Tomato root specific promoter pSlPOD and application thereof

Abstract

The invention discloses a tomato root specific promoter pSlPOD and application of the tomato root specific promoter pSlPOD. The invention provides a DNA (Deoxyribonucleic Acid) molecule which is any one of the following: (A1) a DNA molecule as shown in SEQ ID No.1; (A2) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of homology with the nucleotide sequence limited by (A1) and has a promoter function; and (A3) a DNA molecule which can be hybridized with the nucleotide sequence limited by (A1) or (A2) under strict conditions and has a promoter function. The DNA molecule is a root specific expression promoter, and a key promoter resource is provided for accurately improving the tomato rhizosphere stress resistance through genetic engineering.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a tomato root-specific promoter pSlPOD and application thereof. BACKGROUND

[0002] Root is the only way for plants to absorb water and nutrients from the soil, and is also an important organ for plants to cope with drought, salinity and heavy metal environment, and to protect the aboveground part from stress. Using genetic engineering technology can effectively enhance the ability of root nutrient absorption and resistance to adversity, which depends on the development and utilization of root-specific expression genes and their promoter regulatory mechanisms. Promoter is a DNA sequence located upstream of the 5' end of the structural gene, which can activate RNA polymerase, make it accurately bind to the template DNA and has the specificity of transcription initiation, and is divided into three types of constitutive, tissue-specific and inducible. Although the constitutive promoter can drive the transcription of genes in a wide range of tissues, making the target protein express in large quantities, it will cause energy waste and metabolic burden, which is not conducive to the normal growth and development process of plants. The mining of tissue-specific promoter can make the target gene express in specific tissues, which can not only reduce the energy consumption in the plant body, but also avoid the negative effects caused by the excessive accumulation of target proteins in inappropriate development period or unnecessary tissue parts, providing a new idea for the application of transgenic technology in tomato breeding, and having important application value for plant stress resistance improvement and yield increase.

[0003] Peroxidase (POD) widely exists in animals, plants and microorganisms, as an important part of endogenous antioxidant system, which can effectively remove intracellular peroxides and coordinate accurate transmission of intracellular signals. Plant peroxidase has many functions, as an important regulatory factor for plant growth and development, it is involved in plant stress response, such as drought resistance, salt resistance, frost resistance, disease resistance and other processes. Studies have shown that plants can protect cell membrane structure and enhance resistance by activating the antioxidant system under stress, and the POD activity in tomato seedling leaves increases after polyethylene glycol simulates drought stress treatment.

[0004] However, there is no report on the function of POD gene in tomato roots, and the expression characteristics and regulatory mechanism of its promoter are not clear. This technology has application value in plant resistance to pests and diseases, improving plant adaptation to adverse environment, and improving quality. SUMMARY

[0005] The purpose of the present application is to provide a tomato root-specific promoter pSlPOD and application thereof.

[0006] In a first aspect, the present application claims a.

[0007] The DNA molecule claimed in the present application can be any one of the following: (A1) a DNA molecule represented by SEQ ID No. 1; (A2) a DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the nucleotide sequence defined in (A1) and having a promoter function; (A3) a DNA molecule capable of hybridizing to the nucleotide sequence defined in (A1) or (A2) under stringent conditions and having a promoter function. In the above DNA molecules, the homology of the nucleotide sequence can be determined using the homology search site on the Internet, such as the BLAST page of the NCBI homepage. For example, the homology of one pair of nucleotide sequences can be calculated by using blastn as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a search in High-Stat BLAST 2.1, and then the value of the homology (%) can be obtained.

[0008] In the above DNA molecules, the 95% or more homology can be at least 96%, 97%, 98%, or 99% homology. The 90% or more homology can be at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology. The 85% or more homology can be at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology. The 80% or more homology can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology.

[0009] The stringent conditions can be as follows: hybridization at 50℃ in a mixture of 7% sodium dodecyl sulfate (SDS), 0.5M Na3PO4 and 1mM EDTA, and rinsing at 50℃ in 2xSSC, 0.1% SDS; or as follows: hybridization at 50℃ in a mixture of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and rinsing at 50℃ in 1xSSC, 0.1% SDS; or as follows: hybridization at 50℃ in a mixture of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and rinsing at 50℃ in 0.5xSSC, 0.1% SDS; or as follows: hybridization at 50℃ in a mixture of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and rinsing at 50℃ in 0.1xSSC, 0.1% SDS; or as follows: hybridization at 50℃ in a mixture of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and rinsing at 65℃ in 0.1xSSC, 0.1% SDS; or as follows: hybridization at 65℃ in a solution of 6xSSC, 0.5% SDS, and rinsing the membrane once each with 2xSSC, 0.1% SDS and 1xSSC, 0.1% SDS.

[0010] In a second aspect, the present application claims protection of an expression cassette or a recombinant vector or a recombinant bacterium or a transgenic plant cell line or a transgenic plant tissue or a transgenic plant organ containing the DNA molecule as described in the first aspect above.

[0011] Further, the expression cassette is composed of the DNA molecule having the function of a promoter, a gene of interest expressed under the control of the promoter, and a transcription termination sequence; the DNA molecule is functionally linked to the gene of interest, and the gene of interest is linked to the transcription termination sequence.

[0012] In some embodiments of the present application, the gene of interest is a GUS gene; and the transcription termination sequence is a NOS transcription terminator.

[0013] Further, the recombinant vector can be either a recombinant expression vector or a recombinant cloning vector.

[0014] Further, the recombinant vector is a recombinant vector containing the expression cassette.

[0015] In some embodiments of the present application, the recombinant vector is specifically a recombinant plasmid obtained by inserting the DNA molecule (SEQ ID No. 1) into the restriction enzyme site between the p1300GN vector Xba I and Bam H I.

[0016] Further, the recombinant bacterium can be a recombinant Agrobacterium.

[0017] In one embodiment of the present application, the recombinant bacteria is Agrobacterium GV3101 containing the recombinant vector.

[0018] Further, the transgenic plant cell line, the transgenic plant tissue and the transgenic plant organ all exclude reproductive material.

[0019] In a third aspect, the present application claims the use of the DNA molecule as described in the first aspect above to initiate expression of a target gene.

[0020] In the use of the DNA molecule to initiate expression of a target gene, the expression of the target gene is initiated in plants.

[0021] Further, the expression is root-specific expression.

[0022] In a fourth aspect, the present application claims a method for specifically expressing a target gene in the roots of a plant.

[0023] The method for specifically expressing a target gene in the roots of a plant claimed by the present application can comprise the following steps: introducing the expression cassette or the recombinant vector as described in the second aspect above carrying the target gene into a plant, thereby achieving specific expression of the target gene in the roots of the plant.

[0024] The promoter sequence of the present application can be constructed upstream of any target gene using a plant expression vector, introduced into plant cells, and a transgenic plant with root-specific expression can be obtained. The plant expression vector carrying the promoter sequence of the present application can be transformed into plant cells or tissues by using Ti plasmid, direct DNA transformation, microinjection, Agrobacterium-mediated transformation, and other conventional biological methods, and the transformed plant tissues can be cultivated into plants.

[0025] In each of the above-mentioned related aspects, the plant can be any of the following: (B1) a dicotyledonous plant; (B2) a Solanaceae plant or a Brassicaceae plant; (B3) tomato or Arabidopsis thaliana.

[0026] In one embodiment of the present application, the plant is Arabidopsis thaliana Col-0.

[0027] In a fifth aspect, the present application claims a primer pair for amplifying the full length or a partial fragment of the DNA molecule as described in the first aspect above.

[0028] In some embodiments of the present application, the primer pair is a primer pair consisting of two single-stranded DNAs as shown in SEQ ID No. 2 and SEQ ID No. 3.

[0029] The application preliminarily obtains a tomato root-specific expression gene through transcriptome data analysis SlPOD , and the specific expression of the gene in the tomato root is confirmed by fluorescence quantitative PCR. SlPOD The sequence of 2000 bp upstream of ATG is shown as SEQ ID NO:1. SlPOD The GUS histochemical staining proves that the promoter (SEQ ID NO:1) drives the GUS reporter gene to be specifically expressed in the root of Arabidopsis. The application provides a key promoter resource for precisely improving the rhizosphere stress resistance of tomatoes through genetic engineering.

[0030] The application has the following beneficial effects: 1. The promoter of the application can specifically drive the downstream target gene to be expressed in the root of a plant, but not in other tissues and organs, and has tissue expression specificity.

[0031] 2. The root of a plant is an important nutrient organ, and breeding experts can use the promoter of the application to specifically express a target gene in the root to improve the resistance of a plant to diseases and pests, the salt and alkali tolerance, change the metabolic pathway of the plant, and improve and enhance the yield and nutritional components of the plant. The promoter of the application provides a key promoter resource for improving the expression and accumulation level of an exogenous gene in a specific tissue of a tomato through a genetic engineering means and precisely improving the rhizosphere stress resistance of a tomato through a transgenic method. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 It is a schematic diagram of a p1300GN-pSlPOD expression vector.

[0033] Figure 2 It is SlPOD The expression amount of the gene in different tissues of a tomato.

[0034] Figure 3 It is the construction of a p1300GN-pSlPOD expression vector. SlPOD PCR identification of the gene promoter (i.e. pSlPOD), the marker III marker is in lane M, and the bands from top to bottom are 4500 bp, 3000 bp, 2000 bp, 1200 bp, 800 bp, 500 bp and 200 bp, the PCR product (2000 bp) of pSlPOD is in lane 1, and the negative control with water as a template is in lane 2; B is the identification of the p1300GN-pSlPOD plasmid, the marker III marker is in lane M, the p1300GN-pSlPOD plasmid is in lane 1, and the p1300GN-pSlPOD plasmid is digested by X ba I and B am H I double enzymes.

[0035] Figure 4 PCR detection of T1 generation transgenic Arabidopsis. Lane M is marker III marker, lane 1 is a positive control with p1300GN-pSlPOD vector as a template, lane 2 is a negative control with water as a template, and lanes 3-14 are different transgenic Arabidopsis positive plants.

[0036] Figure 5 SlPOD Promoter expression pattern analysis. WT-1 and WT-2 are two wild-type Arabidopsis repeats; pSlPOD-1 and pSlPOD-2 are two trans-p1300GN-pSlPOD recombinant vector Arabidopsis repeats; 35S-1 and 35S-2 are two trans-p1300GN vector Arabidopsis repeats. DETAILED DESCRIPTION

[0037] The application will be further described in detail below in conjunction with the specific embodiments, and the examples given are only to illustrate the application, not to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0038] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0039] In the following examples, the primers and sequencing were completed by Beijing Meijiesi Nuobio Technology Co., Ltd. The fast endonuclease was a product of Thermo Fisher Scientific Company. The DNA gel recovery kit, DNA marker, etc. were products of Dalian Baosheng Company and Tian Gen Biochemical Technology (Beijing) Co., Ltd. The tomato 'Micro-Tom', wild-type Arabidopsis Col-0, E. coli competent strain TOP10, Agrobacterium competent strain GV3101, and modified plant genetic expression vector p1300GN used in the experiment were all preserved in the laboratory. p1300GN vector: described in the article "Wang Jia Yin, et al. Cloning and functional analysis of tomato phosphate transporter SlPT2 gene promoter. Henan Agricultural Science, 2024, 53 (6): 120-127", available to the public from the applicant, only for repeated use in the experimental application, not for other purposes.

[0040] ​Tomato variety 'Micro-Tom': recorded in the article "Wang Jiaoyin et al. Cloning and functional analysis of the promoter of phosphate transporter gene SlPT2 in tomato. Henan Agricultural Sciences, 2024, 53(6): 120-127", available to the public from the applicant, only for repeated experiments of the invention, not for other purposes.

[0041] Example 1, tomato root-specific promoter pSlPOD and its application I. Materials and methods 1. Materials The tomato material used in the present application is 'Micro-Tom' (Solanum lycopersicum cv. Micro-Tom), and the Arabidopsis material is Col-0 (Arabidopsis thaliana Col-0). Solanum lycopersicum Arabidopsis thaliana Tomato and Arabidopsis thaliana for Agrobacterium infection are planted in small black square containers filled with German K brand Dahan nutrient soil. Arabidopsis T1 generation seeds for GUS staining are sown on MS solid medium. The culture conditions are as follows: tomato at 25°C, light intensity 90 μmol·m -2 ·s -1 , 16 h light / 8 h dark; Arabidopsis at 23°C, light intensity 70 μmol·m -2 ·s -1 , 12 h light / 12 h dark. The binary expression vector p1300GN, E. coli TOP10 and Agrobacterium GV3101 used in the experiment are preserved in the laboratory.

[0042] 2. Methods (1) Tomato total RNA extraction and cDNA synthesis By analyzing the gene expression transcriptome data of the four organs of tomato variety Micro-Tom root, leaf, flower and fruit, a root-specific SlPOD ( Solyc02g064970 ) 。 Total RNA extraction of tomato root, stem, leaf, flower, green mature period and color breaking period fruit tissue, according to the instructions of RNA simple Total RNA Kit (DP419, Tiangen Biochemical Technology Co., Ltd., Beijing). First-strand cDNA synthesis, according to the instructions of PrimeScript™ RT reagent Kit with gDNAEraser (RR037A, Baosheng Bioengineering Co., Ltd., Dalian).

[0043] (2) SlPOD Analysis of gene expression patterns According to SlPOD gene and internal reference gene actin-7 ​Based on the sequence information of (GenBank accession number: NM_001308447.1), primers SlPOD-qRT F / R and Slactin-qRT F / R were designed for quantitative real-time PCR (Table 1). The reaction system consisted of: 10 μL 2×SYBR qPCR Mix (LowROX), 0.5 μL 10 μM forward primer, 0.5 μL 10 μM reverse primer, 1 μL cDNA template, and 8 μL ddH2O. The reaction program was 95℃ for 3 min; 95℃ for 10 s, 60℃ for 10 s, 72℃ for 20 s, for 40 cycles; finally, a melting curve analysis program was set. The experiment was performed in triplicate, using 2... -ΔΔCt Methods for calculating different tissues SlPOD The expression levels were analyzed using Excel 2010 and SPSS 8.5 software to create bar charts and perform significance analysis.

[0044] Table 1. Primers used in the experiment

[0045] Note: Underscores indicate... Xba I and Bam The HI restriction site sequence. The sequence before the "-" in SlPOD-proF / R is the vector sequence, used for homologous recombination.

[0046] (3) SlPOD Gene promoter cloning Download from the tomato genome website (http: / / solgenomics.net / ) SlPOD Primers SlPOD-proF and SlPOD-proR were designed based on the 2000 bp sequence upstream of the gene start codon (Table 1). In upstream primer F, the first 15 bases TGCCTGCAGGTCGAC represent the homologous recombination arm on the p1300GN vector, and the subsequent TCTAGA represents... Xba I restriction site, the remaining sequence is SlPOD Upstream primer sequences for promoter amplification; in downstream primer R, the first 15 bases GGACTGACCACCCGG represent the homologous recombination arm on the p1300GN vector, followed by GGATCC... Bam HI restriction site, the remaining sequence is SlPOD Amplification primer sequences downstream of the promoter.

[0047] The genomic DNA of tomato variety 'Micro-Tom' was extracted by CTAB method, and used as a template. The reaction system was as follows: 5 μL 10×buffer, 2 μL dNTPs, 2 μL DNA template, 1 μL DMSO, 1.5 μL upstream primer F and downstream primer R, 0.5 μL KOD Plus-Neo polymerase, and 36.5 μL ddH2O. The reaction conditions were as follows: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 54℃ annealing for 30 s, 68℃ extension for 1 min, 30 cycles; 68℃ incubation for 10 min. After the PCR, 5 μL reaction solution was taken for agarose gel electrophoresis.

[0048] (4) SlPOD Construction of promoter plant expression vector Use Xba I, Bam HI double enzyme digestion p1300GN vector and purification of vector skeleton. The double enzyme digestion system was as follows: Xba I 2 μL, Bam HI 2 μL, p1300GN plasmid 20 μL, FastDigest green buffer 4 μL, and ddH2O 12 μL. 37℃ enzyme digestion for 1 h, and the enzyme digestion product was purified according to the DNA gel recovery kit instruction after agarose gel electrophoresis. The 2×EasyGeno Single Assembly Mix (Tiangen) was used for homologous recombination SlPOD The promoter fragment and the recovered vector skeleton were used to construct the plant expression vector p1300GN-pSlPOD for Arabidopsis genetic transformation Figure 1 ). The recombination system was as follows: vector skeleton 2.5 μL, purified PCR fragment 2.5 μL, and 2×EasyGeno Assembly Mix 5 μL, 50℃ water bath for 30 min. Then, the TOP10 strain was transformed, and was inoculated on kanamycin-containing LB medium for culture, and then colony PCR amplification was carried out. The PCR amplification reaction system was as follows: 1 μL SlPOD-proF, 1 μL SlPOD-proR, 10 μL 2×A8 FastHiFi PCR MasterMix (Aidley, PC8201), single colony, and ddH2O 8 μL. The PCR reaction conditions were as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 2 min, 35 cycles; 72℃ incubation for 5 min.

[0049] The plasmid DNA of the clone with 2000 bp band amplified by PCR was extracted, Xba I, Bam HI double enzyme digestion verification and sequencing results correct recombinant named p1300GN-pSlPOD.

[0050] (5) Genetic transformation of Arabidopsis thaliana and detection of positive plants The p1300GN-pSlPOD and p1300GN control vectors (containing) were prepared using a freeze-thaw method. 35S The starter, which initiates the process. GUS Gene expression was introduced into Agrobacterium GV3101. The cells were cultured overnight at 28℃ and 200 r / min, and positive Agrobacterium single colonies were screened by colony PCR. The cells were resuspended in 1 / 2 MS liquid medium containing 5% sucrose and 0.05% Silwet L-77. The flower-dipping method was used to infect Arabidopsis thaliana plants with decapitated and open flower buds removed. The harvested T0 generation seeds were treated with 30 mg·L⁻¹... -1 T1 generation seedlings were obtained through hygromycin screening. Genomic DNA was extracted from positive seedlings and used... Hyg PCR detection was performed using primers Hyg-F / R (Table 1). The PCR amplification reaction system was as follows: 1 μL template, 0.5 μL primer Hyg-F, 0.5 μL primer Hyg-R, 10 μL 2×A8 FastHiFi PCRMasterMix (Adley, PC8201), and 8 μL ddH2O.

[0051] A fragment of approximately 917 bp was obtained and tested positive.

[0052] (6) Histochemical staining analysis of transgenic plants using GUS staining Wild-type, p1300GN-transformed, and p1300GN-pSlPOD-transformed Arabidopsis thaliana seedlings were immersed in 500 μL of GUS staining solution (GUS staining kit (SL7160-2) was Coolaber product) and stained at 37°C for 3 h. After removing the staining solution, the seedlings were destained with 70% ethanol until they turned white, and then scanned and photographed using a SteREO Discovery V.12 stereomicroscope.

[0053] II. Results and Analysis 1. SlPOD Gene expression pattern analysis Real-time PCR analysis SlPOD The expression levels of the gene in various parts of tomato, including roots, stems, leaves, flowers, green-ripe stage, and fruit at the color-breaking stage, showed that... SlPOD Gene-specific expression in tomato roots ( Figure 2 ).

[0054] 2. SlPOD Gene promoter cloning and expression vector construction Using tomato 'Micro-Tom' leaf genomic DNA as a template, PCR amplification was performed, yielding a 2.0 kb target band. Figure 3(A) Xba I, Bam The p1300GN vector was digested with HI double enzyme and the vector backbone was purified. The PCR results were then processed. SlPOD The fragment was homologously recombinated with the purified vector backbone and transformed into TOP10. Colony cloning plasmids that tested positive by PCR were extracted. The recombinant, verified by double enzyme digestion and sequencing, was named p1300GN-pSlPOD. Figure 3 (B)

[0055] Structural description of the recombinant expression vector p1300GN-pSlPOD: The fragment shown in SEQ ID No. 1 was inserted into the restriction enzyme site of the p1300GN vector via homologous recombination. Xba I and Bam The recombinant plasmid obtained after HI was analyzed. SEQ ID No. 1 is... SlPOD Promoter sequence.

[0056] 3. Genetic transformation of Arabidopsis thaliana and acquisition of positive plants The p1300GN-pSlPOD expression vector and the control vector p1300GN were transformed into GV3101 cells, which were then infected with Arabidopsis thaliana. T0 generation seeds were collected, and positive seedlings were obtained after selection culture with hygromycin. Genomic DNA was extracted from their leaves and used... Hyg PCR amplification was performed using primers, yielding a fragment of approximately 917 bp (consistent with the expected size), confirming the formation of transgenic Arabidopsis thaliana. Figure 4 ).

[0057] 4. Detection of GUS activity in transgenic Arabidopsis thaliana Wild-type, p1300GN-transformed, and p1300GN-pSlPOD-transformed Arabidopsis thaliana seedlings were selected for GUS histochemical staining. The results showed that wild-type Arabidopsis thaliana plants did not show a blue stain, while p1300GN-transformed Arabidopsis thaliana plants showed a blue stain, and the roots of p1300GN-pSlPOD-transformed Arabidopsis thaliana plants showed a blue stain. GUS Gene expression is strong and appears blue, while it is not expressed in the above-ground parts and appears colorless. Figure 5 (The blue area against the white background in the image represents the GUS expression site). This result indicates that the pSlPOD promoter (SEQ ID No. 1) drives the downstream gene to be specifically expressed in roots, but not in other tissues and organs; that is, this promoter is a root-specific promoter. This invention provides a key promoter resource for precisely improving tomato quality and enhancing rhizosphere stress resistance through genetic engineering.

[0058] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the application.

Claims

1. A DNA molecule, being any one of: (Al) a DNA molecule as set forth in SEQ ID No. 1; (A2) a DNA molecule having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the nucleotide sequence as defined in (Al) and having a promoter function; (A3) a DNA molecule capable of hybridizing to the nucleotide sequence as defined in (Al) or (A2) under stringent conditions and having a promoter function.

2. An expression cassette or a recombinant vector or a recombinant bacterium or a transgenic plant cell line or a transgenic plant tissue or a transgenic plant organ comprising the DNA molecule of claim 1. The expression cassette consists of the DNA molecule having a promoter function, a gene of interest expressed under the control of the promoter, and a transcription termination sequence; the DNA molecule is functionally linked to the gene of interest, and the gene of interest is linked to the transcription termination sequence.

4. Use of the DNA molecule of claim 1 in initiating expression of a gene of interest. The initiation of expression of the gene of interest is initiation of expression of the gene of interest in a plant.

3. The expression cassette of claim 2, wherein: The expression is root-specific expression.

7. A method for specifically expressing a gene of interest in the roots of a plant, comprising the step of: introducing the expression cassette or the recombinant vector of claim 2 or 3 carrying the gene of interest into a plant, thereby achieving specific expression of the gene of interest in the roots of the plant.

5. Use according to claim 4, characterized in that: The plant is any one of: (Bl) a dicotyledonous plant; (B2) a plant of the Solanaceae family or a plant of the Brassicaceae family; (B3) tomato or Arabidopsis.

6. Use according to claim 5, characterized in that:

9. A primer pair for amplifying the full length or a partial fragment of the DNA molecule of claim 1. The primer pair is a primer pair consisting of two single-stranded DNAs as set forth in SEQ ID No. 2 and SEQ ID No.

3.

8. Use or method according to any one of claims 5 to 7, characterized in that: ​ ​ ​ ​ ​ 10. The primer pair according to claim 9, characterized in that: ​

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