CN109750039B - Plant touch response promoter and application thereof - Google Patents

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

A plant touch response promoter and its application

technical field

The invention belongs to the field of biotechnology, in particular to a plant touch response promoter and its application.

Background technique

A promoter is a DNA sequence located upstream of the gene initiation codon (ATG), which can bind to RNA polymerase and initiate gene transcription, which is equivalent to a "switch" for gene transcription. The promoter mainly contains two regions: the core promoter region and the non-core promoter region. The core promoter region forms a universal transcription structure, usually composed of some short conserved sequences (such as transcription initiation site, TATA-box, CAAT-box, etc.). The structural features of these sequences determine the recognition and binding of the promoter to RNA polymerase and the initiation of transcription. The non-core promoter region includes the proximal regulatory region and the distal regulatory region, and there are often some specific transcription factor binding sites to regulate the temporal and spatial specificity of gene transcription. Promoters are classified into three categories: constitutive, inducible, and tissue-specific promoters according to their transcriptional patterns. Constitutive promoters can continuously and efficiently drive gene transcription in different types of cells and at different stages of cell development, and the transcriptional activity is relatively constant. Genes driven by constitutive promoters are often called housekeeping genes. Tissue-specific promoters are efficiently transcribed only in certain types of cells (tissues) or at a certain stage of cell (tissue) development, and contain cis-regulatory elements that determine the specificity of the promoter, which is related to the tissue. The promoter activity can be exhibited only when the specific transcription factor is combined, and the gene driven by the tissue-specific promoter is often closely related to the function of this type of cell (tissue). Inducible promoters require inducible signal stimulation to initiate gene transcription. Inducible promoters are more common in plants, and the genes they drive usually play an important role in the regulation of plant growth and development and in response to adverse environments.

Plants face many complex and changeable environmental factors in their life cycle, such as changes in basic conditions such as temperature, light, and water to maintain normal plant growth and development. The stimulation caused by the touch of objects) is also an important signal in the external environment, which widely affects the growth and development of plants. The process in which plants respond to mechanical stimuli and change their morphological development is called triggered morphogenesis, which is mainly reflected in the reduction of the leaf area of plants and the inhibition of the radial elongation of stems by mechanical stimulation, resulting in the formation of short and stout plants. There is a long-standing interest in plant responses to mechanical stimuli, the most well-known examples being the tactile folding of the leaves of Mimosa and the tactile curling of tendrils of vines. However, the current research on plant response to mechanical stimuli is mainly reflected in the research on morphogenesis, developmental patterns and physiological states. In addition to the research at the tissue and organ level, the mechanical response of plants is also manifested at the cell and organelle level. However, little research has been done on how plants respond to and transmit mechanical stimuli and the molecular mechanisms by which plants respond to mechanical stimuli.

Luciferase is a general term for enzymes that can produce bioluminescence in nature. It can catalyze the oxidation of luciferin or firefly aldehyde to emit light in vivo. The luciferase that exists in nature comes from fireflies, luminous bacteria, luminous starfish, luminous beetles, luminous fish, luminous beetles, etc. Among them, the most representative one is the luciferase in a firefly named Photinus pyralis. The 550-amino acid luciferase protein is a monomeric enzyme of 61kDa, which directly has complete enzymatic activity without post-expression modification. Luciferase can be genetically engineered and has been used in various experiments. The gene sequence encoding luciferase has been identified and has been widely used in many fields of biology to study the regulation of genes and the detection of expression levels. As a fluorescent marker, the gene encoding luciferase can be synthesized and inserted into organisms or transfected into cells, used to label target proteins, and track the localization and dynamic process of proteins. It is common to insert the gene encoding luciferase into the downstream of the promoter of the gene to be studied, and detect the expression level of the gene encoding luciferase under specific spatiotemporal conditions, so as to study the mode of action of the target protein. The luciferase label has strong specificity and emits light due to enzyme and substrate specificity. In addition, luciferase also has the characteristics of high sensitivity and good stability. Therefore, luciferase is of great significance in biological research.

SUMMARY OF THE INVENTION

The purpose of the present invention is to initiate the expression of a gene of interest by touching (eg, mechanical pressure).

The present invention first protects a specific DNA molecule, which can be as follows (a1) or (a2) or (a3):

(a1) the DNA molecule shown in Sequence 4 of the Sequence Listing;

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

(a3) A DNA molecule that hybridizes to the nucleotide sequence defined in (a1) or (a2) under stringent conditions and has a promoter function.

One of ordinary skill in the art can readily mutate the nucleotide sequence of the specific DNA molecule of the present invention using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides with 75% or higher identity to the nucleotide sequence of the specific DNA molecule provided by the present invention, as long as they have a promoter function, are derived from the nucleotide sequence of the present invention and Equivalent to the sequences of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or higher, 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher to the nucleotide sequence of a specific DNA molecule of the invention Nucleotide sequences of identity. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

An expression cassette containing any of the above-mentioned specific DNA molecules also belongs to the protection scope of the present 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 of interest region, a transcription termination region and optionally a translation termination region. The promoter region and the target gene region can be natural/similar to the host cell, or the promoter region and the target gene region can be natural/similar to each other, or the promoter The regions and/or gene regions of interest are heterologous to the host or to each other. "Heterologous" refers to a sequence that is derived from a foreign species, or, if from the same species, substantially modified in composition and/or genomic locus from the native form by deliberate human intervention. The optionally included transcription termination region can be homologous to the transcription initiation region, homologous to the operably linked target gene region, and homologous to the host; or; the target gene region and host are foreign or heterologous.

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

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

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 selectable markers listed above are not limiting. Any selectable marker gene can be used in the present invention.

Recombinant plasmids containing any of the above-mentioned specific DNA molecules also belong to the protection scope of the present invention.

The recombinant plasmid can be a recombinant plasmid obtained by inserting any of the above-mentioned specific DNA molecules into the starting plasmid. Specifically, the recombinant plasmid can be a recombinant plasmid obtained by inserting any of the above-mentioned specific DNA molecules into the multiple cloning site of the starting plasmid.

The recombinant plasmid may include any of the above-mentioned expression cassettes containing the specific DNA molecule.

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

Transgenic cell lines containing any of the above-mentioned specific DNA molecules also belong to the protection scope of the present invention.

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

None of the transgenic plant cell lines included propagation material. The transgenic plant is understood to include 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 can be propagated in that species, or conventional breeding techniques can be used to transfer the specific DNA molecule into other varieties of the same species, including in particular commercial varieties. The transgenic plants include seeds, callus, whole plants and cells.

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

The application of any of the above-mentioned specific DNA molecules as a promoter or a touch-responsive promoter also falls within the protection scope of the present invention.

The application of any of the above-mentioned specific DNA molecules, any of the above-mentioned expression cassettes or any of the above-mentioned recombinant plasmids in initiating the expression of a target gene also belongs to the protection scope of the present invention.

The present invention also protects methods for expressing a gene of interest.

The method for expressing a target gene protected by the present invention is specifically method one, and the method one can use any of the specific DNA molecules described above as a promoter or a touch-responsive promoter to initiate the expression of the target gene.

The method for expressing a target gene protected by the present invention may specifically be a second method, and the second method may include the following steps: inserting any of the above-mentioned specific DNA molecules into the upstream of any target gene or enhancer to activate expression of the gene of interest.

The method for expressing a target gene protected by the present invention may specifically be method three, and the method three may include the following steps: inserting the target gene into the downstream of the specific DNA molecule in any of the above-mentioned expression cassettes, and using the Specific DNA molecules initiate the expression of the gene of interest.

The method for expressing a target gene protected by the present invention is specifically method 4, and the method 4 may include the following steps: inserting the target gene into the downstream of the specific DNA molecule in any of the above-mentioned recombinant plasmids, and using the Specific DNA molecules initiate the expression of the gene of interest.

The expression of any of the above-mentioned target genes may specifically be the expression of the target gene when touched (eg, mechanical pressure).

Any of the above-mentioned specific DNA molecules can be used as a promoter (specifically, a touch-responsive promoter) to express genes (eg, exogenous genes) in plants. The plant may be a cruciferous plant. The cruciferous plant can specifically be Arabidopsis thaliana. The Arabidopsis thaliana may specifically be the wild-type Arabidopsis thaliana Columbia-0 subtype.

Any of the above-mentioned gene of interest may be a gene encoding luciferase. The amino acid sequence of the luciferase can be shown as sequence 2 in the sequence listing. The encoding gene of the luciferase can be shown as sequence 1 in the sequence listing.

Any of the above mentioned touching may be mechanical pressure. In one embodiment of the present invention, the mechanical pressure can be formed by covering the surface of plants (specifically, Arabidopsis) with quartz sand with a certain thickness (eg, 5 mm). In another embodiment of the present invention, the mechanical pressure can be formed by a glass plate covering a certain thickness (eg, 5 mm) on the surface of the plant (specifically, Arabidopsis thaliana). The covering time can be more than 20min (eg 30min).

Experiments show that the specific DNA molecule provided by the present invention can initiate the expression of the target gene (such as the gene encoding luciferase) when touched (such as mechanical pressure), indicating that the specific DNA molecule is a touch-responsive promoter. The invention has important application value.

Description of drawings

Figure 1 shows the luminescence detection results of TCH1p and TCH3p-driven luciferase.

Figure 2 shows the results of luminescence detection of luciferase driven by the CaMV 35S promoter.

Figure 3 shows the detection results of the transcription levels of TCH1 gene and TCH3 gene before and after touch stimulation in wild-type Arabidopsis thaliana.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention.

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

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

The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.

The wild-type Arabidopsis thaliana Columbia-0 subtype is described in: Kim H, Hyun Y, Park J, Park M, Kim M, Kim H, Lee M, Moon J, Lee I, Kim J. A genetic linkbetween cold responses and flowering time through FVE in Arabidopsisthaliana. Nature Genetics. 2004, 36:167-171. Wild-type Arabidopsis thaliana Columbia-0 subtype is hereinafter referred to as wild-type Arabidopsis or Arabidopsis thaliana.

In the following examples, the conditions of light-dark alternate cultivation (ie, light and dark cultivation alternate) are: 22±2°C; 16h light cultivation/8h dark cultivation; the light intensity during light cultivation is 80-100 μE/m ² /s .

In the following examples, the diameter of the sand grains of the quartz sand is 1-2 mm.

Example 1. Screening of Arabidopsis touch-responsive genes

The inventors of the present invention use the RNA-seq method to detect the differentially expressed genes of Arabidopsis thaliana after touch stimulation, and then screen to obtain the touch-responsive genes of Arabidopsis thaliana. Specific steps are as follows:

1. Take Arabidopsis thaliana seeds, sterilize with 75% (v/v) ethanol aqueous solution containing 0.05% (v/v) triton X-100 for 15 minutes, and then wash with sterilized water for 3-5 times.

2. After completing step 1, seeds of Arabidopsis thaliana were sown in 1/2 MS solid medium and vernalized at 4°C for 3 days.

3. Place the 1/2 MS solid medium obtained in step 2 in an incubator, and alternately cultivate with light and dark for 5 days to obtain Arabidopsis seedlings.

Part of Arabidopsis thaliana seedlings (ie untreated Arabidopsis seedlings) (whole plants) were snap-frozen in liquid nitrogen and stored at -80°C.

4. After completing step 3, cover the surface of the remaining Arabidopsis seedlings with quartz sand (with a thickness of 5 mm) for 30 minutes to obtain mechanically stimulated Arabidopsis seedlings. The quartz sand on the mechanically stimulated Arabidopsis thaliana seedlings was removed, and then the mechanically stimulated Arabidopsis thaliana seedlings (whole plants) were quickly frozen in liquid nitrogen and stored at -80°C.

5. Extract total RNA from untreated Arabidopsis seedlings or mechanically stimulated Arabidopsis seedlings, and then purify and isolate poly(A)-containing mRNA with a column coupled with oligo(dT) (product of Annoroad Company). .

6. After completing step 5, take the mRNA containing poly(A), add mRNA fragmentation buffer (product of Annoroad Company), and treat at 94° C. for 2 min to obtain fragmented RNA fragments.

7. After completing step 6, using the fragmented RNA fragments as templates, firstly use random primers to reverse-transcribe to synthesize first-strand cDNA; then use DNA polymerase I to synthesize double-stranded DNA, and add RNaseH to digest the RNA template at the same time. The reverse transcribed cDNA product was recovered and purified with QiaQuick PCR purification kit (product of Qiagen Company). Finally, the cDNA fragment was amplified by PCR with adapter primers, and sequenced by HiSeq2500 of Illumina.

8. After completing step 7, RNA-seq analysis was performed on untreated Arabidopsis seedlings or mechanically stimulated Arabidopsis seedlings to obtain differentially expressed genes (ie, Arabidopsis touch-responsive genes) that were significantly up-regulated after mechanical stimulation treatment. .

The results showed that TCH1 gene (gene ID: AT5G37780) and TCH3 gene (gene ID: AT2G41100) were Arabidopsis touch-responsive genes.

9. Taking the expression level of the Arabidopsis touch-responsive gene (TCH1 gene or TCH3 gene) in the untreated Arabidopsis seedlings as 1, detect the relative expression of the Arabidopsis touch-responsive genes in the mechanically stimulated Arabidopsis thaliana seedlings quantity.

The results showed that the expression levels of Arabidopsis touch-responsive genes were significantly increased in mechanically stimulated Arabidopsis seedlings compared with untreated Arabidopsis seedlings.

Example 2. Cloning and luminescence detection of the promoter of the Arabidopsis touch-responsive gene

1. Construction of recombinant plasmids

1. Construction of recombinant plasmid pLUC

(1) Using pGreenII 0800-LUC plasmid (product of BioVector Company) as template, primer F: 5'-CG GGATCC ATGGAAGACGCCAAAAACATAAAG-3' (underlined is the recognition site of restriction endonuclease BamHI) and primer R: 5 PCR amplification was performed with a primer pair consisting of '-GG GGTACC TTACAATTTGGACTTTCCGCC-3' (underlined as the recognition site of the restriction endonuclease KpnI), and a PCR amplification product with a size of about 1700 bp was recovered.

Reaction program: 94°C for 3 min; 94°C for 15 seconds, 57°C for 30 seconds, 68°C for 2 min, 35 cycles; 68°C for 10 min.

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

The sequencing results showed that the PCR amplification product contained the DNA fragment shown in SEQ ID NO: 1 in the sequence listing. The DNA fragment shown in Sequence 1 in the Sequence Listing encodes the protein shown in Sequence 2 in the Sequence Listing (ie, luciferase).

(3) After step (1) is completed, the PCR amplification product is taken and digested with restriction endonucleases BamHI and KpnI, and the digested product of about 1.7 kb is recovered.

(4) Take plasmid pBI121 (product of Beijing Biotech Co., Ltd., product catalog number is MP-091), digest with restriction enzymes SacI and EcoRI, and recover a DNA fragment of about 277 bp. This DNA fragment is the Noster poly A termination sequence.

(5) Take the vector pUC19 (product of Beijing Biotech Biotechnology Co., Ltd., product catalog number DP7801), digest it with restriction enzymes SacI and EcoRI, and recover the vector backbone of about 2686 bp.

(6) Connect the DNA fragment obtained in step (4) with the vector backbone obtained in step (5) to obtain a recombinant plasmid pUC19-Noster.

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

(8) linking the digested product obtained in step (3) and the digested product obtained in step (7) to obtain an intermediate plasmid.

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

(10) The plasmid pCAMBIA1301 (product of Biovector Co.) was taken and digested with restriction enzymes BamHI and EcoRI to recover a vector backbone of about 10 kb.

(11) Connect the DNA fragment obtained in step (9) with the vector backbone obtained in step (10) to obtain a recombinant plasmid pLUC.

2. Cloning of Arabidopsis touch-responsive gene promoters

(1) Cloning of the promoter of the TCH1 gene (hereinafter abbreviated as TCH1p)

a. Extract the genomic DNA of Arabidopsis thaliana and use it as a template, using upstream primer: 5'-CC AAGCTT agcttattactctcttccttggttttg-3' (underlined is the recognition site of restriction endonuclease HindIII) and downstream primer: 5'-CG A primer pair consisting of GGATCC agcttcttcgagaaatcgtctttc-3' (underlined is the recognition site of the restriction endonuclease BamHI) was used for PCR amplification, and the PCR amplification product with a size of about 1374 bp was recovered. The PCR amplification product contains TCH1p.

The reaction system is 30 μL, consisting of 0.6 μL KOD (concentration of 5U/μL) (KOD is a product of TOYOBO Company), 3 μL 10×buffer (10×buffer is a component of KOD), 3 μL dNTPs (concentrations of dATP, dTTP, dGTP and dCTP) 2.0 mM), 2.4 μL MgSO4 solution (25 mM concentration), 0.9 μL upstream primer (10 μM concentration), 0.9 μL downstream primer (10 μM concentration), 3 μL Arabidopsis genomic DNA and 16.2 μL ddH ₂ O.

Reaction program: 94°C for 2 min; 94°C for 15 seconds, 56°C for 30 seconds, 68°C for 2 min, 35 cycles; 68°C for 10 min.

b. After step a is completed, the PCR amplification product is taken and sequenced.

The sequencing results show that the nucleotide sequence of TCH1p is shown in sequence 3 in the sequence listing.

(2) Cloning of the promoter of the TCH3 gene (hereinafter abbreviated as TCH3p)

a. Extract the genomic DNA of Arabidopsis thaliana and use it as a template, using the upstream primer: 5'-CC AAGCTT CATTAGGGTCTGGCTTGTATG-3' (underlined is the recognition site of the restriction endonuclease HindIII) and the downstream primer: 5'-CG PCR amplification was performed with a primer pair consisting of GGATCC ACCCGAATTATTTGAAATGACG-3' (underlined as the recognition site of the restriction endonuclease BamHI), and a PCR amplification product with a size of about 1400 bp was recovered. The PCR amplification product contains TCH3p.

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

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

b. After step a is completed, the PCR amplification product is taken and sequenced.

The sequencing results show that the nucleotide sequence of TCH3p is shown in sequence 4 in the sequence listing.

3. Cloning of CaMV 35S promoter (hereinafter referred to as 35Sp)

a. Using the pGreenII 0800-LUC plasmid as the template, the upstream primer: 5'-CC AAGCTT TGAGACTTTTCAACAAAGGGTAATTTC-3' (underlined is the recognition site of the restriction endonuclease HindIII) and the downstream primer: 5'-CG GGA TCC TGTCCTCTCCAAATGAAATGAACTTC- The primer pair consisting of 3' (underlined is the recognition site of the restriction endonuclease BamHI) was PCR amplified, and the PCR amplification product with a size of about 346 bp was recovered. The PCR amplification product contains 35Sp.

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

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

b. After step a is completed, the PCR amplification product is taken and sequenced.

The sequencing results show that the nucleotide sequence of 35Sp is shown in sequence 5 in the sequence listing.

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) Take the recombinant plasmid pLUC constructed in step 1, digest with restriction enzymes HindIII and BamHI, and recover the vector backbone of about 10 kb.

(1-2) Take the PCR amplification product amplified in step 2 (1), digest with restriction endonucleases HindIII and BamHI, and recover a digested fragment of about 1374 bp.

(1-3) Connect the vector backbone obtained in step (1-1) with the restriction fragment obtained in step (1-2) to obtain a recombinant plasmid pLUC-TCH1p.

The recombinant plasmid pLUC-TCH1p was sequenced. According to the sequencing results, the structure of the recombinant plasmid pLUC-TCH1p is described as follows: the small DNA fragment between the restriction enzymes HindIII and BamHI of the recombinant plasmid pLUC is replaced with the DNA molecule shown in sequence 3 in the sequence table, and the resulting recombinant plasmid. In the recombinant plasmid pLUC-TCH1p, the expression of luciferase is initiated by TCH1p.

(2) Construction of recombinant plasmid pLUC-TCH3p

According to the method of step (1), in step (1-2), replace "PCR amplification product amplified in (1) in step 2" with "PCR amplification product amplified in (2) in step 2", other The steps remain unchanged to obtain the recombinant plasmid pLUC-TCH3p.

The recombinant plasmid pLUC-TCH3p was sequenced. According to the sequencing results, the structure of the recombinant plasmid pLUC-TCH3p is described as follows: the small DNA fragment between the restriction enzymes HindIII and BamHI of the recombinant plasmid pLUC is replaced with the DNA molecule shown in sequence 4 in the sequence table, and the resulting recombinant plasmid. In the recombinant plasmid pLUC-TCH3p, the expression of luciferase is initiated by TCH3p.

(3) Construction of recombinant plasmid pLUC-35Sp

According to the method of step (1), in step (1-2), replace "PCR amplification product amplified in step 2 (1)" with "PCR amplification product amplified in step 3", and other steps remain unchanged , the recombinant plasmid pLUC-35Sp was obtained.

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

Second, the acquisition of recombinant Agrobacterium

The recombinant plasmid pLUC-TCH1p was introduced into Agrobacterium tumefaciens GV3101 to obtain a recombinant Agrobacterium, which was named GV3101/pLUC-TCH1p.

The recombinant plasmid pLUC-TCH3p was introduced into Agrobacterium tumefaciens GV3101 to obtain a recombinant Agrobacterium, which was named GV3101/pLUC-TCH3p.

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

Third, the acquisition of transgenic Arabidopsis

1. Adopt the Arabidopsis thaliana inflorescence soaking transformation method (described in the following documents Clough, SJ, and Bent, AF. Floraldip: asmplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. (1998) 16, 735-743. ), GV3101/pLUC-TCH1p was transfected into wild-type Arabidopsis thaliana to obtain T ₁ generation transgenic TCH1p Arabidopsis thaliana seeds.

2. Sow the TCH1p transgenic Arabidopsis seeds obtained in step ₁ on the _1/2 MS medium containing 30 mg/L hygromycin, and the Arabidopsis that can grow normally (resistant seedlings) is T1 The seeds received from the TCH1p-positive seedlings of the T ₁ generation and the TCH1p-positive seedlings of the T 1 generation were the seeds of Arabidopsis thaliana of the T ₂ generation.

3. Sow the TCH1p Arabidopsis thaliana seeds from different strains screened in step ₂ on 1/2MS medium containing 30 mg/L hygromycin for screening. The ratio of the number of Arabidopsis thaliana (resistant seedlings) to the number of Arabidopsis thaliana that cannot grow normally (non-resistant seedlings) is 3:1, then this line is a line with one copy of TCH1p inserted. The seeds received by the resistant seedlings are the T ₃ generation transgenic TCH1p Arabidopsis seeds.

4. The T ₃ generation transgenic Arabidopsis thaliana seeds screened in step 3 were sown again on 1/2 MS medium containing 30 mg/L hygromycin for screening, and those that were all resistant seedlings were the T ₃ generation pure Synthesized TCH1p Arabidopsis. Two of the T ₃ generation homozygous transgenic TCH1p Arabidopsis lines were named TCH1p-1 and TCH1p-2, respectively, and subsequent experiments were carried out.

According to the above method, GV3101/pLUC-TCH1p was replaced with GV3101/pLUC-TCH3p, and other steps were the same to obtain T ₃ generation homozygous trans-TCH3p Arabidopsis thaliana. Two of the T ₃ generation homozygous transgenic TCH3p Arabidopsis lines were named TCH3p-1 and TCH3p-2, respectively, and subsequent experiments were carried out.

According to the above method, GV3101/pLUC-TCH1p was replaced by GV3101/pLUC-35Sp, and other steps were the same to obtain T ₃ generation homozygous trans-35Sp Arabidopsis. One of the T ₃ generation homozygous transgenic 35Sp Arabidopsis lines was named 35S:LUC, and follow-up experiments were carried out.

4. Luminescence detection

D-Luciferin stock solution: Dissolve 1 g of D-Luciferin powder (MW=318.42 g/mol) in 62.8 mL of water to obtain D-Luciferin stock solution with a concentration of 50 mM, which is stored in aliquots at -80°C and protected from light.

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

The Arabidopsis seeds to be tested are the T 3 generations of TCH1p-1, the T ₃ generations of TCH1p-2, the T ₃ generations of TCH3p-1, the T ₃ generations of TCH3p- ₂ , and the T ₃ generations of 35S:LUC. seeds or wild-type Arabidopsis seeds.

1. Take the Arabidopsis thaliana seeds to be tested, sterilize them with a 75% (v/v) ethanol aqueous solution containing 0.05% (v/v) triton X-100 for 15 minutes, and then wash with sterilized water for 3-5 times.

2. After completing step 1, the Arabidopsis thaliana seeds to be tested were sown in 1/2 MS solid medium, and vernalized at 4°C for 3 days.

3. Place the 1/2 MS solid medium obtained in step 2 in an incubator, and alternately cultivate with light and dark for 5 days to obtain the Arabidopsis thaliana seedling to be tested.

4. After completing step 3, the Arabidopsis thaliana seedlings to be tested were taken, sprayed with D-Luciferin working solution, then left to stand for 24 hours, and photographed with a plant in vivo imager (Berthold LB985).

5. After completing step 4, the surface of part of the Arabidopsis thaliana seedlings to be tested was covered with a glass plate (thickness of 5 mm). After 30 minutes, the glass plate was removed and photographed with a plant in vivo imager (Berthold LB985).

6. After completing step 4, some of the Arabidopsis thaliana seedlings to be tested were not treated, and were photographed with a plant in vivo imager (Berthold LB985) 30 minutes later. as comparison.

Part of the experimental results are shown in Figure 1 (A is the control, in which the 1# in the upper left is the T ₃ generation seed of TCH1p-1, the 1# in the upper right is the T ₃ generation seed of TCH1p-2, and the 2# in the lower left is the T 3 generation seed of TCH3p-1. The _3rd generation seeds, the 2# in the lower right is the T _3rd generation seeds of TCH3p-2; B is the cover glass plate treated for 30min, the 1# in the upper left is the T _3rd generation seeds of TCH1p-1, and the 1# in the upper right is TCH1p-2 The T ₃ generation seeds in the lower left, 2# in the lower left are the T ₃ generation seeds of TCH3p-1, and the 2# in the lower right are the T ₃ generation seeds of TCH3p-2) and Figure 2 (the left picture is the control, the right picture is the cover glass plate After treatment for 30 min, Col is the seed of wild-type Arabidopsis thaliana, and 35S:LUC is the T ₃ generation seed of 35S:LUC). The results showed that TCH1p-1, TCH1p-2, TCH3p-1 and TCH3p-2 had basically no fluorescence before touch stimulation (ie, without covering the glass plate); The fluorescence intensity of TCH1p-2 was weak, while the fluorescence intensity of TCH3p-1 and TCH3p-2 was stronger; the fluorescence intensity of 35S:LUC before and after touch stimulation was strong and did not change significantly; There was no fluorescent signal in wild-type Arabidopsis before stimulation and after touch stimulation.

Thus, both TCH1p and TCH3p can respond to touch stimuli (eg, mechanical pressure). Both TCH3p and TCH1p can drive luciferase to produce fluorescence under touch stimuli such as mechanical pressure.

5. Detection of transcription levels of TCH1 and TCH3 genes

The Arabidopsis thaliana seedling to be tested is the wild-type Arabidopsis seedling whose surface is not covered with glass plate in step 6 of step 4 or the wild-type Arabidopsis thaliana seedling whose surface is covered with glass plate in step 5 of step 4.

1. Take the Arabidopsis thaliana seedling to be tested, and extract the total RNA by using a plant total RNA kit (product of SIGMA-ALDRICH company) to obtain the total RNA of the Arabidopsis thaliana to be tested.

2. Take the total RNA of the Arabidopsis to be tested, and synthesize the cDNA of the Arabidopsis to be tested by referring to the operation steps of the RT-PCR kit (product of Tiangen Biochemical Technology (Beijing) Co., Ltd.).

(1) Take 2 μg of total RNA of Arabidopsis to be tested, add 2 μL of 5×gDNA Buffer, then make up to 10 μL with RNase-Free ddH ₂ O, mix well, centrifuge briefly, and let stand at 42°C for 3 min.

(2) Take the system that has completed step (1), add 10 μL of reverse transcription mixture (consisting of 2 μL 10×King RT Buffer, 1 μL FastKing RT Enzyme Mix, 2 μL FQ-RT Primer Mix and RNase-Free ddH2O), mix well; Incubate at 42°C for 15 minutes, then at 95°C for 3 minutes and place on ice to obtain the cDNA of Arabidopsis to be tested.

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

3. After completing step 2, use TB Green ^™ Fast qPCR Mix kit (product of Takara) to perform Real Time PCR (qPCR) detection.

(1) Take the cDNA of Arabidopsis to be tested and dilute it 20 times with ddH ₂ O to obtain a template solution.

(2) Prepare a reaction system. The reaction system was 20 μL, consisting of 5 μL template solution, 10 μL TB Green Fast qPCRMix (2×), 0.8 μL upstream primer (10 μM concentration), 0.8 μL downstream primer (10 μM concentration), 0.4 μL ROXReference Dye (50×) and ddH ₂ O composition.

TB Green Fast qPCR Mix (2×) and ROX Reference Dye (50×) are both components in the TB Green ^™ Fast qPCR Mix kit.

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

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

(3) After completing step (2), take the reaction system and carry out Real-time PCR detection.

The reaction program was: 95°C for 20s; 95°C for 3s, 60°C for 30s, 40 cycles; 95°C for 15s; 60°C for 1 min; 95°C for 15s; 60°C for 15s.

The real-time PCR detection results are shown in Figure 3 (0h means the surface is not covered with a glass plate, and 0.5h means the surface is covered with a glass plate for 30 minutes). The results showed that before the touch stimulation (ie, the glass plate was not covered), the expression levels of TCH1 and TCH3 genes were taken as 1; 2 times, the expression of TCH3 gene was up-regulated 31 times). It can be seen that both the TCH1 gene and the TCH3 gene have obvious responses to touch stimuli (such as mechanical pressure), which further indicates that both the TCH1 gene promoter and the TCH3 gene promoter have obvious responses to touch stimuli (such as mechanical pressure). response.

<110> Peking University

<120> A plant touch response promoter and its application

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 1653

<212> DNA

<213> Artificial sequences

<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 sequences

<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

<400> 3

agcttattac tctcttcctt ggttttgttt tatttttatc tagttttagt tttgaaattt 60

attttttttta ttatgaattt taaaaattta atttttattt gtgttggaat ttttttattt 120

tctttgtaac ctatatgaac ttggatattt ttttttctttc 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 ttttttccgac 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

<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 sequences

<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 (10)

1. A specific DNA molecule, which is the DNA molecule shown in Sequence 4 of the Sequence Listing. 2. An expression cassette comprising the specific DNA molecule of claim 1. 3. A recombinant plasmid containing the specific DNA molecule of claim 1. 4. A transgenic cell line comprising the specific DNA molecule of claim 1. 5. The application of the specific DNA molecule of claim 1 as a promoter or a touch-responsive promoter. 6. The application of the specific DNA molecule of claim 1, the expression cassette of claim 2 or the recombinant plasmid of claim 3 in initiating the expression of a target gene. 7. A method for expressing a target gene, comprising using the specific DNA molecule of claim 1 as a promoter or a touch-responsive promoter to initiate the expression of the target gene. 8. A method for expressing a target gene, comprising the steps of: inserting the specific DNA molecule of claim 1 into the upstream of any target gene or enhancer, so as to initiate the expression of the target gene. 9. A method for expressing a target gene, comprising the steps of: inserting the target gene into the downstream of the specific DNA molecule in the expression cassette of claim 2, and starting the expression of the target gene by the specific DNA molecule. 10. A method for expressing a target gene, comprising the steps of: inserting the target gene into the downstream of the specific DNA molecule in the recombinant plasmid of claim 3, and starting the expression of the target gene by the specific DNA molecule.

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