A plant constitutive expression promoter and its application

Abstract

The present application relates to the field of genetic engineering, in particular to a plant constitutive expression promoter and application thereof. The plant constitutive expression promoter provided by the present application comprises a DNA sequence as shown in SEQ ID NO. 1. Through DNA manipulation and / or recombination in vivo or in vitro, the plant constitutive expression promoter provided by the present application can drive the expression of a target gene in various tissues of a plant after being introduced into the plant, and the expression activity is equivalent to that of a commonly used constitutive promoter CaMV 35S. The plant constitutive expression promoter can be applied to plant genetic engineering improvement, such as stress resistance and quality improvement, and provides an efficient regulatory element for plant molecular breeding, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to a plant constitutive expression promoter and its applications. Background Technology

[0002] Plant genetic engineering technology, as a core branch of modern biotechnology, has become a key means for crop genetic improvement, biological sex transformation regulation, and molecular fields. Its core is to achieve precise improvement of plant quality by directionally introducing exogenous functional genes.

[0003] In plant gene expression systems, promoters are generally classified into three categories based on their expression patterns: constitutive promoters, inducible promoters, and tissue-specific promoters. Constitutive promoters can drive the continuous and stable expression of target genes throughout the entire growth period of plants and in all tissues and organs, without relying on external environmental signals for induction or activation by tissue-specific regulatory factors. Therefore, they have irreplaceable applications in plant genetic engineering.

[0004] In stress resistance gene engineering, constitutive promoters are used to drive the sustained expression of genes for insect resistance, drought resistance, salt resistance, and herbicide resistance in various plant tissues, forming a plant's stress resistance protection mechanism. In quality improvement engineering, constitutive expression promoters drive nutrient synthesis-related genes, enabling the efficient synthesis of downstream products in the plant and improving crop nutritional quality.

[0005] Currently, the most widely used constitutive promoters in plant genetic engineering are the CaMV 35S promoter and the ubiquitin promoter, both derived from Cauliflower Mosaic Virus (CaMV). The CaMV 35S promoter, in particular, possesses strong transcriptional driving capabilities and is widely used in the construction of gene expression vectors for dicotyledonous plants and some monocotyledonous plants. However, because this promoter is derived from a virus, its long-term stability in the rice genome has not been verified, thus affecting its application effectiveness. While the Ubiquitin promoter can be expressed in various rice tissues, its expression level is significantly influenced by the growth and development stage, potentially affecting the optimal expression of the gene at specific growth and developmental stages.

[0006] Therefore, discovering a constitutive expression promoter derived from rice that can be efficiently and stably expressed in all rice tissues is of great application value for breaking through the current bottlenecks in rice genetic engineering technology, promoting the molecular breeding process and commercial application of transgenic rice. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a plant constitutive expression promoter and its applications. After being introduced into a plant through DNA manipulation and / or recombination in or outside the plant, this plant constitutive expression promoter can regulate the strong expression of genes in various tissues of the plant. This promoter is derived from the natural plant genome and has efficient and stable transcriptional activity, making it suitable for various species and tissues.

[0008] Therefore, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides, in optional embodiments, a plant constitutive expression promoter comprising the DNA sequence shown in SEQ ID NO.1. In this invention, it is denoted as PosHA1 or promoter PosHA1.

[0010] Preferably, the DNA sequence of the plant constitutive expression promoter has at least 80% homology with the DNA sequence shown in SEQ ID NO.1; or, the plant constitutive expression promoter is a mutant, allele, or derivative generated by adding, substituting, inserting, or deleting one or more nucleotides in the DNA sequence shown in SEQ ID NO.1; or, the plant constitutive expression promoter has a product that hybridizes with the DNA sequence shown in SEQ ID NO.1.

[0011] In this invention, these plant constitutive promoters have the same function as the DNA sequence shown in SEQ ID NO.1, namely, driving the expression of target genes in plants.

[0012] Secondly, in an optional embodiment, the present invention provides an expression cassette containing the above-mentioned plant constitutive expression promoter, which is used to introduce the gene into the plant via in vivo or in vitro DNA manipulation techniques to control the expression intensity and spatiotemporal expression characteristics of the driven gene.

[0013] Thirdly, in optional embodiments, the present invention provides a recombinant expression vector comprising the aforementioned plant constitutive expression promoter; in the recombinant expression vector, the plant constitutive expression promoter is linked upstream of the gene sequence to be expressed in the vector. Preferably, the gene to be expressed is the GUS gene, the vector is pCAMBIA1305, and the recombinant expression vector is obtained by constructing the promoter PosHA1 in pCAMBIA1305 and linking the promoter PosHA1 upstream of the GUS gene, denoted as pCAMBIA1305-PosHA1.

[0014] Fourthly, in optional embodiments, the present invention provides a host bacterium comprising the aforementioned plant constitutive expression promoter, the aforementioned expression cassette, or the aforementioned recombinant expression vector. Preferably, the host bacterium is Agrobacterium tumefaciens.

[0015] Fifthly, in optional embodiments, the present invention provides a transformant comprising the above-described plant constitutive expression promoter, the above-described expression cassette, the above-described recombinant expression vector, or the above-described host bacteria. The transformant is preferably a transgenic cell, callus tissue, or plant.

[0016] Sixthly, in an optional embodiment, the present invention provides an application of the above-mentioned plant constitutive expression promoter in the cultivation of genetically engineered plants. The application includes: linking or recombining the above-mentioned plant constitutive expression promoter upstream of a gene sequence to be expressed, thereby driving the expression of the target gene in the plant. The plant is a monocotyledonous plant, including rice, wheat, corn, barley, sorghum, or oats, preferably rice.

[0017] Furthermore, the DNA sequence of the plant constitutive expression promoter provided by this invention can be linked to a plant binary expression vector to replace the constitutive promoter. Moreover, the DNA sequence of this plant constitutive expression promoter can be linked to a desired target gene to construct a recombinant plant expression vector. After transformation, this constitutive promoter can drive the expression of the target gene in the plant.

[0018] The nucleotide sequence shown in SEQ ID NO.1 is as follows:

[0019]

[0020] Compared with the prior art, the present invention has one of the following beneficial effects:

[0021] 1. After being introduced into a plant through DNA manipulation and / or recombination in or outside the plant, the plant constitutive expression promoter provided by this invention can regulate the strong expression of genes in various tissues of the plant. This promoter is derived from the natural plant genome and has efficient and stable transcriptional activity, making it suitable for a variety of species and tissues. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the construction of the promoter PosHA1 in the pCAMBIA1305 vector plasmid in Examples 1-2 of the present invention, wherein... Figure 1 A is a gel electrophoresis image of promoter PosHA1 amplification. Figure 1 Image B is a gel electrophoresis result of pCAMBIA1305-PosHA1 double enzyme digestion. Figure 1 C is a schematic diagram of the pCAMBIA1305-PosHA1 vector, showing the expression of the Gus gene located downstream of it driven by the PosHA1 promoter;

[0023] Figure 2 This is a schematic diagram showing the Gus staining results of different parts of the transgenic rice plant in Example 3 of this invention, where the PosHA1 promoter drives Gus gene expression. The Gus gene is present in the roots of the transgenic plant (…). Figure 2 A), Leaf ( Figure 2 B), stem ( Figure 2 C) Leaf sheath ( Figure 2 D), chrysanthemum ( Figure 2 E) and callus tissue ( Figure 2 As shown in F), the scale bar in the figure is 2mm.

[0024] Figure 3 This is a schematic diagram showing the results of real-time quantitative PCR detection of the expression level of the PosHA1-driven GUS gene in roots, stems, leaves, leaf sheaths, and glumes in Example 3 of the present invention.

[0025] Figure 4 This is a schematic diagram showing the results of real-time quantitative PCR detection of the expression level of the PosHA1-driven GUS gene during the callus stage in Example 3 of the present invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0028] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0029] Example 1

[0030] This embodiment provides a method for preparing the PosHA1 promoter containing an enzyme cleavage site, including the following steps:

[0031] Based on the whole genome sequence of the rice variety Nipponbare provided in NCBI, amplification primers were designed according to the promoter sequence of the PosHA1 gene, and the restriction enzyme sites of the primers were designed according to the characteristics of the selected vector and the target gene.

[0032] In this embodiment, the rice binary expression vector pCAMBIA1305 was used, the marker gene was the GUS gene, and the primers were designed as follows: the forward primer, i.e., the upstream primer with EcoRI at the 5' end and the restriction enzyme site (GAATTC), and the reverse primer, i.e., the downstream primer with NcoI at the 5' end and the restriction enzyme site (CCATGC).

[0033] The forward and reverse primers were synthesized by Hefei Youkang Biotechnology Co., Ltd.

[0034] Using DNA from the rice variety Nipponbare as a template, the promoter PosHA1 was amplified using forward and reverse primers, following the amplification procedure according to a standard PCR system:

[0035] Pre-denaturation at 95℃ for 5 min, denaturation at 90℃ for 30 s, annealing at 58℃ for 30 s, extension at 72℃ for 2 min, 35 cycles from pre-denaturation at 95℃ to extension at 72℃, and finally extension at 72℃ for 5 min.

[0036] The target fragment amplified by PCR was recovered. The target fragment was 2000 bp in length. Figure 1 As shown in Figure A, the fragment was ligated into the blunT-SimpleT vector (purchased from Transgene, mixed according to the instructions). After transformation of *E. coli* XL-Blue competent cells using the heat shock method to activate the competent cells, the target fragment was then transferred into the activated competent cells. Positive clones were obtained through colony PCR screening. Plasmids were extracted from single clones via shaking and verified by double digestion with EcoRI and NcoI. Figure 1 As shown in B, the identified positive clones were sequenced for verification, and the correct positive clone was confirmed to be the promoter PosHA1 to be obtained, whose nucleotide sequence is shown in SEQ ID NO.1.

[0037] Example 2

[0038] This embodiment provides a method for constructing a plant expression vector and transforming it with Agrobacterium, specifically:

[0039] Plasmids were extracted from the positive clones prepared in Example 1, and double-digested with EcoRI and NcoI to recover the PosHA1 promoter fragment. Simultaneously, pCAMBIA1305 was linearized using EcoRI and NcoI, and pCAMBIA1305 was recovered. The PosHA1 fragment and pCAMBIA1305 fragment were ligated using a fast ligase (purchased from NEB) to obtain the plant expression vector pCAMBIA1305-PosHA1, which fused the PosHA1 promoter with the Gus gene (see [link to example]). Figure 1 C), the plant expression vector pCAMBIA1305-PosHA1 was transformed into Agrobacterium tumefaciens EHA105 (preserved by the Rice Group of the Transgenic Organism Product Component Supervision and Testing Center of the Ministry of Agriculture, Anhui Academy of Agricultural Sciences) using a freeze-thaw method. Positive plasmids were extracted from the freeze-thaw product and verified by EcoRI and NcoI restriction enzyme digestion. The verification results are as follows: Figure 1 As shown in B.

[0040] Example 3

[0041] This embodiment provides a method for driving the expression of the GUS reporter gene in rice using the PosHA1 promoter, specifically as follows:

[0042] (1) After removing the husks from mature seeds, soak the seeds in 70% alcohol for 1 min, discard the alcohol, and then soak the seeds in a 50% sodium hypochlorite solution containing 1 drop of Tween 20 (the effective chlorine concentration of the original solution is greater than 4%) for 40 min (150 r / min). Then discard the sodium hypochlorite and wash the seeds 5 times with sterile water until the solution is clear and has no sodium hypochlorite odor. Soak the seeds in sterile water overnight, and use a scalpel to peel off the aleurone layer of the pretreated seeds to remove the embryo. Inoculate the embryo onto the callus induction medium. After dark culture at 30℃ for 11 days, separate the callus from the endosperm and embryo. Pre-culture the primary callus tissue that is in good condition and dividing vigorously for 3-5 days before using it for Agrobacterium transformation. Agrobacterium tumefaciens transformed with the recombinant expression vector prepared in Example 2 was subjected to Agrobacterium-mediated genetic transformation. The genetic transformation, transformant selection, and transgenic plant regeneration were performed according to the methods proposed by Yongbo Duan (Yongbo Duan, Chenguang Zhai, et al. An efficient and high-throughput protocol for Agrobacterium mediated transformation based on phosphomannose isomerase positive selection in Japonica rice (Oryza sativa L.)[J]. Plant Cell Report, 2012.DOI 10.1007 / s00299-012-1275-3.). A total of 18 pCAMBIA1305-PosHA1 plants (PosHA1::GUS transgenic rice plants) were obtained.

[0043] (2) Following the method proposed by Jefferson (Jefferson RA et al. GUS fusion: β-Glucuronidase as a sensitive and versatile gene fusion marker in higher plant[J].EMBO J., 1987, 6:3901-3907), the tissue to be stained was vacuum-sealed and then immersed in the staining solution for 24 hours at 37°C. For destaining, 95% ethanol was used at 37°C until the negative control material turned white. After staining the PosHA1::GUS transgenic plant tissue, the transgenic root ( Figure 2 A), stem ( Figure 2 B), Leaf Figure 2 C) Leaf sheath ( Figure 2 D), chrysanthemum ( Figure 2 E) Callus tissue ( Figure 2 F) is expressed, and the GUS staining results are shown in [F]. Figure 2 .

[0044] Example 3

[0045] Quantitative PCR analysis of promoter activity

[0046] The qualitative results of GUS staining indicate that PosHA1 is a constitutive promoter. To verify the activity of PosHA1, we extracted RNA from 10-day-old transgenic seedlings, reverse transcribed it into cDNA, and used real-time quantitative PCR (RT-qPCR) to detect changes in PosHA1-driven GUS gene expression. Transgenic plants with CaMV 35S-driven GUS expression were used as controls.

[0047] The total RNA extraction kit from Tiangen Plant Materials Co., Ltd. (Beijing) (TIANGEN, centrifuge column type, DP432) was used. The obtained RNA was used for cDNA reverse transcription according to the following procedure: 5 μL RNase-Free ddH2O, 2 μL 5×gDNA buffer, and 3 μL RNA were added to an RNase-free centrifuge tube and incubated at 42℃ for 3 min, then placed on ice. To the above reaction solution, 5 μL RNase-Free ddH2O, 2 μL FQ-RT Primer Mix, 2 μL 10×Fast RT Buffer, and 1 μL RTEnzyme Mix were added sequentially and mixed thoroughly. The mixture was incubated at 42℃ for 15 min, then at 95℃ for 3 min, and finally placed on ice. This yielded cDNA.

[0048] RT-qPCR was performed using the SuperReal real-time quantitative PCR kit (TIANGEN, SYBR Green, FP205) from Tiangen Biotech (Beijing). The rice ACTIN gene was used as an internal control to quantify the amount of RNA template used. Two... –ΔΔCT (ΔCT = CT target gene – CT internal reference gene; ΔΔCT = ΔCT treated gene – ΔCT control gene) The obtained signals and data were processed. Each gene was tested in triplicate. The quantitative primers used in this experiment were:

[0049] Actin-FP, 5'-CCTGACGGAGCGTGGTTAC-3'; and Actin-RP, 5'-CCAGGGCGATGTAGGAAAGC-3' were used for ACTIN amplification;

[0050] GUS-FP, 5'-TACGGCAAAGTGTGGGTCAATAATCA-3' and GUS-RP, 5'-CAGGTGTTCGGCGTGGTGTAGAG-3' are used for GUS amplification.

[0051] Quantitative PCR results as follows Figure 3 and Figure 4 As shown, with the expression level of the GUS gene driven by CaMV 35S as 1, the expression levels of the GUS gene in various tissues of the PosHA1::GUS transgenic plants were detected. The results showed that the expression levels in the roots, stems, leaves, leaf sheaths, and glumes of the transgenic plants were comparable to those of CaMV 35S::GUS. This indicates that PosHA1::GUS is a constitutive expression promoter. Detection of gene expression levels during callus formation revealed that PosHA1 was highly expressed in callus, exhibiting an expression level 2.8 times higher than that of CaMV 35S. By utilizing the callus-specific expression promoter PosHA1, efficient and high-volume gene expression can be achieved at the callus stage. The PosHA1 promoter of this invention exhibits higher expression efficiency in callus tissue, which serves as an intermediate recipient material for plant tissue culture, gene gun transformation, and Agrobacterium-mediated transformation. More efficient expression in callus tissue allows for better screening of marker genes. For example, placing antibiotic or herbicide resistance genes under the drive of this promoter enables rapid and high-intensity expression of resistance proteins at the callus stage, thereby rigorously screening successfully transformed cells, preventing false positives, and greatly improving screening accuracy. For many medicinal plants or special economic crops, the target product is often found in the highest concentration in callus tissue or cell suspension. Using this promoter to drive synthetic pathway genes allows for the specific enrichment of products in callus tissue without interfering with normal plant growth, and subsequent extraction and separation are more convenient.

[0052] Although the principles of the present invention have been described in detail above with reference to preferred embodiments, those skilled in the art should understand that the above embodiments are merely illustrative explanations of the implementation of the present invention and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute a limitation on the scope of the present invention. Any obvious changes, such as equivalent transformations or simple substitutions, based on the technical solutions of the present invention without departing from the spirit and scope of the present invention fall within the protection scope of the present invention.

Claims

1. A plant constitutive expression promoter, characterized in that, The plant constitutive expression promoter contains a DNA sequence as shown in SEQ ID NO.

1.

2. The plant constitutive expression promoter according to claim 1, characterized in that, The DNA sequence of the plant constitutive expression promoter has at least 80% homology with the DNA sequence shown in SEQ ID NO.1; or, The plant constitutive expression promoter is a mutant, allele, or derivative generated by adding, substituting, inserting, or deleting one or more nucleotides in the DNA sequence shown in SEQ ID NO.1; or, The plant constitutive expression promoter has a product that hybridizes with the DNA sequence shown in SEQ ID NO.

1.

3. An expression box, characterized in that, The plant constitutive expression promoter contained in claim 1 or 2 is used to be introduced into a plant via in vivo or in vitro DNA manipulation techniques to control the expression intensity and spatiotemporal expression characteristics of the driven gene.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the plant constitutive expression promoter as described in claim 1 or 2; In the recombinant expression vector, the plant constitutive expression promoter is linked upstream of the gene sequence to be expressed in the vector.

5. A host bacterium, characterized in that, It includes the plant constitutive expression promoter of claim 1 or 2, the expression cassette of claim 3, or the recombinant expression vector of claim 4.

6. A transformant, characterized in that, It comprises the plant constitutive expression promoter of claim 1 or 2, the expression cassette of claim 3, the recombinant expression vector of claim 4, or the host bacterium of claim 5.

7. The application of a plant constitutive expression promoter as described in claim 1 or 2 in the cultivation of genetically engineered plants, characterized in that, The application includes: linking or recombining the plant constitutive expression promoter of claim 1 or 2 upstream of the gene sequence to be expressed, thereby driving the expression of the target gene in the plant.

8. The application according to claim 7, characterized in that, The plant is a monocotyledonous plant, including rice, wheat, corn, barley, sorghum, or oats.

9. The application of a plant constitutive expression promoter as described in claim 1 or 2 in improving plant growth characteristics.

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