Promoters with specific driving action and their use

Abstract

The application discloses a promoter with specific driving action and application thereof, and relates to the technical field of genetic engineering.The nucleotide sequence of the promoter provided by the application is shown in any one of SEQ ID NO.1-8.Eight novel tissue-specific promoters from Glycyrrhiza uralensis are successfully obtained, the promoters have clear and stable expression patterns and high driving activity, and can realize precise and directional expression of target genes in plant roots, stems, leaves and flower organs, and cover various types of multi-tissue coordinated expression.The promoters of the application are all endogenous sequences of plants, have stable driving activity and strong species adaptability, can construct specific expression cassettes and recombinant vectors, and are suitable for transformation of plants such as Arabidopsis thaliana and tobacco.The application enriches the resources of plant-specific promoters, solves the problems of single expression profile and insufficient precision of traditional promoters, and provides efficient, safe and diversified regulation tools for plant functional gene research, molecular breeding and improvement of medicinal plant traits.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to promoters with specific driving effects and their applications. Background Technology

[0002] Precise spatiotemporal regulation of plant gene expression is a core technological aspect of modern molecular breeding and synthetic biology. Tissue-specific promoters, as key regulatory elements, enable the targeted expression of target genes in specific organs, tissues, or cell types, avoiding the energy waste, metabolic burden, and trait interference caused by constitutive promoter-driven gene expression. This significantly improves the targeting and safety of genetic engineering. In fields such as crop quality improvement, stress resistance breeding, secondary metabolism regulation of medicinal plants, creation of male-sterile lines, and floral organ development research, promoters with clear tissue specificity have become a scarce and crucial resource.

[0003] In current plant genetic engineering applications, commonly used constitutive promoters such as CaMV 35S, Ubquitin, and Actin, while exhibiting high expression efficiency, lack spatiotemporal selectivity. This can lead to the continuous expression of exogenous genes throughout all tissues and growth stages, causing problems such as abnormal plant growth, increased resistance costs, and biosafety controversies, making it difficult to meet the needs of precise regulation. As research deepens, the market demand for specific promoters that enable the expression of genes in specific tissues or multiple tissues in synergistic ways, such as roots, stems, leaves, flowers, and fruits, is becoming increasingly urgent. Especially in scenarios involving the joint regulation of vegetative and reproductive organs, promoters that can stably drive gene expression specifically in stems, leaves, and flowers, and can finely distinguish the structure of flower parts, have significant application value for elucidating flower development mechanisms, regulating flowering time, improving plant morphology and photosynthetic efficiency, and targeted enrichment of bioactive components.

[0004] Medicinal plants such as Ural licorice possess both ecological and economic value, but the development of their genetic resources is relatively lagging. This invention aims to discover novel endogenous promoters in Ural licorice and establish a toolkit for targeted expression covering stems, leaves, floral organs, and different floral tissues. This is of great significance for promoting molecular breeding, functional gene research, and synthetic biology applications in medicinal plants. Summary of the Invention

[0005] The purpose of this invention is to provide promoters with specific driving effects and their applications to solve the problems existing in the prior art. The promoters provided by this invention have clear and stable expression patterns and efficient driving activities, enabling precise and targeted expression of target genes in plant roots, stems, leaves, and floral organs, covering multiple multi-tissue co-expression types.

[0006] To achieve the above objectives, the present invention provides the following solution: The present invention provides a promoter with specific driving function, the nucleotide sequence of which is shown in any one of SEQ ID NO.1-8.

[0007] The present invention also provides a gene expression cassette, including the promoter described above.

[0008] The present invention also provides a recombinant expression vector comprising the gene expression cassette described above.

[0009] The present invention also provides a recombinant host cell comprising the above-described recombinant expression vector.

[0010] The present invention also provides the application of a promoter with a nucleotide sequence as shown in SEQ ID NO.2 in driving the expression of a target gene in plants, wherein the expression is specific in roots, stems and leaves and other floral tissues except for the stigma.

[0011] The present invention also provides the application of a promoter with a nucleotide sequence as shown in SEQ ID NO.4 in driving the expression of a target gene in plants, wherein the expression is specific in stems, leaves and flowers.

[0012] The present invention also provides the application of a promoter with a nucleotide sequence as shown in SEQ ID NO.5 in driving the expression of a target gene in plants, wherein the expression is specific in root tissues other than the taproot tip and lateral roots, stems and leaves, and stigmas and filaments of flowers.

[0013] The present invention also provides the application of a promoter with a nucleotide sequence as shown in SEQ ID NO.6 in driving the expression of a target gene in plants, wherein the expression is specific in true leaves and in the stigma and anther of flowers.

[0014] The present invention also provides the application of a promoter with a nucleotide sequence as shown in SEQ ID NO.7 in driving the expression of a target gene in plants, wherein the expression is specific in the primary root, stem and leaves, and stigma and anther of the flower.

[0015] The present invention also provides the application of a promoter with a nucleotide sequence as shown in SEQ ID NO.8 in driving the expression of a target gene in plants, wherein the expression is specific in the leaf tip and the stigma of the flower.

[0016] The present invention discloses the following technical effects: This invention successfully obtained eight novel tissue-specific promoters derived from Ural licorice, possessing clear and stable expression patterns and highly efficient driving activities. These promoters enable precise targeted expression of target genes in plant roots, stems, leaves, and floral organs, covering various multi-tissue co-expression types. All promoters in this invention are endogenous plant sequences, exhibiting stable driving activities and strong species adaptability. They can be used to construct specific expression cassettes and recombinant vectors, suitable for transformation in plants such as Arabidopsis thaliana and tobacco. This invention enriches plant-specific promoter resources and solves the problems of limited expression profiles and insufficient precision of traditional promoters, providing efficient, safe, and diverse regulatory tools for plant functional gene research, molecular breeding, and medicinal plant trait improvement. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a PCR electrophoresis image of the GuCPKs promoters; where M represents a 2000 bp marker; GuCPK1, GuCPK5, GuCPK7, GuCPK8, GuCPK10, GuCPK12, GuCPK13, and GuCPK16 represent the promoters pGuCPK1, pGuCPK5, pGuCPK7, pGuCPK8, pGuCPK10, pGuCPK12, pGuCPK13, and pGuCPK16, respectively. Figure 2 The agarose gel electrophoresis diagram of the PCR products in pGuCPK1::GUS is shown; where M represents a 2000 bp marker; 1-10 represent pGuCPK1::GUS. Figure 3 GUS staining image of Arabidopsis seedlings overexpressing pGuCPKs::GUS; where GuCPK1, GuCPK5, GuCPK7, GuCPK8, GuCPK10, GuCPK12, GuCPK13, and GuCPK16 represent the plant expression vectors pGuCPK1::GUS, pGuCPK5::GUS, pGuCPK7::GUS, pGuCPK8::GUS, pGuCPK10::GUS, pGuCPK12::GUS, pGuCPK13::GUS, and pGuCPK16::GUS driven by the pGuCPK promoter, respectively; scale bar is 1 mm. Figure 4GUS staining of flowers of Arabidopsis thaliana overexpressing pGuCPKs::GUS; where GuCPK1, GuCPK5, GuCPK7, GuCPK8, GuCPK10, GuCPK12, GuCPK13, and GuCPK16 represent the plant expression vectors pGuCPK1::GUS, pGuCPK5::GUS, pGuCPK7::GUS, pGuCPK8::GUS, pGuCPK10::GUS, pGuCPK12::GUS, pGuCPK13::GUS, and pGuCPK16::GUS driven by the pGuCPK promoter, respectively; the scale bar is 1 mm. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] The promoter sequence involved in this invention is as follows: The nucleotide sequences of promoters pGuCPK1, pGuCPK5, pGuCPK7, pGuCPK8, pGuCPK10, pGuCPK12, pGuCPK13 and pGuCPK16 are shown in SEQ ID NO.1-8, respectively.

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[0033] Example 1 1. Experimental Methods 1.1 Promoter Cloning DNA was extracted from *Glycyrrhiza uralensis* seedlings using a DNA extraction kit. Promoter sequences were cloned using *Glycyrrhiza uralensis* genomic DNA as a template; the promoter primers are shown in Table 1.

[0034] Table 1 Promoter primers 1.2 Connection and conversion of recovered products with pMD19-T After the PCR reaction was completed, the products were separated by agarose gel electrophoresis, and the target DNA fragment was purified using a DNA gel recovery kit. Subsequently, the purified PCR product was ligated into the pMD19-T vector (purchased from Takara Bio Inc.). Following the operating procedures of the pMD19-T Vector Kit, the target fragment was inserted into the vector, mixed thoroughly, and reacted at 16°C for 12 h.

[0035] 1.3 Identification, extraction and sequencing analysis of recombinant plasmids Competent *E. coli* cells were prepared using the CaCl2 method. The transformed *E. coli* cells were first inoculated onto LB agar plates containing the corresponding antibiotics and cultured overnight. Single colonies were then picked from the plates for subsequent colony PCR analysis. Agarose gel electrophoresis was used to identify positive strains. Positive colonies were then picked and placed in liquid LB agar containing the corresponding antibiotics, and cultured at 37°C and 200 rpm for 12 hours. Plasmids were then extracted using a kit and sent to Shanghai Sangon Biotech for sequencing. The sequencing results were assembled using MEGA11 and analyzed using DNAMAN software to obtain the full-length sequence.

[0036] 1.4 Construction of pGuCPKs::GUS plant expression vector This method uses overlap extension PCR (OE-PCR). Using a recombinant plasmid verified by sequencing as a template, PCR amplification was performed using primers containing the homologous recombination arm of pCAMBIA1301. After amplification, the gel-recovered product was ligated to the linearized pCAMBIA1301 vector and incubated overnight at 16°C. The ligation product was then transformed using DH5α competent cells. An appropriate amount of the transformation product was evenly spread onto the surface of an LB agar plate supplemented with kanamycin. The plate was then inverted and incubated overnight at 37°C.

[0037] Five single-clone colonies were randomly selected and inoculated into LB liquid medium containing kanamycin, and cultured at 37°C with shaking for 8–10 h. Subsequently, the bacterial cultures were identified by PCR, and the correctly identified cultures were mixed with 50% glycerol and sent to Shanghai Sangon Biotech for sequencing analysis.

[0038] 1.5 Transformation of Agrobacterium with plant expression vector After expanding the recombinant bacterial culture with correct sequencing, the recombinant plasmid was extracted using a plasmid miniprep kit and detected by agarose gel electrophoresis. The recombinant plasmid was transformed into Agrobacterium GV3101 competent cells, and the transformation product was plated on LB solid medium containing rifampin, gentamicin, and kanamycin, and incubated at 28°C for 48–72 h. Five single colonies were randomly selected and inoculated into LB liquid medium containing triple antibodies (50 μg / mL rifampin, 50 μg / mL gentamicin, and 50 μg / mL kanamycin), and cultured at 28°C for 16–24 h. Subsequently, bacterial culture was identified by PCR, and correctly identified bacterial cultures were stored in a mixture with 50% glycerol.

[0039] 1.6 Flower Drop Infection of Arabidopsis thaliana Take a small amount of wild-type Arabidopsis seeds into a 1.5 mL EP tube, wash 5-7 times with sterile distilled water in a clean bench, disinfect with 75% alcohol for 3 min, wash again with sterile distilled water 5-7 times, then disinfect with 10% sodium hypochlorite for 3 min, and finally wash 5-7 times with sterile distilled water. Sow the seeds on 1 / 2 MS solid medium plates and vernalize at 4℃ for 2-3 days. Then transfer them to a 20℃ light incubator and culture for about 10 days under 16 h light and 8 h darkness conditions. After the seedlings have developed 3-4 rosette leaves, transplant them into potting soil (nutrient soil:vermiculite:perlite = 3:1:1) and continue to culture under the same light conditions.

[0040] One week after the first flowering of Arabidopsis thaliana, when it reaches its peak flowering period, remove the existing siliques. Pick a single colony from an Agrobacterium plate carrying the recombinant plasmid and inoculate it into 3 mL of LB broth containing the appropriate antibiotic. Incubate at 28°C with shaking at 200 rpm. Transfer 30 μL of the culture to 30 mL of LB broth with the same formulation and continue culturing until the OD of the bacterial culture reaches its maximum. 600 The bacterial culture concentration was set at 0.6-0.8. The cells were collected by centrifugation, and the supernatant was discarded. The cells were then resuspended in a mixed solution containing 5% sucrose and 0.02% Silwet L-77. The bacterial suspension was added dropwise to Arabidopsis inflorescences and incubated in the dark for 24 hours before being transferred to normal light conditions. Infections were performed every 5-7 days for a total of three infections. Once the plants matured, the T0 generation seeds were harvested for subsequent experiments.

[0041] 1.7 Preliminary screening and DNA identification of transgenic Arabidopsis thaliana The harvested T0 generation seeds were sown on 1 / 2 MS medium containing kanamycin (Kan) or hygromycin (Hyg), and these Arabidopsis thaliana seedlings at this stage were designated as the T1 generation. Once the seedlings had developed four green true leaves and were growing normally, genomic DNA was extracted from leaves of 3-week-old wild-type and T1 generation transgenic Arabidopsis thaliana strains using standard molecular biology methods. The extracted DNA was used as a template for PCR detection. T1 generation seeds were harvested from Arabidopsis thaliana seedlings that tested positive for PCR.

[0042] 1.8 GUS histochemical detection of transgenic Arabidopsis thaliana Seeds of each T1 generation line that tested positive for DNA were sown in 1 / 2 MS medium containing hygromycin. GUS Gene expression levels were analyzed using quantitative real-time analysis (upstream primer sequence 5'-ACGGGGAAACTCAGCAAGC-3', SEQ ID NO.25; downstream primer sequence 5'-TGAGCGTCGCAGAACATTACAT-3', SEQ ID NO.26). Lines with high expression levels were selected for transplantation, and the harvested seeds were designated as the T2 generation. T2 generation seeds were sown in 1 / 2 MS medium containing hygromycin. For each promoter expression vector, a stable transgenic Arabidopsis thaliana expression line was selected. 15-day-old seedlings, flowering flowers, and mature siliques were soaked in GUS staining solution at a temperature maintained between 25-37°C for 1 hour to overnight. If the material consisted of green leaves, for easier observation, the samples could be destained in 70% ethanol, repeated 2-3 times until the negative control material was completely destained and turned white. Subsequently, the blue spots appearing against a white background, observed visually or under a microscope, indicated the specific locations of GUS reporter gene expression.

[0043] 2. Experimental Results and Analysis 2.1 Cloning of the GuCPKs promoter This invention uses genomic DNA from *Glycyrrhiza uralensis* as a template to amplify specific bands: pGuCPK1 (1997 bp), pGuCPK5 (1988 bp), pGuCPK7 (1998 bp), pGuCPK8 (2001 bp), pGuCPK10 (2005 bp), pGuCPK12 (2074 bp), pGuCPK13 (1982 bp), and pGuCPK16 (2007 bp). These bands were sequenced, and the sequencing results were compared with known sequences to confirm that this fragment is the GuCPK promoter sequence. Figure 1 ).

[0044] 2.2 Analysis of the expression pattern of GUS reporter genes driven by GuCPKs promoter This experiment constructed a plant expression vector pGuCPKs::GUS (GUS) driven by the GuCPKs promoter to express the GUS reporter gene. Figure 2 ), and then transferred to Agrobacterium GV3101.

[0045] The expression patterns of genes regulated by the GuCPKs promoter were investigated using GUS histochemical staining. T3 generation transgenic Arabidopsis plants containing the promoter fragment were planted on 1 / 2 MS medium. Untransformed and transformed seedlings grown for 15 days were subjected to GUS histochemical staining and observed. The results showed that no stained tissue was detected in untransformed wild-type Arabidopsis plants. In transgenic Arabidopsis containing the promoter fragment, GUS expression regulated by all eight promoters was high, but the expression differed significantly between roots and leaves. (See Table 2). Figure 3 As shown, when using the pGuCPK1 promoter, GUS is expressed in roots, stems, and leaves, with low expression levels in the primary root tip and lateral roots. When using the pGuCPK5 promoter, GUS is expressed in roots, stems, and leaves, with high expression levels in the root tip. When using the pGuCPK7 promoter, GUS is expressed in roots, stems, and leaves. When using the pGuCPK8 promoter, GUS is highly expressed in stems and leaves, but not in roots. When using the pGuCPK10 promoter, GUS is expressed in roots, stems, and leaves, but not in the primary root tip or lateral roots, and has high expression levels in the primary root. When using the pGuCPK12 promoter, GUS is expressed only in true leaves. When using the pGuCPK13 promoter, GUS is expressed in roots, stems, and leaves, with high expression levels in the primary root and no expression in lateral roots. When using the pGuCPK16 promoter, GUS is expressed only in leaf tips.

[0046] Afterwards, the seedlings were transplanted into the substrate for further culture. Later, flowers and siliques were collected for GUS histochemical staining. GUS was not expressed in the siliques. The eight promoters regulated GUS expression differently in flowers (Table 2 and...). Figure 4When using the pGuCPK1 promoter, GUS expression is high in the flower. When using the pGuCPK5 promoter, GUS is not expressed in the stigma, but is highly expressed in other parts. When using the pGuCPK7 promoter, GUS expression is high in the flower. When using the pGuCPK8 promoter, GUS expression is high in the stigma, low in the anther, and not expressed in the style, filament, or petals. When using the pGuCPK10 promoter, GUS expression is high in the stigma, low in the filament, and not expressed in the style, anther, or petals. When using the pGuCPK12 promoter, GUS expression is high in the stigma, low in the anther, and not expressed in the style, filament, or petals. When using the pGuCPK13 promoter, GUS expression is high in the stigma, low in the anther, and not expressed in the style, filament, or petals. When the pGuCPK16 promoter is used, GUS is highly expressed in the stigma, but not in the anther, style, filament, or petals.

[0047] Table 2. Expression of GUS driven by the GuCPKs promoter in Arabidopsis thaliana. The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A promoter with specific driving effect, characterized in that, The nucleotide sequence of the promoter is shown in any one of SEQ ID NO.1-8.

2. A gene expression cassette, characterized in that, Includes the promoter described in claim 1.

3. A recombinant expression vector, characterized in that, Includes the gene expression cassette as described in claim 2.

4. A recombinant host cell, characterized in that, Includes the recombinant expression vector as described in claim 3.

5. The application of a promoter with the nucleotide sequence shown in SEQ ID NO.2 in driving the expression of a target gene in plants, characterized in that, The expression is specific in roots, stems, leaves, and other floral tissues except for the stigma.

6. The application of a promoter with the nucleotide sequence shown in SEQ ID NO.4 in driving the expression of a target gene in plants, characterized in that, The expression is specific to stems, leaves, and flowers.

7. The application of a promoter with the nucleotide sequence shown in SEQ ID NO.5 in driving the expression of a target gene in plants, characterized in that, The expression is specifically expressed in root tissues other than the taproot tip and lateral roots, stems and leaves, and the stigma and filaments of flowers.

8. The application of a promoter with the nucleotide sequence shown in SEQ ID NO. 6 in driving the expression of a target gene in plants, characterized in that, The expression is specifically expressed in true leaves, as well as in the stigma and anthers of flowers.

9. The application of a promoter with the nucleotide sequence shown in SEQ ID NO.7 in driving the expression of a target gene in plants, characterized in that, The expression is specifically expressed in the taproot, stems and leaves, and stigmas and anthers of the flowers.

10. The application of a promoter with the nucleotide sequence shown in SEQ ID NO. 8 in driving the expression of a target gene in plants, characterized in that, The expression is specifically expressed in the leaf tip and the stigma of the flower.

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