CN109750039B

CN109750039B - Plant touch response promoter and application thereof

Info

Publication number: CN109750039B
Application number: CN201910052428.3A
Authority: CN
Inventors: 钟上威; 陈丽萍; 施慧; 申醒
Original assignee: Peking University
Current assignee: Peking University
Priority date: 2019-01-21
Filing date: 2019-01-21
Publication date: 2022-09-20
Anticipated expiration: 2039-01-21
Also published as: CN109750039A

Abstract

The invention discloses a plant touch response promoter and application thereof. The nucleotide sequence of the specific DNA molecule provided by the invention is shown as a sequence 4 in a sequence table. Experiments prove that the specific DNA molecule provided by the invention can start the expression of a target gene (such as a luciferase encoding gene) when touching (such as mechanical pressure), and the specific DNA molecule is a touch response promoter. The invention has important application value.

Description

Plant touch response promoter and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a plant touch response promoter and application thereof.

Background

A promoter is a DNA sequence located upstream of the initiation codon (ATG) of a gene, and is capable of binding RNA polymerase and initiating transcription of the gene, and corresponds to a "switch" for gene transcription. Promoters contain mainly two regions: a core start area and a non-core start area. The core promoter region forms a universal transcription structure and usually consists of several short conserved sequences (e.g., transcription start site, TATA-box, CAAT-box, etc.). The structural characteristics of these sequences determine the recognition, binding and initiation of the transcription process of the promoter and the RNA polymerase. The non-core promoter region includes proximal and distal regulatory regions, often presenting specific transcription factor binding sites, regulating the temporal and spatial specificity of gene transcription. The promoters are classified into constitutive promoters, inducible promoters and tissue-specific promoters according to the transcription mode of the promoters. Constitutive promoters drive gene transcription continuously and efficiently in different types of cells and at different stages of cell development, and the transcription activity is relatively constant, and genes driven by constitutive promoters are often called housekeeping genes (housekeeping genes). A tissue-specific promoter is a promoter that is efficiently transcribed only in a certain cell (tissue) type or a certain stage of cell (tissue) development, and contains cis-regulatory elements that determine the specificity of the promoter, and the promoter activity is expressed by binding with a transcription factor specifically existing in the tissue, and a gene driven by the tissue-specific promoter is often closely related to the function of the cell (tissue) type. Inducible promoters, which are more common in plants, require an inducing signal to stimulate in order to initiate transcription of a gene, and the gene driven by the inducible promoters generally plays an important role in plant growth and development regulation and adverse environmental response.

The plant is exposed to a plurality of complex and changeable environmental factors in the life cycle, such as basic condition changes of temperature, illumination, moisture and the like for maintaining the normal growth and development of the plant, and in addition, stimulation caused by touch (such as touch of gravity, wind, rain, snow and obstacles) is also an important signal in the external environment, and the growth and development process of the plant is widely influenced. The process of plant response to mechanical stimulation and further morphological development change is called as triggered morphogenesis, which is mainly characterized in that mechanical stimulation can reduce the leaf area of the plant, and the radial extension of the stem is inhibited, so that a short, small and stout plant is formed. There has been a long felt interest in plants responding to mechanical stimuli, most commonly known as tactile closure of mimosa leaves, tactile curling of tendrils, and the like. However, the current research on plant response to mechanical stimuli is mainly embodied in the aspects of morphogenesis, developmental patterns and physiological states, and in addition to the research on tissue and organ levels, the mechanical response of plants is also expressed on cell and organelle levels. However, there is little research on how plants respond to and transmit mechanical stimulus signals, and the molecular mechanisms by which plants respond to mechanical stimuli.

Luciferase (Luciferase) is a general term for enzymes capable of generating bioluminescence in nature, and can catalyze the oxidative luminescence of luciferin (luciferin) or fatty aldehyde (firefly aldehyde) in an organism. The luciferase existing in nature is from firefly, luminous bacteria, luminous starfish, luminous festival worm, luminous fish, luminous beetle and the like, wherein the most representative is the luciferase in the firefly with the chemical name of Photinus pyralis, the gene can code the luciferase protein with 550 amino acids, and the luciferase protein is a 61kDa monomeric enzyme which does not need to be modified after expression and directly has complete enzyme activity. Luciferases can be generated by genetic engineering methods and have been used in different experiments. The sequence of a gene encoding luciferase has been identified and has been widely applied to many fields of organisms for studying the regulation of genes and the detection of expression levels. As a fluorescent marker, a gene encoding luciferase can be synthesized and inserted into an organism or transfected into cells, and used to mark a target protein, track protein localization and dynamic processes. It is common to study the mode of action of a protein of interest by inserting a gene encoding luciferase downstream of the promoter of the gene to be studied and detecting the expression level of the gene encoding luciferase under specific spatiotemporal conditions. Luciferase markers have strong specificity and emit light by specific action of enzymes and substrates. In addition, the luciferase has the characteristics of high sensitivity and good stability. Therefore, luciferases have important significance in research in the field of biology.

Disclosure of Invention

The present invention aims to initiate the expression of a target gene by touch (e.g., mechanical pressure).

The invention firstly protects a specific DNA molecule which can be (a1) or (a2) or (a 3):

(a1) DNA molecules shown in sequence 4 of a sequence table;

(a2) a DNA molecule which has 75% or more identity with the nucleotide sequence defined in (a1) and has a promoter function;

(a3) and (b) a DNA molecule which hybridizes with the nucleotide sequence defined in (a1) or (a2) under stringent conditions and has a promoter function.

The nucleotide sequence of a specific DNA molecule of the invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which have been artificially modified to have an identity of 75% or more with the nucleotide sequence of the specific DNA molecule provided by the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they have a promoter function.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, 80% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a specific DNA molecule of the invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to assess the identity between related sequences.

Expression cassettes containing any of the specific DNA molecules described above are also within the scope of the invention.

The expression cassette (from 5 'to 3') may include a promoter region (consisting of the specific DNA molecule), a transcription initiation region, a gene region of interest, a transcription termination region, and optionally a translation termination region. The promoter region and the gene region of interest may be native/analogous to the host cell, or the promoter region and the gene region of interest may be native/analogous to each other, or the promoter region and/or the gene region of interest may be heterologous to the host cell or to each other. "heterologous" means that the sequence is a sequence derived from a foreign species, or, if from the same species, substantially modified in composition and/or genomic locus in nature by deliberate human intervention. Optionally a transcription termination region homologous to the transcription initiation region, to the operably linked gene region of interest, and to the host; or; the target gene region and the host are exogenous or heterologous.

The expression cassette may also include a 5' leader sequence. The 5' leader sequence may enhance translation.

In preparing the expression cassette, adaptors or linkers may be used to join the DNA fragments, or other manipulations may be involved to provide appropriate restriction sites, remove excess DNA, remove restriction sites, and the like. To achieve this, in vitro mutations, primer repair, restriction, annealing, re-substitutions, such as transitions and transversions, can be made.

The expression cassette may also include a selectable marker gene for screening transformed cells. Selectable marker genes can be used to screen transformed cells or tissues. Marker genes include genes encoding antibiotic resistance. Other selectable markers include phenotypic markers such as fluorescent proteins. The selection markers listed above are not limiting. Any selectable marker gene may be used in the present invention.

Recombinant plasmids containing any of the specific DNA molecules described above also fall within the scope of the present invention.

The recombinant plasmid can be obtained by inserting any one of the specific DNA molecules into a starting plasmid. The recombinant plasmid can be specifically a recombinant plasmid obtained by inserting any one of the specific DNA molecules into a multiple cloning site of a starting plasmid.

The recombinant plasmid may comprise any of the expression cassettes described above containing the specific DNA molecule.

The recombinant plasmid may be specifically the recombinant plasmid pULC-TCH 3p mentioned in the examples. The recombinant plasmid pLUC-TCH3p is obtained by replacing a small DNA fragment between restriction enzymes HindIII and BamHI of the recombinant plasmid pLUC with a DNA molecule shown in a sequence 4 in a sequence table.

Transgenic cell lines containing any of the specific DNA molecules described above are also within the scope of the invention.

The transgenic cell line can be a transgenic plant cell line.

None of the transgenic plant cell lines includes propagation material. The transgenic plants are understood to comprise not only the first generation of transgenic plants obtained by transforming the recipient plant with the specific DNA molecule, but also the progeny thereof. For transgenic plants, the specific DNA molecule may be propagated in that species, or may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

In one embodiment of the present invention, the plant may be a crucifer. The cruciferous plant may specifically be arabidopsis thaliana. The Arabidopsis thaliana can be a wild type Arabidopsis thaliana (Arabidopsis thaliana) Columbia-0 subtype.

The application of any one of the specific DNA molecules as a promoter or a touch response promoter also belongs to the protection scope of the invention.

The application of any one of the above specific DNA molecules, any one of the above expression cassettes or any one of the above recombinant plasmids in promoting the expression of target genes also belongs to the protection scope of the invention.

The invention also protects a method for expressing the target gene.

The method for expressing a target gene protected by the invention can be specifically the first method, and the first method can start the expression of the target gene by using any one of the specific DNA molecules as a promoter or a touch response promoter.

The method for expressing a target gene protected by the invention can be specifically a second method, and the second method can comprise the following steps: the expression of a gene of interest is initiated by inserting any of the specific DNA molecules described above upstream of any gene of interest or enhancer.

The method for expressing the target gene protected by the invention can be specifically a third method, and the third method can comprise the following steps: inserting a target gene into any one of the expression cassettes downstream of the specific DNA molecule, and promoting the expression of the target gene by the specific DNA molecule.

The method for expressing the target gene protected by the invention can be specifically a method four, and the method four can comprise the following steps of: inserting a target gene into any one of the recombinant plasmids downstream of the specific DNA molecule, and initiating the expression of the target gene by the specific DNA molecule.

The expression of any of the above target genes may be specifically the expression of the target gene when touched (e.g., by mechanical pressure).

Any of the above specific DNA molecules can be used as a promoter (specifically, a touch response promoter) to express a gene (e.g., a foreign gene) in a plant. The plant may be a crucifer. The cruciferous plant may specifically be arabidopsis thaliana. The Arabidopsis thaliana can be a wild type Arabidopsis thaliana (Arabidopsis thaliana) Columbia-0 subtype.

Any of the above target genes may be a gene encoding luciferase. The luciferase has an amino acid sequence shown as a sequence 2 in a sequence table. The encoding gene of the luciferase can be shown as a sequence 1 in a sequence table.

Any of the above-mentioned contact may be mechanical pressure. In one embodiment of the invention, the mechanical pressure may be formed by covering the surface of the plant (which may be specifically Arabidopsis thaliana) with quartz sand to a certain thickness (e.g. 5 mm). In another embodiment of the invention, the mechanical pressure may be formed by covering the surface of the plant (which may be specifically Arabidopsis thaliana) with a glass plate having a certain thickness (e.g. 5 mm). The coverage time may be 20min or more (e.g., 30 min).

Experiments prove that the specific DNA molecule provided by the invention can start the expression of a target gene (such as a luciferase encoding gene) when touching (such as mechanical pressure), and the specific DNA molecule is a touch response promoter. The invention has important application value.

Drawings

FIG. 1 shows the results of luminescence detection of TCH1p and TCH3 p-driven luciferases.

FIG. 2 shows the result of luminescence detection of luciferase driven by CaMV 35S promoter.

FIG. 3 shows the results of the measurement of the transcription levels of TCH1 gene and TCH3 gene before and after the touch stimulation of wild type Arabidopsis thaliana.

Detailed Description

The following examples are intended to facilitate a better understanding of the invention, but are not intended to limit the invention thereto.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

The Columbia-0 subtype of wild type Arabidopsis thaliana (Arabidopsis thaliana) is described in the following references: kim H, Hyun Y, Park J, Park M, Kim M, Kim H, Lee M, Moon J, Lee I, Kim J.A genetic link between colored responses and flowing time through FVE in Arabidopsis thaliana Nature genetics.2004, 36: 167-. The Columbia-0 subtype of wild type Arabidopsis (Arabidopsis thaliana) is hereinafter referred to simply as wild type Arabidopsis thaliana or Arabidopsis thaliana.

In the following examples, the conditions of light-dark alternate culture (i.e., light culture and dark culture alternate) are: 22 +/-2 ℃; culturing for 16h in light/8 h in dark; the illumination intensity during illumination culture is 80-100 mu E/m ² /s。

In the following examples, the silica sand had a sand grain diameter of 1 to 2 mm.

Example 1 screening of Arabidopsis thaliana touch response Gene

The inventor of the invention adopts an RNA-seq method to detect the differential expression gene of arabidopsis thaliana after touch stimulation, and further screens and obtains the touch response gene of arabidopsis thaliana. The method comprises the following specific steps:

1. taking Arabidopsis seeds, sterilizing with 75% (v/v) ethanol aqueous solution containing 0.05% (v/v) triton X-100 for 15min, and washing with sterilized water for 3-5 times.

2. After completion of step 1, Arabidopsis seeds were sown in 1/2MS solid medium and vernalized at 4 ℃ for 3 d.

3. And (3) placing the 1/2MS solid culture medium which completes the step 2 into an incubator, and culturing for 5d in light and dark alternately to obtain the arabidopsis seedlings.

A part of Arabidopsis seedlings (i.e., untreated Arabidopsis seedlings) (whole plants) was snap-frozen with liquid nitrogen and stored at-80 ℃.

4. And (3) covering quartz sand (the thickness is 5mm) on the surfaces of the other arabidopsis seedlings for 30min after the step 3 is finished, so as to obtain the mechanically stimulated arabidopsis seedlings. The mechanically stimulated Arabidopsis seedlings were freed of silica sand and then the mechanically stimulated Arabidopsis seedlings (whole plants) were flash frozen with liquid nitrogen and stored at-80 ℃.

5. Total RNA from untreated or mechanically stimulated Arabidopsis seedlings was extracted, and then mRNA containing poly (A) was purified and isolated using a column coupled with oligo (dT) (product of Annuoda).

6. After the step 5 is completed, the mRNA containing poly (A) is taken, mRNA fragmentation buffer (a product of Annuodda) is added, and the mixture is treated for 2min at 94 ℃ to obtain fragmented RNA fragments.

7. After the step 6 is finished, the fragmented RNA fragments are taken as templates, and random primers are adopted to perform reverse transcription to synthesize first strand cDNA; then, double-stranded DNA is synthesized with DNA polymerase I, and RNA template is digested by adding RNaseH. The reverse transcribed cDNA product was recovered and purified using QiaQuick PCR purification kit (Qiagen). Finally, the cDNA fragments were amplified by PCR with an adaptor primer and sequenced using HiSeq2500 from Illumina.

8. After step 7 is completed, RNA-seq analysis is carried out on the untreated Arabidopsis seedlings or the mechanically stimulated Arabidopsis seedlings to obtain the differential expression gene (namely the Arabidopsis touch response gene) which is obviously up-regulated after the mechanical stimulation treatment.

The results showed that the TCH1 gene (gene ID: AT5G37780) and TCH3 gene (gene ID: AT2G41100) are Arabidopsis touch response genes.

9. The expression level of the arabidopsis touch response gene (TCH1 gene or TCH3 gene) in the untreated arabidopsis seedlings was taken as 1, and the relative expression level of the arabidopsis touch response gene in the mechanically stimulated arabidopsis seedlings was examined.

The results show that the expression level of the arabidopsis touch response gene in the mechanically stimulated arabidopsis seedlings is remarkably improved compared with untreated arabidopsis seedlings.

Example 2 cloning and luminescence detection of promoters of Arabidopsis thaliana touch response genes

Construction of recombinant plasmid

1. Construction of recombinant plasmid pLUC

(1) pGreenII 0800-LUC plasmid (Biovector) was used as a template, and primer F: 5' -CGGGATCCATGGAAGACGCCAAAAACATAAAG-3' (recognition site for restriction enzyme BamHI is underlined) and primer R: 5' -GGGGTACCTTACAATTTGGACTTTCCGCC-3' (recognition sites for the restriction enzyme KpnI are underlined) was subjected to PCR amplification, and a PCR amplification product having a size of about 1700bp was recovered.

Reaction procedure: 3min at 94 ℃; 15 seconds at 94 ℃, 30 seconds at 57 ℃, 2min at 68 ℃ and 35 cycles; 10min at 68 ℃.

(2) After step (1) is completed, sequencing the PCR amplification product.

The sequencing result shows that the PCR amplification product contains a DNA fragment shown in a sequence 1 in a sequence table. The DNA fragment shown in the sequence 1 in the sequence table encodes the protein (namely luciferase) shown in the sequence 2 in the sequence table.

(3) After completion of step (1), the PCR amplification product was digested with restriction enzymes BamHI and KpnI, and an digested product of about 1.7kb was recovered.

(4) Plasmid pBI121 (product of Beijing Bylendi Biotechnology Co., Ltd., catalog No. MP-091) was digested with restriction enzymes SacI and EcoRI, and a DNA fragment of about 277bp was recovered. The DNA fragment is a Noster poly A termination sequence.

(5) The vector pUC19 (product of Beijing Baitacg Biotechnology Co., Ltd., catalog No. DP7801) was digested with restriction enzymes SacI and EcoRI, and the vector backbone of about 2686bp was recovered.

(6) And (3) connecting the DNA fragment obtained in the step (4) with the vector framework obtained in the step (5) to obtain a recombinant plasmid pUC 19-Noster.

(7) The recombinant plasmid pUC19-Noster was digested with restriction enzymes BamHI and KpnI, and the digested product of about 2950bp was recovered.

(8) And (4) connecting the enzyme digestion product obtained in the step (3) with the enzyme digestion product obtained in the step (7) to obtain an intermediate plasmid.

(9) The intermediate plasmid was digested with restriction enzymes BamHI and EcoRI, and a DNA fragment of about 2kb was recovered.

(10) The plasmid pCAMBIA1301 (product of Biovector Co.) was digested with restriction enzymes BamHI and EcoRI, and the vector backbone of about 10kb was recovered.

(11) And (3) connecting the DNA fragment obtained in the step (9) with the vector skeleton obtained in the step (10) to obtain a recombinant plasmid pLUC.

2. Cloning of the promoter of the Arabidopsis thaliana touch response Gene

(1) Cloning of promoter of TCH1 Gene (hereinafter abbreviated as TCH1p)

a. Extracting the genome DNA of arabidopsis thaliana and taking the genome DNA as a template, adopting an upstream primer: 5' -CCAAGCTTagcttattactctcttccttggttttg-3' (recognition site for restriction enzyme HindIII is underlined) and downstream primers: 5' -CGGGATCCagcttcttcgagaaatcgtctttc-3' (recognition site for the restriction enzyme BamHI is underlined) was subjected to PCR amplification, and a PCR amplification product of about 1374bp in size was recovered. The PCR amplification product contained TCH1 p.

The reaction system was 30. mu.L composed of 0.6. mu.L KOD (concentration 5U/. mu.L) (KOD is a product of TOYOBO Co.), 3. mu.L 10 xbuffer (10 xbuffer is a component of KOD), 3. mu.L dNTPs (concentrations of dATP, dTTP, dGTP and dCTP are each 2.0mM), 2.4. mu.L MgSO4 solution (concentration 25mM), 0.9. mu.L upstream primer (concentration 10. mu.M), 0.9. mu.L downstream primer (concentration 10. mu.M), 3. mu.L Arabidopsis genomic DNA and 16.2. mu.L ddH ₂ And (C) O.

Reaction procedure: 94 ℃ for 2 min; 15 seconds at 94 ℃, 30 seconds at 56 ℃, 2min at 68 ℃ and 35 cycles; 10min at 68 ℃.

b. And (c) after the step a is finished, taking the PCR amplification product and sequencing.

The sequencing result shows that the nucleotide sequence of TCH1p is shown as sequence 3 in the sequence table.

(2) Cloning of promoter of TCH3 Gene (hereinafter abbreviated as TCH3p)

a. Extracting the genome DNA of arabidopsis thaliana and taking the genome DNA as a template, adopting an upstream primer: 5' -CCAAGCTTCATTAGGGTCTGGCTGGTATG-3' (recognition site for restriction enzyme HindIII underlined) and downstream primers: 5' -CGGGATCCACCCGAATTATTTGAA primer pair consisting of AATGACG-3' (the recognition site for the restriction enzyme BamHI is underlined) was subjected to PCR amplification, and a PCR amplification product having a size of about 1400bp was recovered. The PCR amplification product contained TCH3 p.

The reaction system is the same as the reaction system of a in the step (1).

The reaction sequence is the same as that of a in step (1).

The sequencing result shows that the nucleotide sequence of TCH3p is shown as sequence 4 in the sequence table.

3. Cloning of CaMV 35S promoter (hereinafter, abbreviated as 35Sp)

a. pGreenII 0800-LUC plasmid is taken as a template, and an upstream primer is adopted: 5' -CCAAGCTTTGAGACTTTTCAACAAAGGGTAATTTC-3' (recognition site for restriction enzyme HindIII is underlined) and downstream primers: 5' -CGGGA TCCTGTCCTCTCCAAATGAAATGAACTTC-3' (recognition site for restriction enzyme BamHI is underlined) was subjected to PCR amplification, and a PCR amplification product of about 346bp in size was recovered. The PCR amplification product contained 35 Sp.

The reaction system is the same as the reaction system of a in the step (1).

The reaction sequence is the same as that of a in the step (1).

The sequencing result shows that the nucleotide sequence of 35Sp is shown as a sequence 5 in the sequence table.

4. Construction of recombinant plasmid pLUC-TCH1p, recombinant plasmid pLUC-TCH3p and recombinant plasmid pLUC-35Sp

(1) Construction of recombinant plasmid pLUC-TCH1p

(1-1) the recombinant plasmid pLUC constructed in step 1 was digested with restriction enzymes HindIII and BamHI, and a vector backbone of about 10kb was recovered.

(1-2) taking the PCR amplification product amplified in the step 2 (1), carrying out enzyme digestion by using restriction enzymes HindIII and BamHI, and recovering an enzyme digestion fragment of about 1374 bp.

(1-3) connecting the vector skeleton obtained in the step (1-1) with the enzyme digestion fragment obtained in the step (1-2) to obtain a recombinant plasmid pLUC-TCH1 p.

The recombinant plasmid pLUC-TCH1p was sequenced. According to the sequencing results, the recombinant plasmid pULC-TCH 1p was structurally described as follows: replacing a small DNA fragment between restriction enzymes HindIII and BamHI of the recombinant plasmid pLUC with a DNA molecule shown as a sequence 3 in a sequence table to obtain the recombinant plasmid. In the recombinant plasmid pLUC-TCH1p, expression of luciferase was initiated by TCH1 p.

(2) Construction of recombinant plasmid pLUC-TCH3p

According to the method of step (1), the "PCR amplification product amplified in step 2 (1)" in step (1-2 was replaced with the "PCR amplification product amplified in step 2 (2)", and the other steps were not changed, to obtain recombinant plasmid pLUC-TCH3 p.

The recombinant plasmid pLUC-TCH3p was sequenced. According to the sequencing results, the recombinant plasmid pULC-TCH 3p was structurally described as follows: replacing the DNA small fragment between restriction enzymes HindIII and BamHI of the recombinant plasmid pLUC with a DNA molecule shown as a sequence 4 in a sequence table to obtain the recombinant plasmid. In the recombinant plasmid pLUC-TCH3p, expression of luciferase was initiated by TCH3 p.

(3) Construction of recombinant plasmid pLUC-35Sp

Replacing the PCR amplification product amplified in the step 2 in the step (1-2) with the PCR amplification product amplified in the step 3 in the step (1-2) according to the method in the step (1), and keeping the other steps unchanged to obtain the recombinant plasmid pLUC-35 Sp.

The recombinant plasmid pLUC-35Sp was sequenced. According to the sequencing results, the recombinant plasmid pLUC-35Sp was structurally described as follows: replacing the DNA small fragment between the restriction enzymes HindIII and BamHI of the recombinant plasmid pLUC with a DNA molecule shown as a sequence 5 in a sequence table to obtain the recombinant plasmid. In the recombinant plasmid pLUC-35Sp, luciferase expression was initiated from 35 Sp.

II, obtaining recombinant agrobacterium

The recombinant plasmid pLUC-TCH1p was introduced into Agrobacterium tumefaciens GV3101 to obtain recombinant Agrobacterium, which was designated GV3101/pLUC-TCH1 p.

The recombinant plasmid pLUC-TCH3p was introduced into Agrobacterium tumefaciens GV3101 to obtain recombinant Agrobacterium, which was designated GV3101/pLUC-TCH3 p.

The recombinant plasmid pLUC-35Sp was introduced into Agrobacterium tumefaciens GV3101 to obtain recombinant Agrobacterium, which was designated GV3101/pLUC-35 Sp.

Third, obtaining transgenic Arabidopsis

1. T3101/pLUC-TCH 1p was transferred to wild type Arabidopsis thaliana by the floral dip transformation method of Arabidopsis thaliana (described in Clough, S.J., and Bent, A.F. Floraldip: amplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. plant J. (1998)16, 735-743.), to obtain T3101/pLUC-TCH 1p ₁ TCH1p Arabidopsis seeds were transferred.

2. The T obtained in the step 1 ₁ Transferring TCH1p Arabidopsis seeds to be sown on 1/2MS culture medium containing 30mg/L hygromycin, and Arabidopsis capable of growing normally (resistant seedlings) is T ₁ Transfer TCH1p positive seedling, T ₁ The seeds received by TCH1p positive seedlings are T ₂ TCH1p Arabidopsis seeds were transferred.

3. The T of different strains screened in the step 2 ₂ TCH1p transgenic Arabidopsis seeds were selected by sowing them on 1/2MS medium containing 30mg/L hygromycin, if the ratio of the number of Arabidopsis seeds capable of growing normally in a line (resistant shoots) to the number of Arabidopsis seeds incapable of growing normally (non-resistant shoots) was 3: 1, the strain is a strain in which TCH1p is inserted into one copy, and the seeds received by the resistant seedlings in the strain are T ₃ TCH1p Arabidopsis seeds were transferred.

4. The T screened out in the step 3 ₃ Transferring TCH1p Arabidopsis seeds, sowing the seeds on 1/2MS culture medium containing 30mg/L hygromycin again for screening, wherein the seeds which are all resistant seedlings are T ₃ The transgenic TCH1p Arabidopsis thaliana was homozygous. 2 of them are T ₃ The generation-homozygous TCH1p Arabidopsis lines are named TCH1p-1 and TCH1p-2 respectively, and subsequent experiments are carried out.

The method is similar to the previous method except that GV3101/pLUC-TCH1p is replaced by GV3101/pLUC-TCH3p to obtain T ₃ Generation homozygous TCH3p arabidopsis thaliana. 2 of them are T ₃ The generation homozygous TCH3p Arabidopsis lines are respectively named TCH3p-1 and TCH3p-2,and subsequent experiments were performed.

The method is followed by replacing GV3101/pLUC-TCH1p with GV3101/pLUC-35Sp, and the other steps are the same to obtain T ₃ The generation was homozygous for 35Sp Arabidopsis. 1 of them is T ₃ The generation-homozygous transgenic 35Sp Arabidopsis lines were designated 35S: LUC, and subsequent experiments were performed.

Fourth, luminescence detection

D-Luciferin mother liquor: dissolving 1g D-Luciferin powder (MW 318.42g/mol) in 62.8mL of water to obtain 50mM D-Luciferin mother liquor, and storing at-80 ℃ in a split way in a dark place.

D-Luciferin working solution: the D-Luciferin stock solution was diluted with water to a concentration of 1 mM.

T with TCH1p-1 serving as arabidopsis seeds to be detected ₃ T of seed generation TCH1p-2 ₃ T of seed generation TCH3p-1 ₃ T of seed generation TCH3p-2 ₃ Seed generation, 35S, T of LUC ₃ Generation seed or wild type arabidopsis seed.

1. Taking to-be-detected Arabidopsis seeds, sterilizing the seeds for 15min by using 75% (v/v) ethanol aqueous solution containing 0.05% (v/v) triton X-100, and then washing the seeds for 3 to 5 times by using sterilized water.

2. After the step 1 is completed, the arabidopsis seeds to be tested are sown in 1/2MS solid culture medium and vernalized for 3d at 4 ℃.

3. And (3) placing the 1/2MS solid culture medium which finishes the step 2 in an incubator, and alternately culturing for 5d in light and dark to obtain the arabidopsis thaliana seedling to be detected.

4. And (3) after the step 3 is finished, spraying the arabidopsis seedlings to be detected with a D-Luciferin working solution, standing for 24 hours, and taking a picture with a plant living body imager (Berthold LB 985).

5. After the step 4 is completed, the surface of part of the arabidopsis seedlings to be tested is covered with a glass plate (the thickness is 5mm), the glass plate is removed after 30min, and a picture is taken by a plant living body imager (Berthold LB 985).

6. After the step 4 is completed, part of the arabidopsis seedlings to be detected are not treated, and a plant living body imager is used for photographing after 30min (Berthold LB 985). As a control.

The results of some of the experiments are shown in FIG. 1(A is a control, where the top left 1# is TCH1 p-1T ₃ Generation seed, top right 1# is TCH1 p-2T ₃ Generation seed, the lower left 2# is TCH3 p-1T ₃ Generation seed, right lower 2# is T of TCH3p-2 ₃ Seed generation; b is a T for covering the glass plate for 30min, wherein the 1# at the upper left is TCH1p-1 ₃ Generation seed, top right 1# is TCH1 p-2T ₃ Generation seed, the lower left 2# is TCH3 p-1T ₃ Generation seed, right lower 2# is TCH3 p-2T ₃ Generation seed) and FIG. 2 (left panel is control, right panel is cover glass plate treatment for 30min, Col is seed of wild type Arabidopsis thaliana, 35S: LUC is T of 35S: LUC ₃ Seed generation). The results show that TCH1p-1, TCH1p-2, TCH3p-1 and TCH3p-2 are essentially free of fluorescence before touching the stimulus (i.e., not covering the glass sheet); after the touch stimulus (the cover glass plate is treated for 30min), the fluorescence intensity of TCH1p-1 and TCH1p-2 is weaker, and the fluorescence intensity of TCH3p-1 and TCH3p-2 is stronger; the fluorescence intensity of 35S LUC is stronger and has no obvious change before and after touch stimulation; both before and after touch stimulation, wild type arabidopsis thaliana had no fluorescent signal.

It can be seen that both TCH1p and TCH3p are capable of responding to touch stimuli (e.g., mechanical pressure). Under touch stimulus (e.g., mechanical pressure), both TCH3p and TCH1p can drive luciferase to produce fluorescence.

Fifthly, detecting the transcription level of TCH1 gene and TCH3 gene

The arabidopsis seedlings to be detected are wild type arabidopsis seedlings with surfaces not covered by the glass plates in step four 6 or wild type arabidopsis seedlings with surfaces covered by the glass plates in step four 5.

1. Taking to-be-detected arabidopsis thaliana seedlings, and extracting total RNA by using a plant total RNA kit (a product of SIGMA-ALDRICH company) to obtain to-be-detected arabidopsis thaliana total RNA.

2. The cDNA of the arabidopsis thaliana to be detected is synthesized by taking the total RNA of the arabidopsis thaliana to be detected according to the operation steps of the specification of an RT-PCR kit (a product of Tiangen Biochemical technology (Beijing) Co., Ltd.).

(1) 2 mu.g of Arabidopsis thaliana total RNA to be tested is taken, 2 mu.L of 5 XgDNA Buffer is added, and RNase-Free ddH is used ₂ Supplementing O to 10 μ L, mixing, centrifuging for a short time, and standing at 42 deg.C for 3 min.

(2) Taking the system which finishes the step (1), adding 10 mu L of reverse transcription mixture (consisting of 2 mu L of 10 xKing RT Buffer, 1 mu L of LFastKing RT Enzyme Mix, 2 mu L of FQ-RT Primer Mix and RNase-Free ddH 2O), and uniformly mixing; and (3) incubating at 42 ℃ for 15min, then incubating at 95 ℃ for 3min, and then placing on ice to obtain the cDNA of the arabidopsis to be detected.

5×gDNA Buffer、RNase-Free ddH ₂ O, 10 XKing RT Buffer, FastKing RT Enzyme Mix and FQ-RT Primer Mix are all components of the RT-PCR kit.

3. After step 2 is completed, TB Green is adopted ^TM Real Time PCR (qPCR) detection was performed using Fast qPCR Mix kit (product of Takara).

(1) Taking cDNA of arabidopsis thaliana to be tested, and using ddH ₂ Diluting by 20 times with O to obtain a template solution.

(2) Preparing a reaction system. The reaction system was 20. mu.L from 5. mu.L of template solution, 10. mu.L of TB Green Fast qPCR Mix (2X), 0.8. mu.L of forward primer (10. mu.M in concentration), 0.8. mu.L of reverse primer (10. mu.M in concentration), 0.4. mu.L of ROX Reference Dye (50X) and ddH ₂ And (C) O.

TB Green Fast qPCR Mix (2X) and ROX Reference Dye (50X) were both TB Green ^TM Components in the Fast qPCR Mix kit.

The nucleotide sequence of an upstream primer for detecting TCH1 gene is 5'-CCCGAGTTCCTGAACCTGAT-3', and the nucleotide sequence of a downstream primer is 5'-CAGCCTCACGGATCATCTCT-3'.

The nucleotide sequence of an upstream primer for detecting TCH3 gene is 5'-ATGGTGACGGAACCATCAGT-3', and the nucleotide sequence of a downstream primer is 5'-CGCTATGTCCCTCTCCCAAT-3'.

(3) And (3) after the step (2) is completed, taking the reaction system, and carrying out Real-time PCR detection.

The reaction procedure is as follows: 20s at 95 ℃; 3s at 95 ℃, 30s at 60 ℃ and 40 cycles; 15s at 95 ℃; 1min at 60 ℃; 15s at 95 ℃; 60 ℃ for 15 s.

The results of Real-time PCR are shown in FIG. 3(0h for a surface-uncoated glass plate and 0.5h for a surface-coated glass plate for 30 min). The results show that the expression level of TCH1 gene and TCH3 gene is 1 before touch stimulation (i.e. uncovered glass plate); after touch stimulation, the expression levels of the TCH1 gene and the TCH3 gene were significantly up-regulated (2-fold up-regulation of the TCH1 gene and 31-fold up-regulation of the TCH3 gene). Therefore, the TCH1 gene and the TCH3 gene have obvious response to touch stimulus (such as mechanical pressure), and further the TCH1 gene promoter and the TCH3 gene promoter have obvious response to touch stimulus (such as mechanical pressure).

<110> Beijing university

<120> plant touch response promoter and application thereof

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 1653

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 1

atggaagacg ccaaaaacat aaagaaaggc ccggcgccat tctatcctct agaggatgga 60

accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt 120

gcttttacag atgcacatat cgaggtgaac atcacgtacg cggaatactt cgaaatgtcc 180

gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta 240

tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt 300

gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag tatgaacatt 360

tcgcagccta ccgtagtgtt tgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa 420

aaaaaattac caataatcca gaaaattatt atcatggatt ctaaaacgga ttaccaggga 480

tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat 540

tttgtaccag agtcctttga tcgtgacaaa acaattgcac tgataatgaa ttcctctgga 600

tctactgggt tacctaaggg tgtggccctt ccgcatagaa ctgcctgcgt cagattctcg 660

catgccagag atcctatttt tggcaatcaa atcattccgg atactgcgat tttaagtgtt 720

gttccattcc atcacggttt tggaatgttt actacactcg gatatttgat atgtggattt 780

cgagtcgtct taatgtatag atttgaagaa gagctgtttt tacgatccct tcaggattac 840

aaaattcaaa gtgcgttgct agtaccaacc ctattttcat tcttcgccaa aagcactctg 900

attgacaaat acgatttatc taatttacac gaaattgctt ctgggggcgc acctctttcg 960

aaagaagtcg gggaagcggt tgcaaaacgc ttccatcttc cagggatacg acaaggatat 1020

gggctcactg agactacatc agctattctg attacacccg agggggatga taaaccgggc 1080

gcggtcggta aagttgttcc attttttgaa gcgaaggttg tggatctgga taccgggaaa 1140

acgctgggcg ttaatcagag aggcgaatta tgtgtcagag gacctatgat tatgtccggt 1200

tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct 1260

ggagacatag cttactggga cgaagacgaa cacttcttca tagttgaccg cttgaagtct 1320

ttaattaaat acaaaggata tcaggtggcc cccgctgaat tggaatcgat attgttacaa 1380

caccccaaca tcttcgacgc gggcgtggca ggtcttcccg acgatgacgc cggtgaactt 1440

cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga gatcgtggat 1500

tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac 1560

gaagtaccga aaggtcttac cggaaaactc gacgcaagaa aaatcagaga gatcctcata 1620

aaggccaaga agggcggaaa gtccaaattg taa 1653

<210> 2

<211> 550

<212> PRT

<213> Artificial sequence

<220>

<223>

<400> 2

Met Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr Pro

1 5 10 15

Leu Glu Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met Lys Arg

20 25 30

Tyr Ala Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala His Ile Glu

35 40 45

Val Asn Ile Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala

50 55 60

Glu Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg Ile Val Val

65 70 75 80

Cys Ser Glu Asn Ser Leu Gln Phe Phe Met Pro Val Leu Gly Ala Leu

85 90 95

Phe Ile Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu Arg

100 105 110

Glu Leu Leu Asn Ser Met Asn Ile Ser Gln Pro Thr Val Val Phe Val

115 120 125

Ser Lys Lys Gly Leu Gln Lys Ile Leu Asn Val Gln Lys Lys Leu Pro

130 135 140

Ile Ile Gln Lys Ile Ile Ile Met Asp Ser Lys Thr Asp Tyr Gln Gly

145 150 155 160

Phe Gln Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro Pro Gly Phe

165 170 175

Asn Glu Tyr Asp Phe Val Pro Glu Ser Phe Asp Arg Asp Lys Thr Ile

180 185 190

Ala Leu Ile Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys Gly Val

195 200 205

Ala Leu Pro His Arg Thr Ala Cys Val Arg Phe Ser His Ala Arg Asp

210 215 220

Pro Ile Phe Gly Asn Gln Ile Ile Pro Asp Thr Ala Ile Leu Ser Val

225 230 235 240

Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu

245 250 255

Ile Cys Gly Phe Arg Val Val Leu Met Tyr Arg Phe Glu Glu Glu Leu

260 265 270

Phe Leu Arg Ser Leu Gln Asp Tyr Lys Ile Gln Ser Ala Leu Leu Val

275 280 285

Pro Thr Leu Phe Ser Phe Phe Ala Lys Ser Thr Leu Ile Asp Lys Tyr

290 295 300

Asp Leu Ser Asn Leu His Glu Ile Ala Ser Gly Gly Ala Pro Leu Ser

305 310 315 320

Lys Glu Val Gly Glu Ala Val Ala Lys Arg Phe His Leu Pro Gly Ile

325 330 335

Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Leu Ile Thr

340 345 350

Pro Glu Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val Pro Phe

355 360 365

Phe Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr Leu Gly Val

370 375 380

Asn Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met Ile Met Ser Gly

385 390 395 400

Tyr Val Asn Asn Pro Glu Ala Thr Asn Ala Leu Ile Asp Lys Asp Gly

405 410 415

Trp Leu His Ser Gly Asp Ile Ala Tyr Trp Asp Glu Asp Glu His Phe

420 425 430

Phe Ile Val Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly Tyr Gln

435 440 445

Val Ala Pro Ala Glu Leu Glu Ser Ile Leu Leu Gln His Pro Asn Ile

450 455 460

Phe Asp Ala Gly Val Ala Gly Leu Pro Asp Asp Asp Ala Gly Glu Leu

465 470 475 480

Pro Ala Ala Val Val Val Leu Glu His Gly Lys Thr Met Thr Glu Lys

485 490 495

Glu Ile Val Asp Tyr Val Ala Ser Gln Val Thr Thr Ala Lys Lys Leu

500 505 510

Arg Gly Gly Val Val Phe Val Asp Glu Val Pro Lys Gly Leu Thr Gly

515 520 525

Lys Leu Asp Ala Arg Lys Ile Arg Glu Ile Leu Ile Lys Ala Lys Lys

530 535 540

Gly Gly Lys Ser Lys Leu

545 550

<210> 3

<211> 1374

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 3

agcttattac tctcttcctt ggttttgttt tatttttatc tagttttagt tttgaaattt 60

atttttttta ttatgaattt taaaaattta atttttattt gtgttggaat ttttttattt 120

tctttgtaac ctatatgaac ttggatattt tttttctttc ttacattaat taagtacgta 180

agtttcgaca atcaagaata gggttttgtt gattccaatc gtgttaccca aaaattagaa 240

taaggtttgt ccaacaattt attaaatttg gtcttagagt tgtatacgag cctacgaggt 300

atcttttaga tatgccgaag agatggtagt gcgaagaaga aaagaaacct cattttccat 360

ggtattaagg atttcaagct attcaaactt ttgtagtgac ccaaatacgt gtagaactgg 420

cccaaagaca aatagcttaa ttaagattat cacctaatct taggtattgg tgataggtga 480

ttcgctcaag ccttatgatc aatattagct ccatattatg gagagtgagc agcacatact 540

cgtagataga aaagtattgt tgctcgagta atatttttct gatgataatt gtgtttcgac 600

cactgttggc atctttcaca catgaataca tgatgtacat gtacgacatg caatctttct 660

cttaacgctc gactgctgtc tgctgtgatg gatcatcaca agtaaacttt gtgaagcata 720

tttcgtgagc caaatgatgt tagaaaacaa tcgtgtgagc caaaggcgtg agagcgcttg 780

tagaaagtgg tttatgttgt agttaaaata atccgaatat tcatcttccg gaaaaaagtg 840

aagttcaacc tctcaacggt acgtttttat acctcaacta agttacattg tgtatatcaa 900

catcagctaa ctttttatta atatttcggt tcacaatatc attatctttt tcaaaattta 960

ttaagatttc ttttgctcat tttatttatg tgaattttaa aaataaaata atttttataa 1020

tatagaaaca aaatattact aaaagtaatt tttaaaaaat aaataaaatt aatgtcatat 1080

caacgaaaaa ttcatagttt attaaagtag taaatataaa agtaataata ttttaattat 1140

tttggtatta aattatacac ttttattaat atatgtattt tcttctaaaa attaaaaatt 1200

aattgggttt ttgtttggaa tttttccgac ttaccttttc tccttctttt tgtcgttttg 1260

cttagatatt tgtttgcatc attttcaaag agagacgact ctgaatccaa aaaaaaaaaa 1320

aactcattaa gtaaagacaa aggaagagaa gaaagacgat ttctcgaaga agct 1374

<210> 4

<211> 1400

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 4

cattagggtc tggctggtat gttagatctc tctaaaaagg cgtttgatct ttgaaaataa 60

tttctatgta ataaatttat tgtgttacca tcttttcttg catgcaattt attaaaatga 120

gtttgtaata tggtttcagt atcgaatata attggggtat caatgtatct tttgagaaaa 180

acatttaaaa cgtagcagaa ctaattattg aaaactggta gcattactaa aaattgatct 240

aaatcggtta atgatttggc acatacagtg atcaaatcag ctaagcctca tatctcaccg 300

tcccagagtt cttcaagact cttataagga ctcatctaat gaataatgac atgcctcgca 360

ccaacctgcc ctgcaacatt gaaaacacac gtctacaatt tcgataaccc aagtgttttg 420

agcaaaacaa aggacgccct ttgacccatt tggactacaa tgtgtggaaa attactgcat 480

gttcaatatc agaaaacttg gataacatgt cttcgatatg ggcttttctt attttgtacc 540

cgtgtaattt cgctggccca aaatccaaag caacaagggt aaatttgtat gcagtacatg 600

tggcatgttg ggtcatatgg aaacaggtca caagactcag attctttaca ttgcggcggg 660

tagtagtagc tttctagtct accttgagat tgcccctgcc cgtgattctt gagtcgggca 720

caggaagtgg ttttctgtca actttacttg ctcaagccat ccccgcaagg tagaaacttc 780

tgtggtcttt ctcgttattt cttatgtacc atgtttttgg tcgtttgtgt agtttcttct 840

aaccttccac acacttgtct tgcgaattcc ttccacagca gagatgttaa atcaggaata 900

ttcaggatgt agaaacttct ctgctctttc tcaagtagaa tgcacttgtg gaatccctca 960

gatcagattt cattggtaac gttcgttaac ttcaacaaca acactttact aaacatgtgc 1020

taatcaattg tcatacgctt gtatttcttc gattcatttt tatcttatgc aagtgttttc 1080

tctattatga tttatttagt tttgcataaa taaggacact ccttcggagt cagtataaga 1140

aaactataac ctaaaatata aattaggcaa atgtccactc acccatccaa aattccattg 1200

taaataaatg tttctttcaa ataaatgaaa aaaaaaaaaa aaaacaagtt ggaagaagga 1260

accaaccaag taattgccag tcaaaggagg cgactcacac ttgaaaggat aagctgacat 1320

ggcagacaac gatcctgcgt agaaattgcg tgaacgtgga aaagtttgag gatatagccg 1380

tcatttcaaa taattcgggt 1400

<210> 5

<211> 346

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 5

tgagactttt caacaaaggg taatttcggg aaacctcctc ggattccatt gcccagctat 60

ctgtcacttc atcgaaagga cagtagaaaa ggaaggtggc tcctacaaat gccatcattg 120

cgataaagga aaggctatca ttcaagatgc ctctgccgac agtggtccca aagatggacc 180

cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt 240

ggattgatgt gacatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca 300

agacccttcc tctatataag gaagttcatt tcatttggag aggaca 346

Claims

1. The specific DNA molecule is shown in sequence 4 in the sequence table.

2. An expression cassette comprising the specific DNA molecule of claim 1.

3. A recombinant plasmid comprising the specific DNA molecule of claim 1.

4. A transgenic cell line comprising the specific DNA molecule of claim 1.

5. Use of the specific DNA molecule of claim 1 as a promoter or a touch-responsive promoter.

6. Use of a specific DNA molecule according to claim 1, an expression cassette according to claim 2 or a recombinant plasmid according to claim 3 for promoting expression of a gene of interest.

7. A method for expressing a target gene, which comprises using the specific DNA molecule of claim 1 as a promoter or a touch-responsive promoter to promote the expression of the target gene.

8. A method of expressing a gene of interest comprising the steps of: the specific DNA molecule of claim 1 inserted upstream of any desired gene or enhancer to initiate expression of the desired gene.

9. A method of expressing a gene of interest comprising the steps of: inserting a gene of interest downstream of said specific DNA molecule in said expression cassette of claim 2, whereby expression of said gene of interest is initiated by said specific DNA molecule.

10. A method of expressing a gene of interest comprising the steps of: inserting a target gene into the recombinant plasmid of claim 3 downstream of the specific DNA molecule, and promoting the expression of the target gene by the specific DNA molecule.