blunt-SlWRKY8P clone containing the promoter and pBI121K Escherichia coli were expanded and cultured to extract the plasmid. The solution I, solution II, solution III, Buffer HB, Wash Buffer and ElutionBuffer used are all from the small batch plasmid extraction kit of Omega Bio-Tek Company.

(1) In a 10-20mL test tube, inoculate Escherichia coli carrying the desired plasmid into 5mL LB liquid medium containing ampicillin, and culture with shaking at 37°C for 12-16 hours.

(2) Take 1.5-5mL bacterial liquid and centrifuge at 10000×g for 1 minute at room temperature.

(3) Remove the supernatant, add 250 μL solution I, and vortex until the cells are completely suspended.

(4) Add 250 μL of solution II, gently invert the centrifuge tube 4-6 times to obtain a clear lysate, and incubate at room temperature for 2 minutes.

(5) Add 350 μL of Solution III, and gently invert several times to mix until a white flocculent precipitate appears.

(6) Centrifuge at 13000×g for 10 minutes at room temperature.

(7) Aspirate the supernatant carefully, and transfer it to a clean absorption column equipped with a 2mL centrifuge tube. Centrifuge at 10,000 x g for 1 min at room temperature.

(8) Discard the filtrate, add 500 μL Buffer HB, and centrifuge at 10,000×g for 1 minute.

(9) Discard the filtrate, wash the absorption column with 700 μL Wash Buffer, and centrifuge at 10000×g for 1 minute.

(10) Repeat step (9).

(11) Put the absorption column back into the 2mL centrifuge tube and centrifuge at 13000×g for 2 minutes.

Put the absorption column into a clean 1.5mL centrifuge tube, add 30-50μL ElutionBuffer to the center of the membrane of the adsorption column, and centrifuge at 13000×g for 1 minute.

4. Enzyme digestion and gel recovery of SlWRKY8P fragment and pBI121K vector

The extracted pEASY-Blunt-SlWRKY8P and pBI121K plasmids were simultaneously digested with KpnI and BamHI. The reaction system is shown in Table 1 below, and the reaction conditions were 37°C for 2 hours.

After the digested product was subjected to agarose gel electrophoresis, the SlWRKY8P promoter fragment and the pBI121K vector fragment were respectively recovered.

Table 1 reaction system

Added reagents pESAY-Blunt-SlWRKY8P pBI121K 10×Buffer 5 5 plasmid 10 43 KpnI 1 1 BamHI 1 1 ddH ₂ O 33 0 total capacity 50 50

5. Connection of SlWRKY8P fragment and pBI121K vector

Use T4 DNA ligase to connect the SlWRKY8P promoter fragment and the pBI121K vector fragment. The reaction system is: 10×T4 ligase buffer 1 μL, SlWRKY8P promoter fragment 5 μL, pBI121K vector fragment 3 μL, T4ligase 1 μL. The reaction conditions are: 4°C refrigerator overnight connection for 16-18 hours.

6. Transformation of Escherichia coli

The ligation product was transformed into E. coli.

(1) Take out the Escherichia coli competent cells from the ultra-low temperature refrigerator, and place them on ice to thaw.

(2) The ligation product was added to the thawed competent cells, mixed carefully, and kept on ice for 30 minutes.

(3) Place the centrifuge tube containing the competent cells at 42°C and heat shock for 90 seconds.

(4) Quickly transfer the heat-shocked centrifuge tube to an ice bath and cool for 2-3 minutes.

(5) Add 800 μL of SOC to the cooled centrifuge tube, mix well, and place in a shaker at 37°C at 120 rpm for 1 hour.

(6) Centrifuge the bacterial solution at 4000 rpm for 1 minute, discard part of the supernatant, keep 100-150 μL, flick the suspended bacterial cells, take all the bacterial solution to spread on the plate, and incubate overnight at 37°C.

(7) Pick a single clone of Escherichia coli for colony PCR, inoculate the positive clone into LB liquid medium containing kanamycin sulfate, place it at 37°C, and expand it in a shaker at 150 rpm for 16-18 hours . The expanded Escherichia coli was sent to Huada Gene Technology Co., Ltd. for sequencing analysis. The clones with correct sequencing were extracted with plasmids and used to transform Agrobacterium tumefaciens EHA105.

7. Transformation of recombinant expression vector into Agrobacterium tumefaciens EHA105

(1) Take out the competent cells of Agrobacterium tumefaciens from the ultra-low temperature refrigerator, and place them on ice to thaw.

(2) Add 10 μL of the recombinant expression plasmid with correct sequencing to the thawed competent cells, mix carefully, and place in ice bath for 30 minutes.

(3) Place the centrifuge tube containing the competent cells in liquid nitrogen and freeze for 1 minute.

(4) Quickly transfer the quick-frozen centrifuge tube to a 37°C water bath and let it stand for 2-3 minutes.

(5) Add 800 μL of SOC to the centrifuge tube, mix well, place in a shaker at 28° C. at 120 rpm and shake for 4 hours.

(6) Centrifuge the bacterial solution at 4000 rpm for 1 minute, discard part of the supernatant, keep 100-150 μL, flick the suspended bacterial cells, take all the bacterial solution to spread on the plate, and incubate at 28°C for 2 days.

(7) A single clone of Agrobacterium was picked for colony PCR.

(8) Pick the positive clones and shake the bacteria, and store the bacteria liquid with glycerol for future use.

Embodiment 2 utilizes promoter SlWRKY8P to drive reporter gene to express in tomato

1. Genetic transformation of tomato mediated by Agrobacterium

The recombinant Agrobacterium containing the pBI121K-SlWRKY8P plasmid was transformed into tomato, and the specific method was as follows.

(1) After sterilizing the tomato seeds, sow them in a culture bottle containing 1/2 MS solid medium, culture them in the dark for about 4 days, and turn them under the light after they turn white. The culture conditions are: 25°C, 16 hours of light, 23°C, After 8 hours of darkness, the light intensity was 80 μmolm ^-2 s ^-1 , and the cotyledon was removed before the first true leaf grew, and cultured on the pre-medium for 2 days.

(2) Inoculate the recombinant Agrobacterium containing the pBI121K-SlWRKY8P plasmid in 5 mL of LB medium containing 50 μg/mL rifampicin and 50 μg/mL kanamycin sulfate, and culture it on a shaker at 28 ° C for 12 hours to obtain 5 mL of pBI121K-S1WRKY8P seed liquid. Inoculate 1 mL of pBI121K-SlWRKY8P seed solution into 50 ml of LB medium containing 50 μg/mL rifampicin and 50 μg/mL kanamycin sulfate, and culture in a shaker at 28°C until the OD600 is about 0.8 to obtain pBI121K-SlWRKY8P culture solution. Centrifuge the pBI121K-SlWRKY8P culture solution at room temperature at 4000 rpm for 4 minutes, discard the supernatant to collect the bacteria, resuspend the bacteria with about 10 mL of induction culture medium, and set aside.

(3) Add 40 mL of induction culture solution into a sterilized culture dish, pipette 800 μL of resuspended bacteria into the culture dish containing induction culture solution, and mix well. The tomato cotyledons after pre-cultivation for 2 days were immersed in the mixture of induction culture medium and bacteria for 10 minutes, during which time the cotyledons could be punched vertically with the tip of sterile tweezers.

Take out the cotyledons and place them on sterile filter paper to blot the liquid, then place the cotyledons on the co-culture medium, culture them at 22±1°C in the dark for 2 days, then transfer them to the differentiation medium; then, culture them in the light In the box, the culture conditions are: 25°C, 16 hours of light, 23°C, 8 hours of darkness, and the light intensity is 80 μmol m ^-2 s ^-1 . Medium was changed every 2 weeks. When the callus grows about 2.0 cm new shoots, cut the shoots and transfer them to the rooting medium to take root, and after cultivating for about 4 weeks, complete kanamycin sulfate-resistant plants are obtained. After the root system develops, transfer it to a pot, and cultivate it in a greenhouse at 24±1°C, 16 hours of light, and 8 hours of darkness until the tomato fruit matures, and the mature tomato seeds (ie, T1 generation transgenic seeds) are harvested. The components of each medium are listed in Table 2.

Table 2 Media used for tomato tissue

Note: 6-BA: 6-Benzylaminopurine (6-Benzylaminopurine), IAA: Indoleacetic Acid (Indoleacetic Acid), KT: Kinetin (Kinetin), 2,4-D: 2,4-dichlorophenoxyacetic acid (2,4-Dichlorophenoxyacetic Acid).

(5) Sterilize the T1 generation transgenic tomato seeds to obtain sterile T1 generation tomato seeds. Sow sterile T1 generation tomato seeds on 1/2MS solid selective culture containing 50 μg/mL kanamycin sulfate, and place them in a light incubator. The culture conditions are: 25°C, 16 hours of light, 23°C, 8 hours In the dark, the light intensity is 80 μmol m ^-2 s ^-1 . After selection and cultivation, transgenic tomato seedlings containing the kanamycin sulfate resistance gene are obtained. The transgenic tomato seedlings containing the kanamycin sulfate resistance gene were transferred to pots, cultivated in the greenhouse at 24±1°C, 16 hours of light, and 8 hours of darkness until the tomato fruit matured, and harvested mature tomato seeds (i.e. T2 transgenic seeds).

2. Drought and high salinity induce SlWRKY8P

The T2 generation transgenic tomato seeds are sterilized to obtain sterile T2 generation tomato seeds. Sow sterile T2 generation tomato seeds on 1/2 MS solid selection culture containing 50 μg/mL kanamycin sulfate. Subsequently, six two-week-old sterile transgenic tomato seedlings were placed in 1/2MS liquid medium containing 300mM Mannitol, six two-week-old sterile transgenic tomato seedlings were placed in 1/2MS liquid medium containing 200mM NaCl In the culture medium, six two-week-old sterile transgenic tomato seedlings were placed in 1/2MS liquid medium, and were respectively incubated at 25°C, 16 hours of light, 23°C, 8 hours of darkness, with an average light intensity of 80 μmol m ^- The tomato seedlings treated with 300mM Mannitol (that is, the tomato seedlings under drought stress for 24 hours), the tomato seedlings treated with 200mM NaCl (that is, the tomato seedlings under salt stress for 24 hours) and the tomato seedlings treated with normal culture for 24 hours were respectively obtained under the condition of ² s ^-1 . Tomato seedlings (as a control) were then subjected to GUS histochemical staining of the material obtained above.

3. GUS histochemical staining

GUS can react with the chromogenic substrate X-gluc to appear blue, so the expression level and expression pattern of GUS can be qualitatively studied by histochemical staining.

(1) Preparation of GUS staining substrate: 50mL 0.5M phosphate buffer (pH7.0), 50μL 100mM potassium ferricyanide K ₃ Fe(CN) ₆ (dissolve 3.2924g potassium ferricyanide in water, dilute to 100mL, stored at 4°C), 50μL 100mM potassium ferrocyanide K ₄ [Fe(CN) ₆ ]·3H ₂ O (dissolve 4.2239g of potassium ferrocyanide in water, dilute to 100mL, store at 4°C), 1mL 0.5M EDTA, 250μL 1mg/mL X-gluc (dissolved in dimethylformamide and stored at -20°C in the dark).

(2) Dyeing step

Dyeing: Dip the sample to be tested into the GUS dye solution, and place it in a 37°C incubator for 24-36 hours.

Decolorization: Decolorize the dyed sample with series concentration of ethanol, the concentrations are: 50%, 75% and 90%, and then reduce the concentration after complete decolorization, in order: 90%, 70% and 50%, and finally save the sample in 50% ethanol solution.

(3) Observation

Use a stereoscope (Leica M205C) to observe the tomato seedlings normally cultivated for 24 hours after decolorization, the tomato seedlings treated with 300mM Mannitol for 24 hours after decolorization, and the tomato seedlings treated with 200mM NaCl for 24 hours after decolorization, and take pictures. The results are shown in the figure 2. The results showed that the roots, stems and leaves of the tomato seedlings that were normally cultured for 24 hours were stained with GUS, and the roots, stems and leaves of the tomato seedlings treated with 300mM Mannitol for 24 hours were all dark blue, and Roots, stems and leaves of tomato seedlings treated with 200 mM NaCl for 24 hours also showed dark blue color. The results of GUS staining experiments showed that compared with normal cultured tomatoes, the expression of GUS in tomatoes treated with 300mM Mannitol or 200mM NaCl was significantly increased in roots, stems and leaves.

4. Determination of GUS enzyme activity

According to the method of Cote et al. (Cote C, Rutledge RG. An improved MUG fluorescent assay for the determination of the GUS activity within transgenic tissue of woodyplants. Plant Cell Report, 2003, 21 (6): 619-624.) normal culture for 24 hours The tomato seedlings treated with 300mM Mannitol for 24 hours and the tomato seedlings treated with 200mM NaCl for 24 hours were subjected to fluorescence quantitative analysis of GUS activity. ^1.16nmol 4 ^- MU min ^-1 mg ^-1 protein, the GUS activity value of tomato treated with 300mM Mannitol for 24 hours 3.14nmol 4-MU min ^-1 mg ^-1 protein, the GUS activity of Mannitol treatment and NaCl treatment for 24 hours was 2.6 times and 2.7 times that of normal culture respectively, showing a very significant difference. The above results indicated that the promoter SlWRKY8P was induced by drought and/or high salt.

The present invention is not limited to the foregoing specific embodiments. The present invention extends to any new feature or any new combination disclosed in this specification, and any new method or process step or any new combination disclosed.

sequence listing

<110> Southwest University of Science and Technology

<120> A Drought and/or High Salt Inducible Promoter SlWRKY8P from Tomato and Its Application

<130> 2017

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 1446

<212>DNA

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<400> 1

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ttgttttatt tttttctacc atttggaaga gagtttagtt ccaacttaca agaatgataa 120

aattctccta gttgtttttc ttttctaatt taaacaactt ttttttttgt aataattaat 180

ataagttagt caagcttaaa atatattgtc aacggaatta tgctggctaa aaccaattga 240

gggggtaaga ctagtaaaat actttataca ttctagaaga agatgaaccc aaccatcaaa 300

gataaaaaat caaacttaaa taattaaagt ggccaaaatg gaattgaaac gaaattcctt 360

ttattcaaaa aagagatgag gacaaatgga attgaaacaa aattccaacc ctgctaagta 420

aattaaatga tataatacaa agacaaaaag attcagaaaa gtcaactaaa atgggagggg 480

aaaaaatgaa gtagaagaag taatacgtaa ccgaaaacca ctaatttata tgcgttttac 540

atctaaatct tacttataac ttataattta gtgataagtt atgttttttt ttaaaaaaaa 600

taaataagta aggacatatc aatgctatta caaagtaatg aaagatcttg aataattttt 660

tcctagatcc ataacaattg tatttttctt tgatgttcta gtatgtatgt taataataat 720

taaaaaaaaa atggttacta agaacaaatc ctccaaatca aacacaagca tctcgtgaat 780

cagacggcgt tgtcacactc ctttgacctg atcagttcaa atttccccaa aagagacccc 840

actcactctt atttccccaa agaaaaggtc aatgaaccat tactctcatg accacccta 900

caccacaaca atctaaatca ttttccccctt ttattaaagc aacataatat tctcttcttt 960

cacaatcctc gagaaagaaaaaataaaagt tacttctcac tatacaataa tttaacaaaa 1020

aaataaagtg acaatttaca agtaaaatta tttagtaatc ttgttttttt ataacttact 1080

cattggcaga atcggagtac tcatataaga atgatagagt atattggata gcatacaaaa 1140

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ttctattttc tgagatcagt atacttctca tttgtctaaa ttagttacta taatatggct 1440

gtggag 1446

<210> 2

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<212>DNA

<213> Artificial sequence

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Claims (10)

1. An inducible promoter S1WRKY8P, characterized in that the nucleotide sequence of said S1WRKY8P is optionally selected from the sequences described in (a), (b) or (c) below: (a) the nucleotide sequence shown in the sequence listing SEQ ID NO:1; (b) a nucleotide sequence having more than 75% identity with SEQ ID NO: 1 in the sequence table and having a promoter function; (c) A nucleotide sequence that hybridizes to the nucleotide sequence described in (a) or (b) under high stringency conditions and has a promoter function. 2. The inducible promoter SlWRKY8P according to claim 1, characterized in that, said (b) is a nucleotide sequence having more than 95% homology with SEQ ID NO: 1 in the sequence table and having a promoter function. 3. An expression cassette comprising the nucleotide sequence according to any one of claims 1-2. 4. A recombinant vector comprising the nucleotide sequence according to any one of claims 1-2, the recombinant vector is preferably pBI121K-SlWRKY8P. 5. A transgenic cell line comprising the recombinant vector according to claim 4. 6. A recombinant microorganism comprising the recombinant vector according to claim 4, said recombinant microorganism preferably Escherichia coli or Agrobacterium tumefaciens EHA105. 7. A pair of primers, said primers are used to amplify the inducible promoter S1WRKY8P described in claim 1, said primers are: Forward primer: 5'-GGTACCAAATTCATTTAGCGTTGCAT-3'; Reverse primer: 5'-GGATCCCTCACACAGCCATATTATAGTAAC-3'. 8. A PCR method for extracting and amplifying tomato inducible promoter S1WRKY8P, comprising the following steps: 1) extracting the genomic DNA of tomato AC ⁺ ; 2) using the genomic DNA of tomato AC ⁺ as a template, using primers, and using the high-fidelity enzyme PrimeSTAR HS to amplify the SlWRKY8P promoter; Among them, the total volume of amplification is 50 μL, including 1 μL of forward primer, 1 μL of reverse primer, 1 μL of DNA template, 25 μL of PrimeSTAR HS, and 22 μL of ddH ₂ O. The amplification program is: 98°C for 10 seconds, 55°C for 5 seconds, 72°C for 5 seconds, 30 cycles; The primers are: Forward primer: 5'-GGTACCAAATTCATTTAGCGTTGCAT-3'; Reverse primer: 5'-GGATCCCTCACACAGCCATATTATAGTAAC-3'. 9. The application of the inducible promoter S1WRKY8P according to claim 1 or 2 to promote the expression of the target gene in tomato, rice, Arabidopsis and/or tobacco under drought and/or high salt induction. 10. The application according to claim 9, characterized in that the target gene is drought tolerance, salt tolerance and stress resistance gene.