Constitutive promoter and use thereof

Abstract

The invention relates to a constitutive promoter and application thereof. Nucleotide sequences of the constitutive promoter comprise: (1) a nucleotide sequence having at least 95% sequence identity with a nucleotide sequence shown in any one of SEQ ID NO: 1-3; and (2) a nucleotide sequence as shown in any one of SEQ ID NO: 1-3. The constitutive promoter provided by the invention has high activity, and can significantly increase the expression level of a target heterologous nucleotide sequence in plants.

Description

Technical Field

[0001] The present invention relates to a constitutive promoter and application thereof, in particular to a constitutive ubiquitin gene promoter and application thereof. Background Art

[0002] Transgenic technology can be used to introduce agronomically desirable traits or characteristics into plant species. Typically, desirable traits include improving nutritional value, increasing yield, imparting pest resistance or disease resistance, increasing drought and stress tolerance, improving horticultural quality (e.g., pigmentation and growth), imparting herbicide tolerance, enabling the production of industrially useful compounds and / or materials by plants, and / or enabling the production of drugs. It is well known that the presence of appropriate regulatory elements is required to obtain efficient expression of heterologous genes in plant cells.

[0003] Regulating elements include enhancers, promoters, leader sequences, introns and 3' untranslated regions, and can be used in the field of plant molecular biology and plant genetic engineering. Promoters play a key role in controlling gene expression, and they can determine the time and position of heterologous DNA sequences in vivo expression. Promoters commonly used in plant genetic engineering can be divided into three categories according to their mode of action and function, for example, inducible promoters that can improve gene transcription levels under the stimulation of certain physical or chemical signals, or tissue-specific promoters that only express genes in plant specific tissues, or constitutive promoters that whole plant tissues are carried out to constitutive expression. Therefore, core promoter sequences can be included in the construct of the transformation vector to cause heterologous DNA sequences to be expressed in different ways in transgenic plants.

[0004] By using genetic engineering technology to change plants genetically and thus produce plant products with specific useful traits, it is necessary to obtain a variety of promoters. The accumulation of promoter numbers will enable the expression of recombinant DNA molecules at the desired level and in the desired cell position during the development of plant gene products, thereby enabling crops to obtain expected traits. For example, the method of producing transgenic plant species containing multiple heterologous genes stacked in a single genomic locus by plant transformation technology is called the gene stacking method, in which multiple genes that confer different purpose traits can be introduced into plants. When multiple genes are introduced into plants, it is important to adjust each gene to obtain optimal expression and to diversify promoters to reduce the possibility of gene silencing. In view of this, promoter diversification is particularly important for producing transgenic plant species with high expression of multiple heterologous gene stacks. At the same time, if it is desired that heterologous DNA sequences are continuously expressed in each cell of the plant, it is necessary to use constitutive promoters.

[0005] As a constitutive promoter, the ubiqutin gene promoter shows significantly higher gene expression activity in most tissues and many cell types of plants. Therefore, when multiple different transgenes need to be superimposed in the same transgenic plant, the same Ubi promoter is often reused. However, homology-dependent gene silencing is prone to occur in transgenic plants with multiple copies of the same promoter. Therefore, it is necessary to develop ubiqutin gene promoters from different sources so that heterologous nucleotide sequences can be efficiently constitutively expressed in plant tissues, thereby giving plants the desired traits or characteristics. Summary of the invention

[0006] The object of the present invention is to provide a novel constitutive promoter and its use, wherein the constitutive promoter can constitutively express a target heterologous nucleotide sequence in a plant.

[0007] To achieve the above object, the present invention provides a constitutive promoter, the nucleotide sequence of the constitutive promoter comprising: (1) a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence shown in any one of SEQ ID NOs: 1-3; or (2) a nucleotide sequence shown in any one of SEQ ID NOs: 1-3.

[0008] To achieve the above object, the present invention also provides an expression cassette, which comprises the constitutive promoter operably linked to a target heterologous nucleotide sequence.

[0009] To achieve the above object, the present invention also provides a DNA construct comprising the expression cassette.

[0010] To achieve the above object, the present invention also provides a recombinant vector comprising the DNA construct.

[0011] To achieve the above object, the present invention also provides a method for expressing a target heterologous nucleotide sequence in a plant, comprising: stably integrating the expression cassette or the DNA construct into a plant cell, so that the target heterologous nucleotide sequence is expressed in the plant.

[0012] Preferably, the heterologous nucleotide sequence of interest encodes an abiotic stress resistance protein or a biotic stress resistance protein.

[0013] Furthermore, the abiotic stress resistance protein is a herbicide tolerance protein. The biotic stress resistance protein is an insect resistance protein.

[0014] To achieve the above object, the present invention also provides a use of the constitutive promoter for constitutively expressing a target heterologous nucleotide sequence in plant tissues.

[0015] Furthermore, the target heterologous nucleotide sequence encodes a target protein.

[0016] Based on the above technical solution, the plants include monocotyledonous plants and dicotyledonous plants.

[0017] Further preferably, the dicotyledonous plant includes but is not limited to alfalfa, kidney bean, cauliflower, cabbage, carrot, celery, cotton, cucumber, eggplant, lettuce, melon, pea, pepper, zucchini, radish, rapeseed, spinach, soybean, pumpkin, tomato, Arabidopsis, peanut or watermelon; preferably, the dicotyledonous plant refers to cucumber, soybean, Arabidopsis, tobacco, cotton, peanut or rapeseed. The monocotyledonous plant includes but is not limited to corn, rice, sorghum, wheat, barley, rye, millet, sugarcane, oats or lawn grass; preferably, the monocotyledonous plant refers to corn, rice, sorghum, wheat, barley, millet, sugarcane or oats.

[0018] The following description and definition are provided to better define the present invention and guide those skilled in the art to practice the present invention. Unless otherwise specified, terms should be understood according to conventional usage by those skilled in the art.

[0019] In the present invention, the term "nucleic acid (sequence)" or "DNA molecule" refers to a double-stranded DNA molecule of genomic or synthetic origin, that is, a polymer or DNA molecule of deoxyribonucleotide bases read from the 5' (upstream) end to the 3' (downstream) end. The term "DNA sequence" refers to the nucleotide sequence of a DNA molecule.

[0020] In the present invention, the term "promoter" refers to a region or sequence located upstream and / or downstream of the transcription start site, which is involved in recognizing and binding RNA polymerase and other proteins to initiate transcription. The promoter contains essential nucleic acid sequences near the transcription start site, such as a TATA element for a polymerase II type promoter. Examples of promoters that direct plant constitutive expression include, but are not limited to, 35S promoters from cauliflower mosaic virus, Ubi promoters from maize, promoters from rice GOS2 gene, etc.; examples of promoters that direct the expression level of coding sequences in some tissues of plants, such as green tissues, to be higher than other tissues of the plant (which can be determined by conventional RNA tests) include, but are not limited to, PEP carboxylase promoters; promoters that direct wound-induced expression patterns refer to promoters that, when plants are subjected to mechanical or insect-induced wounds, the expression of coding sequences under the regulation of the promoter is significantly increased compared to normal growth conditions. Examples of wound-induced promoters include, but are not limited to, promoters of protease inhibitor genes (pinⅠ and pinⅡ) of potatoes and tomatoes and protease inhibitor gene (MPI) of maize.

[0021] The term "promoter activity" in the present invention refers to the ability of a regulatory element to initiate transcription. Promoter activity can occur in vivo (eg, in cells) or in vitro.

[0022] The term "constitutive promoter" in the present invention means that under the control of this type of promoter, the expression of the target heterologous nucleotide sequence is generally constant at a certain level, and there is no obvious difference in the expression level in different tissues and / or different growth and development stages of the plant. The tissue is a structural unit in the plant body composed of one or more types of cells of the same origin and performing the same function, such as protective tissue, conductive tissue, nutritional tissue, mechanical tissue, meristem tissue, and several different tissues are organically coordinated and closely connected to form different organs. Different organs cooperate with each other to more effectively complete the entire life activity process of the organism. The growth and development stage can be divided into embryonic stage, seedling stage, mature stage and aging stage according to the differences in plant morphology and function. The term "constitutive expression" in the present invention means that the target heterologous nucleotide sequence is expressed at a generally consistent level in different tissues and / or different growth and development stages of the plant.

[0023] The term "protein expression" in the present invention is any pattern of translation of transcribed RNA molecules into protein molecules. Protein expression can be characterized by its following properties: temporal, spatial, developmental or morphological, and can also be characterized by quantitative or qualitative indicators.

[0024] When preparing the expression cassette, various DNA fragments can be manipulated to provide a DNA sequence that is in the right direction and in the appropriate reading frame. A reporter gene or a selective marker gene can also be included in the expression cassette. Suitable reporter genes include, but are not limited to, GUS (β-glucuronidase gene), GFP (green fluorescent protein). Selective marker genes for selecting transformed cells or tissues can include genes that confer antibiotic resistance or herbicide resistance. An expression cassette comprising a regulatory element operably linked to a polynucleotide sequence of interest can be used to transform any plant.

[0025] "Introduction" as used herein refers to the delivery of a polynucleotide to a plant in such a way that the sequence enters the interior of a plant cell. The methods disclosed herein do not depend on a particular method for introducing a sequence into a plant, as long as the polynucleotide enters the interior of at least one cell of the plant. Methods for introducing polynucleotides into plants are known in the art, and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. "Stable transformation" is a transformation in which a polynucleotide construct introduced into a plant is integrated into the genome of the plant and can be inherited by its progeny. "Transient transformation" means that a polynucleotide is introduced into a plant and it is not integrated into the genome of the plant.

[0026] In the present invention, "sequence identity" refers to the degree to which two optimally aligned DNA or amino acid sequences remain unchanged over the entire range of the alignment window of components (e.g., nucleotides or amino acids). The "identity score" of the aligned segments of the test sequence and the reference sequence (any of SEQ ID NOs: 1-3 of the present invention) is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence segment (i.e., the complete reference sequence or a smaller defined portion of the reference sequence). "Identity%" is the identity score multiplied by 100. For example, the BLAST algorithm available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) is determined using default parameters.

[0027] The term "effectively linked" in the present invention means the connection of nucleic acid sequences, and the connection allows a sequence to provide the function required for the connected sequence. The "effectively linked" in the present invention can be to connect the combined promoter of the present invention to the target heterologous nucleotide sequence of interest, so that the transcription of the target heterologous nucleotide sequence of interest is controlled and regulated by the constitutive promoter. The nucleic acid sequences that can be "effectively linked" include, but are not limited to: sequences that provide gene expression function (i.e., gene expression elements, such as promoters, 5' untranslated regions, introns, protein coding regions, 3' untranslated regions, polyadenylation sites and / or transcription terminators), sequences that provide DNA transfer and / or integration functions (i.e., T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), sequences that provide selective functions (i.e., antibiotic resistance markers, biosynthetic genes), sequences that provide scorable marker functions, sequences that assist sequence manipulation in vitro or in vivo (i.e., polylinker sequences, site-specific recombination sequences), and sequences that provide replication functions (i.e., bacterial replication origins, autonomous replication sequences, centromere sequences).

[0028] In the present invention, the term "recombination" refers to a non-natural DNA, protein, cell, seed or organism that is caused by genetic engineering and is not usually present in nature. "Recombinant DNA molecule" refers to a DNA molecule that is contained in nature and is produced by human intervention, such as a DNA molecule composed of at least two DNA molecules that are heterologous to each other. Wherein, "genetic engineering" refers to the creation of non-natural DNA, protein or organism that is not usually present in nature and requires human intervention. Genetic engineering can be used to use biotechnology, such as one or more techniques in molecular biology, protein biochemistry, bacterial transformation and plant transformation to conceive and create engineered DNA, protein or organism.

[0029] "Herbicide tolerance" or "tolerance" refers to the ability to be completely or partially unaffected by the presence or application of one or more herbicides, such as the ability to resist the toxic effects of a herbicide when applied. A cell, seed, or plant is "herbicide tolerant" or has "improved tolerance" if it is able to maintain at least some normal growth or phenotype in the presence of one or more herbicides. A trait is a herbicide tolerance trait if the presence of a trait confers improved tolerance to a herbicide to a cell, plant, or seed compared to a wild-type or control cell, plant, or seed. A crop comprising a herbicide tolerance trait can continue to grow and is minimally affected by the presence of a herbicide. A protein confers "herbicide tolerance" if its expression confers improved tolerance to a herbicide to a cell, plant, or seed compared to a wild-type or control cell, plant, or seed. "Herbicide tolerance proteins" include, but are not limited to, acetolactate synthase, aryloxyalkanoate dioxygenase, 5-enolpyruvyl-3-phosphate synthase, hydroxyphenylpyruvate dioxygenase, phosphinothricin acetyltransferase, protoporphyrinogen oxidase, glyphosate oxidoreductase. Herbicide tolerance can be complete or partial insensitivity to a specific herbicide, and can be expressed as a percentage (%) of tolerance or insensitivity to a specific herbicide.

[0030] "Insect resistance proteins" can inhibit (inhibit growth, feeding, fertility or viability), contain (inhibit growth, feeding, fertility or viability), control (control pest infestation, control pest feeding activity on a specific crop containing an effective dose of the insecticidal protein) or kill (cause disease, mortality or reduced fertility) pests. An effective dose of the insecticidal protein is provided to the pest, and the pest is exposed to the insecticidal protein to cause disease, mortality, reduced fertility or stunted development. "Insect resistance proteins" also include the expulsion of pests from plants, plant tissues, plant parts, seeds, plant cells or from a specific geographical location where the plant may grow due to the provision of an effective dose of the insecticidal protein in or on the plant. The insecticidal protein can be produced by the plant, or can be applied to the plant or the environment within the location where the plant is located.

[0031] The term "gene" refers to any DNA fragment that contains a DNA region ("transcribed DNA region") that is transcribed into an RNA molecule (e.g., mRNA) in a cell under the control of appropriate regulatory regions (e.g., a plant-expressible promoter region). Thus, a gene may contain several operably linked DNA fragments, such as a promoter, a 5' untranslated leader sequence, a coding region, and a 3' untranslated region containing a polyadenylation site. An endogenous plant gene is a gene that is naturally found in a plant species. A chimeric gene is any gene that is not normally found in a plant species, or any gene whose promoter is not associated with part or all of the transcribed DNA region or with at least another regulatory region of the gene under natural conditions.

[0032] The term "native" refers to a naturally occurring ("wild-type") nucleic acid sequence.

[0033] The term "heterologous" sequence refers to a sequence that originates from a foreign source or species, or when derived from the same source, a sequence that has been modified from its original form.

[0034] In the present invention, the term "DNA construct" is a recombinant DNA molecule comprising two or more heterologous DNA sequences. The DNA construct can be used for transgenic expression and can be included in vectors and plasmids. The DNA construct can be used in a vector for the purpose of transformation, that is, to introduce heterologous DNA into a host cell to produce transgenic cells and plants, and can also be included in the plasmid DNA or genomic DNA of transgenic cells, plants or parts thereof, seeds. A "plant transformation vector" typically contains a plasmid vector comprising a cis-acting sequence required for T-DNA transfer (such as the left and right borders), a selective marker and a target gene that can be expressed in plant cells through engineering, and the sequence required for bacterial replication is also present on this plasmid vector, and the cis-acting sequence is arranged in a manner that allows effective transfer to plant cells and expression therein. For example, the selective marker gene and the pest-killing gene are located between the left and right borders. As understood in the art, the "plant transformation vector" contains virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer (Hellens and Mullineaux, Trends in Plant Science, (2000) 5:446-451).

[0035] Suitable methods for transforming host plant cells include any method that DNA can be introduced into cells (for example, recombinant DNA constructs are stably integrated into plant chromosomes) and are well known in the art. Exemplary and widely used methods for introducing recombinant DNA constructs into plants are Agrobacterium transformation systems well known to those skilled in the art. Another exemplary method for introducing recombinant DNA constructs into plants is to insert the recombinant DNA constructs into the plant genome at a predetermined site by the method for site-directed integration. Site-directed integration can be achieved by any method known in the art, for example, by using zinc finger nucleases, engineered or natural meganucleases, TALE endonucleases or RNA-guided endonucleases (such as CRISPR / Cas9 systems) to achieve. Transgenic plants can be regenerated from transformed plant cells by plant cell culture methods. Methods for regenerating plants are also well known in the art. For example, Ti plasmid vectors have been utilized for transmitting exogenous DNA, as well as direct DNA intake, liposomes, electroporation, microinjection, and microprojectiles.

[0036] The term "plant part" or "plant tissue" in the present invention includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clusters, and intact plant cells in plants or parts of the following plants, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, kernels, ears, cobs, shells, stems, roots, root tips, anthers, etc.

[0037] The genome of the plant, plant tissue or plant cell mentioned in the present invention refers to any genetic material in the plant, plant tissue or plant cell, and includes the cell nucleus, plastid and mitochondrial genomes.

[0038] The terms "protein" and "protein" are used interchangeably in the present invention to refer to a polymer of amino acid residues. One or more of the amino acid residues in the polymer of amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers. The proteins of the present invention can be produced recombinantly or by chemical synthesis.

[0039] The term "transgene" as used herein refers to a DNA molecule that is incorporated into the genome of an organism as a result of human intervention (eg, plant transformation procedures).

[0040] The articles "a" and "an" used in the present invention refer to one or more than one (ie, at least one). For example, "an element" means one or more elements.

[0041] The invention provides a constitutive promoter and use thereof. The constitutive promoter can drive constitutive expression of exogenous genes in plant tissues and has significantly improved regulatory activity compared with Arabidopsis thaliana ubiquitin gene promoter prAtUbi.

[0042] The technical solution of the present invention is further described in detail below through embodiments. DETAILED DESCRIPTION

[0043] The technical scheme of the constitutive promoter and its use of the present invention is further illustrated by specific examples below. The methods and operations described in the following examples are exemplary and should not be construed as limiting.

[0044] First embodiment: obtaining the constitutive promoter sequence of the present invention

[0045] The constitutive promoter prOeUBI was obtained, and its nucleotide sequence is shown in SEQ ID NO: 1 in the sequence list; the constitutive promoter prMtUBI3-4 was obtained, and its nucleotide sequence is shown in SEQ ID NO: 2 in the sequence list; the constitutive promoter prMgUBI was obtained, and its nucleotide sequence is shown in SEQ ID NO: 3 in the sequence list. The control promoter prAtUbi is the Arabidopsis thaliana ubiquitin gene promoter, and its nucleotide sequence is shown in SEQ ID NO: 4 in the sequence list.

[0046] Second Example: Activity Determination of Constitutive Promoters in Tobacco

[0047] Tobacco DNA constructs containing the constitutive promoter prOeUBI, the constitutive promoter prMtUBI3-4, the constitutive promoter prMgUBI or the control promoter prAtUbi nucleotide sequences are constructed in sequence. The tobacco DNA constructs each contain two tandem expression cassettes: the first expression cassette is sequentially composed of the constitutive promoter prOeUBI, prMtUBI3-4, prMgUBI (any one of SEQ ID NO: 1-3) or the control promoter prAtUbi (SEQ ID NO: 4), which is effectively connected to the β-glucuronidase gene (GUS) and effectively connected to the terminator of the nopaline synthase gene; the second expression cassette is sequentially composed of the Arabidopsis thaliana ACTIN2 promoter, which is effectively connected to the luciferase gene (LUC) and effectively connected to the potato proteinase inhibitor II terminator (the elements involved can all refer to Chinese patent application CN202411218516.3). The above tobacco DNA constructs are respectively connected to plant transformation vectors. The above tobacco plant transformation vectors are transiently expressed in tobacco leaves respectively. The method of transiently expressing the plant transformation vector in tobacco leaves is well known to those skilled in the art, such as the method described in CN118006609A.

[0048] Grind the infected tobacco leaves, add cell lysis solution (Sigma-Aldrich, China, PE0230), extract protein using methods known in the art, and detect LUC enzyme activity and GUS enzyme activity according to the instructions of the commercial detection kit. In order to avoid the influence of factors such as cell activity and infection efficiency on the test, the GUS enzyme activity detection value was corrected by the LUC enzyme activity detection value. The correction formula is: GUS enzyme activity correction value = GUS enzyme activity detection value × LUCa / LUC enzyme activity detection value, where LUCa is the average value of the LUC enzyme activity detection value of all test groups. Each plant transformation vector was repeated five times and the average value was taken.

[0049] To evaluate the activity of the constitutive promoter of the present invention, the corrected mean value of the GUS enzyme activity of the plant transformation vector containing the control promoter prAtUbi (SEQ ID NO: 4) was used as a control and defined as a score of 1, and the corrected mean values ​​of the GUS enzyme activity of other plant transformation vectors were scored, with the lowest score being 1 and the highest score being 4. The corrected mean values ​​of the GUS enzyme activity of each plant transformation vector are shown in Table 1.

[0050] Table 1. Corrected mean scores of GUS enzyme activity of each plant transformation vector in tobacco leaves

[0051] Promoter Score prOUBI 3 prMtUBI3-4 2 prMgUBI 2 prAtUbi 1

[0052] The results in Table 1 show that: (1) compared with the control promoter prAtUbi, the GUS enzyme activity corrected mean scores of the plant transformation vectors containing the constitutive promoter prOeUBI, prMtUBI3-4 or prMgUBI are all higher, indicating that the constitutive promoters prOeUBI, prMtUBI3-4 and prMgUBI have higher activities and can significantly increase the expression level of the effectively linked heterologous nucleotide sequence in plants.

[0053] Third Example: Activity determination of constitutive promoter in Arabidopsis

[0054] Arabidopsis DNA constructs respectively comprising the constitutive promoter prOeUBI, the constitutive promoter prMtUBI3-4, the constitutive promoter prMgUBI or the control promoter prAtUbi nucleotide sequence are constructed in sequence. The Arabidopsis DNA constructs each comprise an expression cassette: the expression cassette is composed of the constitutive promoter prOeUBI, prMtUBI3-4, prMgUBI (any one of SEQ ID NO: 1-3) or the control promoter prAtUbi (SEQ ID NO: 4), effectively connected to the phosphinothricin acetyltransferase gene (PAT), and effectively connected to the terminator of the nopaline synthase gene (wherein the elements involved can refer to Chinese patent application CN202411218516.3). The above-mentioned Arabidopsis DNA constructs are respectively connected to plant transformation vectors. The above-mentioned Arabidopsis plant transformation vectors are respectively transformed into wild-type Arabidopsis plants using Agrobacterium and flower soaking methods known in the art, and Arabidopsis T is harvested. 1 seed.

[0055] The harvested Arabidopsis thaliana T 1 The seeds were germinated and formed seedlings on a solid culture medium containing Basta herbicide (15 μg / mL glufosinate ammonium). 150 surviving seedlings were screened for each plant transformation vector, and the copy number of the PAT gene in the surviving seedlings was detected by Taqman. The number of surviving seedlings with a single copy of the transgene was counted for evaluating the promoter activity.

[0056] The number of surviving seedlings of the single copy transgenic plant transformation vector obtained by transferring the control promoter prAtUbi (SEQ ID NO: 4) was used as a control and defined as a score of 1, and the number of surviving seedlings of the single copy transgenic plant transformation vectors of other plant transformation vectors was scored, and the score increased according to the increase in the number of surviving seedlings of the single copy transgenic plant transformation vectors, wherein the lowest score was 1 and the highest score was 4. The scores of the number of surviving seedlings of the single copy transgenic plant transformation vectors are shown in Table 2.

[0057] Table 2. Scores of surviving seedlings of single-copy transgenic plants of each plant transformation vector in Arabidopsis

[0058] Promoter Score prOUBI 3 prMtUBI3-4 2 prMgUBI 2 prAtUbi 1

[0059] The results in Table 2 show that: (1) considering that Basta herbicide is a contact herbicide, the seedlings containing the constitutive promoter prOeUBI, prMtUBI3-4 or prMgUBI can survive in the solid culture medium containing Basta herbicide, indicating that the constitutive promoters prOeUBI, prMtUBI3-4 and prMgUBI are active in both the root and stem of the plant. (2) compared with the control promoter prAtUbi, the single copy transgenic surviving seedling number score of the constitutive promoter prOeUBI, prMtUBI3-4 and prMgUBI is higher (the number of single copy seedlings is larger), indicating that the constitutive promoters prOeUBI, prMtUBI3-4 and prMgUBI are more active, which can significantly increase the expression level of the PAT gene in plants.

[0060] In summary, the present invention discloses for the first time a novel constitutive promoter, which shows high activity in plants, and compared with the control promoter prAtUbi, the constitutive promoter of the present invention shows a significant improvement in the expression level of the target heterologous nucleotide sequence to which it is effectively connected, and can be used to develop plant genetic engineering products such as herbicide-resistant, insect-resistant, and disease-resistant, thereby more accurately improving plant traits and yields.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A constitutive promoter, characterized in that Its nucleotide sequence includes: (1) a nucleotide sequence having at least 95% sequence identity with the nucleotide sequence shown in any one of SEQ ID NOs: 1-3; or (2) having a nucleotide sequence shown in any one of SEQ ID NOs: 1-3.

2. An expression cassette, characterized in that The invention comprises the constitutive promoter according to claim 1 operably linked to a heterologous nucleotide sequence of interest.

3. A DNA construct, characterized in that Comprising the expression cassette of claim 2.

4. A recombinant vector, characterized in that: Comprising the DNA construct according to claim 3.

5. A method for expressing a heterologous nucleotide sequence of interest in a plant, characterized in that: include: The expression cassette of claim 2 or the DNA construct of claim 3 is stably integrated into a plant cell, so that the target heterologous nucleotide sequence is expressed in the plant.

6. The method for expressing a target heterologous nucleotide sequence in a plant according to claim 5, characterized in that: The heterologous nucleotide sequence of interest encodes an abiotic stress resistance protein or a biotic stress resistance protein.

7. The method for expressing a target heterologous nucleotide sequence in a plant according to claim 6, characterized in that: The abiotic stress resistance protein is a herbicide tolerance protein.

8. The method for expressing a target heterologous nucleotide sequence in a plant according to claim 6 or 7, characterized in that: The biotic stress resistance protein is an insect resistance protein.

9. Use of the constitutive promoter according to claim 1 for constitutively expressing a target heterologous nucleotide sequence in plant tissues.

10. The use according to claim 9, wherein the target heterologous nucleotide sequence encodes a target protein.