A leaf vein-specific promoter, expression vector and application thereof

Abstract

This invention discloses a promoter specifically expressed in leaf veins, its expression vector, and its applications. Belonging to the fields of genetic engineering and biotechnology, this invention uses thermal asymmetric PCR and chromosome walking technology to clone a full-length 1772 bp promoter sequence from *Pteris vittata*. This promoter replaces the CaMV35S promoter on the vector pCAMBIA1381Z, thereby driving the expression of the downstream GUS reporter gene. The recombinant plant expression vector containing this specific expression promoter is transformed into tobacco to verify its function, confirming that this promoter can drive the GUS gene in tobacco leaves. This invention utilizes this promoter to construct a plant expression vector driving the expression of the *Pteris vittata* arsenite reverse transport protein PvACR3. Transforming this vector into tobacco confirms that this specific promoter driving PvACR3 expression can specifically increase the arsenic content in tobacco leaves. The promoter of this invention can be used to cultivate transgenic arsenic-accumulating engineered plants, promoting the development of phytoremediation technology.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and biotechnology, and relates to a promoter specifically expressed in aboveground tissues, its expression vector, and its applications; specifically, it relates to a promoter specifically expressed in leaf veins, its expression vector, and its applications. Background Technology

[0002] A promoter is a specific DNA sequence located upstream of the 5' end of a gene. It activates RNA polymerase, enabling it to bind precisely to the template DNA and possessing transcription initiation specificity. Transcription initiation is a crucial stage in gene expression, and a key issue at this stage is the interaction between RNA polymerase and the promoter. The promoter structure affects its affinity for RNA polymerase, thus influencing the level of gene expression. Specific gene expression in different tissues may imply entirely different functions. Promoters significantly influence, and even determine, gene function. Based on their mode of action and function, promoters can be classified into three main types: constitutive promoters, tissue- or organ-specific promoters, and inducible promoters. Currently, constitutive promoters are commonly used in plant transgenic engineering. For example, the cauliflower mosaic virus promoter (CaMV35S) is highly utilized. It drives gene expression indiscriminately in all plant tissues and organs, inevitably leading to a discrepancy between the gene's function and its actual function. Tissue-specific promoters can drive gene expression in specific plant tissues or organs, more accurately identifying gene function and playing a crucial role in regulating the expression of exogenous genes in transgenic plants.

[0003] Centipede grass (Pteris vittata) is the world's first reported arsenic hyperaccumulating fern and has been widely used in the remediation of arsenic-contaminated soils. Research on the key arsenic accumulation gene ACR3 in Centipede grass plays a crucial role in advancing phytoremediation technology. In this experiment, the self-promoter of the ACR3 gene was cloned from Centipede grass, recombined with an expression vector carrying the GUS reporter gene, and transformed into tobacco using Agrobacterium-mediated transformation. PCR and GUS histochemical staining confirmed that the ACR3 self-promoter can drive the expression of the GUS gene in the leaf veins of tobacco. Summary of the Invention

[0004] To address the above problems, the present invention provides an above-ground tissue-specific expression promoter and its expression vector, as well as the uses of the promoter and expression vector.

[0005] The technical solution of this invention is: a promoter specifically expressed in leaf veins and its expression vector, as described in this invention.

[0006] The promoter described is derived from Centipede Grass and is the upstream sequence / promoter of the PvACR3 coding sequence of the Centipede Grass Arsenite Reversible Transport Protein gene, and its nucleotide sequence is shown in SEQ ID No:1;

[0007] Furthermore, the promoter fragment is 1772 bp in size.

[0008] Furthermore, the expression vector is a recombinant vector obtained by inserting the promoter into the multiple cloning site of a plant expression vector.

[0009] Furthermore, the recombinant vector is the vector pCAMBIA1381Z.

[0010] Furthermore, the promoter of this invention can be used in research related to transgenic engineering to cultivate arsenic hyperaccumulating plants.

[0011] The beneficial effects of this invention are as follows: This invention mainly uses TAIL PCR and chromosome walking to amplify the upstream promoter sequence (PvACR3p) of the PvACR3 gene from Centipede Grass; the target fragment is constructed into the multiple cloning site of the target vector through enzyme digestion and ligation, and finally a plant vector is constructed in which the PvACR3 gene's own promoter (PvACR3p) drives the expression of the GUS reporter gene; this invention further involves transforming the recombinant plant expression vector containing this specific expression promoter into tobacco to verify its function, and confirming that the promoter can drive the expression of the GUS gene in the aboveground parts (leaf veins) of tobacco; using this promoter to drive the expression of the PvACR3 gene in tobacco can significantly increase the arsenic content in tobacco leaves. Attached Figure Description

[0012] Figure 1 The vector spectrum of the present invention is shown in (A), which is the pCAMBIA1381Z vector spectrum used, (B) is a schematic diagram of the constructed pCAMBIA1381Z-PvACR3p-GUS vector, and (C) is a schematic diagram of the pCAMBIA1381Z-PvACR3p-PvACR3 vector.

[0013] Figure 2 This is a histochemical staining result of the GUS gene of the present invention; where A and B are leaves, and C and D are roots;

[0014] Figure 3 This is a schematic diagram showing the arsenic content of PvACR3 transgenic and wild-type tobacco after 1 day of exposure to 10 μM AsV (A: root; B: stem; C: leaf; * indicates a significant difference between the PvACR3 transgenic tobacco line and the WT line).

[0015] Figure 4This is a schematic diagram showing the arsenic content of PvACR3 transgenic and wild-type tobacco after 1 day of exposure to 5 μM AsIII (A: root; B: stem; C: leaf; * indicates a significant difference between the PvACR3 transgenic tobacco line and WT).

[0016] Figure 5 This is a schematic diagram showing the arsenic content of PvACR3 transgenic and wild-type tobacco plants after one month of soil cultivation according to the present invention (A: root; B: stem; C: leaf; * indicates that the PvACR3 transgenic tobacco line has a significant difference compared with WT). Detailed Implementation

[0017] The present invention will be further described in detail below with reference to the embodiments. It should be noted that the scope of protection of the present invention is not limited to the following embodiments. These examples are listed for illustrative purposes only and do not limit the present invention in any way.

[0018] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0019] Example 1: Cloning of the self-promoter of the PvACR3 gene from Centipede Grass:

[0020] The plant genomic DNA was extracted using a plant genomic DNA kit from a certain company. 0.1g of tender leaves of Centipede Grass were taken, added to liquid nitrogen and ground thoroughly. The DNA of Centipede Grass was extracted according to the operation steps of the kit. Using Centipede Grass DNA as a template, thermal asymmetric staggered PCR and chromosome walking amplification were performed. The amplification products were detected by agarose gel electrophoresis, thereby cloning the promoter sequence, which was named PvACR3p.

[0021] Example 2: Construction of a promoter-fused GUS plant expression vector:

[0022] The plant expression vector selected was pCAMBIA1381Z. The cloned promoter sequence was ligated to the multiple cloning site of the target vector via restriction enzyme digestion, thus constructing a plant vector for expressing the GUS reporter gene driven by the ACR3 gene self-promoter of *Pteris vittata*, named pCAMBIA1381Z-PvACR3p-GUS. A schematic diagram is shown below. Figure 1 .

[0023] Example 3: Obtaining genetically modified tobacco:

[0024] (1) The constructed plant expression vector was electroporated to obtain recombinant Agrobacterium tumefaciens and cultured overnight at 28°C.

[0025] (2) Take leaves of sterile seedlings of Tobacco Benedict, cut off the edges and main veins, cut the leaves into small squares and put them into the prepared Agrobacterium tumefaciens solution for 10 minutes. Take out the leaves, use sterile filter paper to absorb the bacterial solution on the plant surface, transfer them to MS medium with a layer of sterile filter paper, and incubate in the dark at 28℃ for 3-7 days. Transfer the material to differentiation medium containing antibiotics for culture. When the resistant teeth grow to 2-3 cm, cut them off and transfer them to 1 / 2 MS solid rooting medium to induce rooting. Then transfer them to nutrient soil. After they mature, harvest the seeds. The harvested seeds are the seeds of T0 generation tobacco.

[0026] (3) T0 generation tobacco seeds and wild type (WT) seeds were disinfected with 10% sodium hypochlorite for 10 min, washed 3 times with sterile water, and then sown on 1 / 2 MS medium containing hygromycin (30 mg / L); after the medium was placed in the dark at 4℃ for two days, it was transferred to a light incubator at 25℃ (14 h light, 10 h dark) for 20 days to obtain T1 generation tobacco with hygromycin resistance;

[0027] (4) When the T1 generation tobacco plants are transferred to nutrient soil and grow to 4-6 leaves, the DNA of the tobacco leaves is extracted and PCR identification is performed to obtain T1 generation positive seedlings.

[0028] Example 4: GUS staining analysis:

[0029] Roots and leaves from T1 generation positive seedlings were collected and stained according to the instructions of the GUS staining kit. The staining results are shown in the figure. Figure 2 The leaves, especially the veins, were stained blue. Figure 2 A, B), while the root was not stained ( Figure 2 (C, D). GUS staining results showed that the PvACR3p promoter can drive the expression of the GUS gene in leaf veins.

[0030] Example 5: A specific promoter-driven expression of the PvACR3 gene increases arsenic content in plant leaves, constructing an arsenic-accumulating engineered plant:

[0031] The PvACR3 gene coding sequence (as shown in No:2, 1200bp in size) was constructed into the pCAMBIA1381Z-PvACR3p-GUS vector to replace the GUS fragment. PvACR3 gene expression was driven by the PvACR3p specific promoter, and the vector was transformed into tobacco to obtain T1 generation positive seedlings (method as in Example 3). The arsenic content in the aboveground tissues of tobacco was analyzed using hydroponics and soil culture methods. Specific methods are as follows:

[0032] Determination of arsenic content in hydroponic tobacco: Positive tobacco seedlings with uniform growth were selected and transferred to 1 / 5 Hoagland-Arnon culture medium containing 5 μM AsIII and 10 μM AsV respectively. After hydroponics for 24 h, tobacco samples were collected and the arsenic content in the aboveground tissues of tobacco was determined.

[0033] Determination of arsenic content in soil-grown tobacco: Selected healthy tobacco seedlings with uniform growth were transferred to contaminated soil (arsenic content of 133 ppm) and collected after 30 days of growth. The arsenic content in the aboveground tissues of the tobacco was then determined.

[0034] Determination of total arsenic content in aboveground plant tissues: Inductively coupled plasma mass spectrometry (ICP-MS) was used. 0.1–0.5 g of dried and pulverized plant samples were weighed and digested in a graphite furnace at 105 °C using an HNO3-H2O2 system (10 mL 1:1 (w / v) HNO3 and 2 mL 30% H2O2). An empty digestion tube was used as a blank control during digestion.

[0035] The digestion process is as follows:

[0036] (1) Add 10 mL of 1:1 (w / v) HNO3 to the digestion tube and digest until about 1 mL of digest remains;

[0037] (2) Add 2 mL of 30% H2O2 and continue digestion until the digestion solution is clear and transparent with no bubbles produced and about 1 mL of solution remains. Remove the digestion tube and cool it to room temperature. Make up the volume of the digestion solution to 50 mL with ultrapure water. After dilution, filter the digestion solution through a 0.22 μM filter membrane and dilute it to less than 20 ppb with 0.1 mol / L ultrapure nitric acid (Sigma). After adding the internal standard indium (In) to the arsenic standard solution, blank sample and sample, determine the arsenic content by inductively coupled plasma mass spectrometry (ICP-MS).

[0038] The PvACR3p-specific promoter drives the expression of the PvACR3 gene in tobacco. The measured arsenic content results are shown below. Figure 3-5 After 1 day of exposure to 10 μM AsV, the arsenic content in the leaves of transgenic tobacco was significantly increased compared to wild-type (WT), by 52-71%. Figure 3 C), while the arsenic content in the roots and stems showed no significant difference compared to WT. Figure 3 A, B); After 1 day of exposure to 5 μM AsIII, the arsenic content in transgenic tobacco leaves increased by 50-68% compared to WT. Figure 4 C), the arsenic content in the roots and stems was not significantly different from that in the WT. Figure 4 A, B); After tobacco was grown in soil for one month, the arsenic content in the leaves of transgenic tobacco increased significantly. Figure 5 C), there was no significant difference in arsenic content between the roots and stems. Figure 5 A, B); The above experimental results show that the expression of the PvACR3 gene driven by the PvACR3p specific promoter in tobacco can significantly increase the arsenic content in the aboveground parts of tobacco. This result provides important gene resources and regulatory elements for the cultivation of engineering plants for arsenic pollution remediation.

[0039] SEQ ID No:1PvACR3p promoter sequence

[0040]

[0041] SEQ ID No:2 PvACR3 gene coding sequence (PvACR3 is the arsenite reverse transport protein gene of Centipede Grass, and the promoter described in this invention is the promoter of this gene)

[0042]

[0043] The embodiments described in this invention are only used to illustrate the principles of the embodiments of this invention; other variations are also within the scope of this invention; therefore, as examples rather than limitations, alternative configurations of the embodiments of this invention can be regarded as consistent with the teachings of this invention; correspondingly, the embodiments of this invention are not limited to the embodiments explicitly described and presented in this invention.

Claims

1. A promoter that is specifically expressed in leaf veins, characterized in that, The promoter is from Pteris vittata, and is the promoter of Pteris vittata arsenite antiporter gene PvACR3, and the nucleotide sequence is shown as SEQ ID No:

1.

2. The leaf vein-specific promoter according to claim 1, wherein The fragment size of the promoter is 1772 bp.

3. An expression vector, characterized by, The expression vector comprises a recombinant vector obtained by inserting the promoter in the multiple cloning site of a plant expression vector.

4. Use of the promoter according to any of claims 1-2 in transgenic engineering for cultivating arsenic hyperaccumulating tobacco.

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