Constitutive promoter and use thereof

Abstract

Provided are a constitutive promoter and a use thereof, wherein a nucleotide sequence of the constitutive promoter comprises SEQ ID NO: 35. The constitutive promoter exhibits activity in almost all tissues and multiple types of cells of plants, particularly in roots, stems, leaves, filaments, pollen, seeds, and rachis tips of plants, and has broad application prospects for plants.

Description

Constitutive promoters and their applications Technical Field

[0001] This invention relates to a constitutive promoter and its use, and more particularly to a constitutive promoter derived from goosegrass and its use. Background Technology

[0002] One of the goals of plant genetic engineering is to modify plants according to human needs, thereby producing plants with desired characteristics or traits. Commonly desired traits include improved nutritional quality, increased yield, conferring resistance to pests and diseases, improved drought and stress tolerance, improved horticultural quality, and conferring herbicide resistance. Currently, researchers are able to obtain exogenous genes (such as heterologous or naturally derived genes) and integrate them into the plant genome for expression to exhibit the corresponding traits.

[0003] Choosing appropriate regulatory signals is crucial for expressing target genes in plants. Typical regulatory signals include promoter regions, 5' untranslated guide sequences, and 3' transcription terminators / polyadenylated sequences. A promoter is a DNA sequence located upstream of the 5' end of a structural gene that can be recognized, bound, and initiate gene transcription by RNA polymerase. In plant genetic engineering, promoters are often classified into three categories based on their mode of action and function: constitutive promoters, specific promoters, and inducible promoters. Constitutive promoters are those that guide RNA synthesis at a certain level of expression in most or all plant tissues and / or during growth and development stages. Based on their effectiveness in guiding RNA synthesis, they can be further classified as strong, moderate, and weak promoters. Constitutive promoters are particularly advantageous in situations where it is necessary to simultaneously express the target gene (or chimeric gene) in different plant tissues to achieve the desired gene function.

[0004] Several constitutive promoters that function in plant cells have been described in existing literature. These promoters include the soybean cell elongation factor gene promoter Gm17gTsf1, the nos promoter carried on the *Agrobacterium tumefaciens* tumor-inducing plasmid, the octopus amino acid synthase (ocs) promoter, and cauliflower mosaic virus (CaMV) promoters, such as the CaMV 19S or 35S promoter, the CaMV 35S promoter with repeat enhancers, and the Scrophularia mosaic virus (FMV) 35S promoter. These promoters have been used in transgenic plant constructs. Although some constitutive promoters have been obtained, the discovery and isolation of more constitutive promoters that are safe, efficient, and capable of controlling the expression of target genes (or chimeric genes) at different levels or applied to the superimposed expression of multiple genes in the same transgenic plant remains a hot topic of ongoing interest in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a novel constitutive promoter and its uses. This promoter is derived from Eleusine indica L. and can regulate the efficient expression of target nucleic acids in plant tissues such as roots, stems, leaves, filaments, pollen, grains, and spikelet tips. It provides a new tool and option for plant genetic engineering and has a very broad application prospect.

[0006] In a first aspect, the present invention provides a constitutive promoter whose nucleotide sequence includes SEQ ID NO:35.

[0007] Preferably, the constitutive promoter has a nucleotide sequence including SEQ ID NO:35, and the constitutive promoter is derived from SEQ ID NO:1.

[0008] More preferably, the constitutive promoter has a nucleotide sequence including SEQ ID NO:35, and is a part or a fragment of SEQ ID NO:1.

[0009] More preferably, the constitutive promoter has a nucleotide sequence including SEQ ID NO:35, and is a truncated sequence of SEQ ID NO:1.

[0010] More preferably, the constitutive promoter has a nucleotide sequence including SEQ ID NO:35, and is a 5' (N-terminus) truncated sequence of SEQ ID NO:1.

[0011] More preferably, the 5' end (N end) is truncated to no more than (less than or equal to) 1671 bp.

[0012] More preferably, the constitutive promoter has the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35.

[0013] Secondly, the present invention also provides a chimeric gene comprising the above-described constitutive promoter operatively linked to a target nucleic acid.

[0014] Preferably, the target nucleic acid encodes the target protein.

[0015] Thirdly, the present invention also provides an expression cassette containing the constitutive promoter or chimeric gene of the present invention.

[0016] Fourthly, the present invention also provides a recombinant vector comprising the constitutive promoter, chimeric gene, or expression cassette of the present invention.

[0017] Fifthly, the present invention also provides a host cell comprising the constitutive promoter, chimeric gene, expression cassette, or recombinant vector of the present invention.

[0018] In a sixth aspect, the present invention also provides a method for expressing a target nucleic acid in a plant, comprising: stably integrating the target nucleic acid operatively linked to the above-described constitutive promoter into a plant cell.

[0019] Preferably, the target nucleic acid is constitutively expressed in plant tissues.

[0020] Preferably, the target nucleic acid encodes the target protein.

[0021] More preferably, the target nucleic acid encodes a herbicide-resistant protein.

[0022] More preferably, the target nucleic acid encodes an insect resistance protein.

[0023] Preferably, the plant is corn, Arabidopsis thaliana, rapeseed, tobacco, soybean, cotton, chili pepper, beet, pumpkin, eggplant, Chinese cabbage, carrot, tomato, pea, spinach, potato, or peanut.

[0024] In a seventh aspect, the present invention also provides a plant or plant part comprising the above-described constitutive promoter, chimeric gene, expression cassette or recombinant vector.

[0025] Eighthly, the present invention also provides a method for obtaining processed agricultural products, comprising processing the harvest of the aforementioned plants or plant parts to obtain processed agricultural products.

[0026] In a ninth aspect, the present invention also provides the use of the above-described constitutive promoter in the constitutive expression of a target nucleic acid in plant tissues.

[0027] Preferably, the target nucleic acid encodes the target protein.

[0028] More preferably, the target nucleic acid encodes a herbicide-resistant protein.

[0029] More preferably, the target nucleic acid encodes an insect resistance protein. Preferably, the plant is corn, Arabidopsis thaliana, rapeseed, tobacco, soybean, cotton, chili pepper, beet, pumpkin, eggplant, Chinese cabbage, carrot, tomato, pea, spinach, potato, or peanut.

[0030] In this invention, the terms "comprising" and "including" mean "including but not limited to".

[0031] In this invention, the term "promoter" refers to a DNA regulatory region, which typically contains a TATA box that guides RNA polymerase II to initiate RNA synthesis at a suitable transcription start site in a specific coding sequence.

[0032] In this invention, the terms "gene" or "nucleic acid" refer to any segment of DNA containing, within a cell, a region of DNA transcribed into RNA molecules (e.g., mRNA) under the control of a suitable regulatory region (e.g., a plant-expressible promoter region) ("transcribed DNA region"). A gene or nucleic acid may contain several operatively linked DNA segments, such as a promoter, a 5' untranslated leader sequence, a coding region, and a 3' untranslated region containing a polyadenylation site. Endogenous plant genes are genes naturally found in plant species. Chimeric genes are any genes not normally found in plant species, or any genes whose promoters, under natural conditions, are independent of partially or entirely transcribed DNA regions or at least one other regulatory region of the gene.

[0033] In this invention, the terms "target gene" or "target nucleic acid" can refer to endogenous genes or nucleic acids, or exogenous or heterologous genes or nucleic acids. Endogenous genes or nucleic acids refer to genes naturally found or present in a plant species or plant host, while exogenous or heterologous genes or nucleic acids refer to any genes that do not belong to or exist in a plant species or plant host. Optionally, the target gene or target nucleic acid can be a natural sequence or a selectively synthesized sequence.

[0034] The target gene or target nucleic acid of this invention may be homologous to the promoter of this application; for example, the target gene or target nucleic acid and the promoter may originate from the same plant species or be naturally linked together. The target gene or target nucleic acid of this invention may also be exogenous or heterologous to the promoter of this application, i.e., the target gene or target nucleic acid and the promoter may originate from different plant species or be non-naturally linked together or coexist. For example, a chimeric gene may contain the promoter sequence of this invention, which is operatively linked to a coding sequence different from the promoter sequence of this invention. Although the target gene or target nucleic acid sequence and the promoter sequence are heterologous, they may be homologous, natural, heterologous, or exogenous to the plant host.

[0035] In this invention, the term "constitutive promoter" refers to a special type of gene regulatory sequence. Under the control of this type of promoter, most or all tissues and / or growth and development stages of an organism exhibit a certain degree of gene / nucleic acid expression. Constitutive promoters are used to operatively link target genes, target nucleic acids, or gene editing system guide RNA (gRNA), enabling their expression in most cells of an organism, and the initiated expression has a certain degree of persistence. It is understood that, for the term "constitutive promoter," there may be some variation in the absolute level of expression or activity between different tissues and developmental stages of an organism. The tissues are structural units in plants composed of one or more types of cells of the same origin and performing the same function, such as protective tissues, vascular tissues, nutritive tissues, mechanical tissues, and meristematic tissues. Several different tissues cooperate organically and are closely connected to form different organs. Different organs cooperate with each other to more effectively complete the entire life process of the organism. The growth and development stages can be divided into embryonic stage, seedling stage, mature stage, and senescence stage according to differences in plant morphology and function. In this invention, the terms "constitutive expression" and "constitutive expression" refer to the relatively stable and continuous expression of the target gene or target nucleic acid in most or all tissues and / or growth and development stages of a plant. For example, the CaMV35S promoter of cauliflower mosaic virus can initiate high-intensity expression of exogenous genes in most organs and different developmental stages in plants. Constitutive promoters also ensure widespread expression of gRNA in host cells, improving the efficiency and accuracy of gene editing. The promoters of this invention can be homologous, exogenous, or heterologous relative to the plant species or plant host. Homologous means the promoter originates from the plant species or plant host into which the transcription start region is introduced; exogenous or heterologous means the promoter or transcription start region is not present in the natural plant or plant host into which the transcription start region is introduced.

[0036] This invention provides a constitutive promoter whose nucleotide sequence includes SEQ ID NO:35. Further, the nucleotide sequence of the constitutive promoter includes SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:1. In a preferred embodiment of this invention, the nucleotide sequence of the constitutive promoter includes SEQ ID NO:35 and is derived from SEQ ID NO:1. Further, the promoter of this invention has a nucleotide sequence including SEQ ID NO:35 and is at least a portion of SEQ ID NO:1 or at least a fragment of SEQ ID NO:1. More preferably, the nucleotide sequence of the constitutive promoter includes SEQ ID NO:35 and is a truncated sequence of SEQ ID NO:1, wherein the truncated sequence is a 5' (N-terminal) truncated sequence of SEQ ID NO:1, and the truncated length does not exceed (is less than or equal to) 1671 bp.

[0037] Specifically, SEQ ID NO:35 is a 1404 bp fragment located at the 3' end of SEQ ID NO:1, which is also the remaining fragment (truncated fragment) after removing 1671 bp from the 5' end of SEQ ID NO:1. The constitutive promoter of the present invention can be extended from the 5' end or the 3' end of SEQ ID NO:35. In a preferred embodiment, the constitutive promoter of the present invention can refer to SEQ ID NO:1, extending from the 5' end of SEQ ID NO:35, but the length of the nucleotide sequence extended to the 5' end cannot exceed the 5' end of SEQ ID NO:1 itself. In other words, the constitutive promoter of this application is a truncated nucleotide fragment obtained by removing or eliminating nucleotides of length less than or equal to 1671 bp from the 5' end of SEQ ID NO:1, and this truncated fragment includes SEQ ID NO:35. All constitutive promoters obtained above are within the protection scope of the present invention.

[0038] For the aforementioned promoters, nucleotides other than SEQ ID NO:35 in their nucleotide sequences do not affect the promoter's activity. Example 5 of this invention also demonstrates this conclusion: the prEiUbi2-04 promoter (SEQ ID NO:35) is active, and promoters containing SEQ ID NO:35, such as prEiUbi2-02 (SEQ ID NO:33), prEiUbi2-03 (SEQ ID NO:34), and prEiUbi2-01 (SEQ ID NO:1), all possess good activity. Those skilled in the art, based on the content described in this application, can reasonably expect that SEQ ID NO:35 is the shortest fragment (smallest unit) for performing promoter function, and that when the nucleotide sequence of a promoter includes SEQ ID NO:35, it possesses promoter function and activity. Similarly, when the nucleotide sequence of a promoter includes SEQ ID NO:35 and is selected from at least a portion of SEQ ID NO:1 or is a truncated sequence of SEQ ID NO:1, those skilled in the art can reasonably expect that these promoters have the same or similar functions and activities as the promoter shown in SEQ ID NO:35.

[0039] Promoter sequences that have promoter activity and are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the promoter sequences of the present invention are included in the present invention, that is, the range of sequence identity is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0040] Separate sequences that have promoter activity and hybridize with the promoter sequence or fragment thereof of the present invention under stringent conditions are also included in the present invention. These sequences are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the sequences of the present invention, i.e., the range of sequence identity is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater.

[0041] The promoter sequence and fragments described in this invention, when assembled into a DNA structure to operatively link the promoter sequence with a target nucleic acid sequence, are used for the genetic manipulation of any plant. "Operationally linked" refers to a functional link between the promoter sequence of this invention and a second sequence, wherein the promoter sequence initiates and regulates transcription of the DNA sequence corresponding to the second sequence. Generally, operative linking means that the linked nucleic acid sequences are contiguous, and if necessary, bind two protein-coding regions adjacently within the same reading frame. In this manner, the promoter nucleotide sequence and the target nucleotide sequence constitute the chimeric gene, which is provided together in an expression cassette for expression in the target plant. This expression cassette provides numerous restriction sites for inserting the target nucleotide sequence, which will be subject to transcriptional regulation by a regulatory region containing the promoter sequence of this invention. The expression cassette may additionally contain at least one additional gene that will be co-transformed into the organism. Alternatively, the additional gene may be provided on multiple expression cassettes.

[0042] The expression cassette may additionally contain optional marker genes. Generally, the expression cassette will contain selective marker genes for selecting transformed cells. These selective marker genes are used to select transformed cells or tissues. These selective marker genes include, but are not limited to, genes encoding antibiotic resistance (such as genes encoding neomycin phosphotransferase II (NPT) and hygromycin phosphotransferase (HPT), and genes conferring herbicide resistance such as glufosinate, bromobenzonitrile, imidazolinones, and 2,4-dichlorophenoxyacetic acid (2,4-D) resistance genes.

[0043] The expression cassette includes the promoter sequence of the present invention transcribed along the 5'-3' direction, the translation initiation region, the target heteronucleotide sequence, and the transcription and translation termination regions that function in plants.

[0044] The termination region may be derived from the promoter sequence of this invention, from the target heteronucleotide sequence that can be operatively linked, or from another source. Conventional termination regions can be obtained from the Ti plasmid of Agrobacterium tumefaciens, such as the termination regions of carnitine synthase and caustic solanine synthase (NOS).

[0045] In expression cassette preparation, different DNA fragments can be manipulated to provide DNA sequences with appropriate orientation and, at the appropriate time, appropriate reading frames. Therefore, acceptors or linkers can be applied to bind DNA fragments, or other manipulations can be performed to provide convenient restriction sites, remove redundant DNA, or eliminate restriction sites. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, and re-substitution, such as transformation and conversion, may be involved.

[0046] Under suitable conditions, the target heteronucleotide sequence can be optimized to increase expression levels in transformed plants. This can be achieved by using plant-preferred codons to synthesize genes and improve expression.

[0047] As is known in the art, additional sequence modifications can enhance gene expression levels in the host cell. These include, but are not limited to, removing signals encoding pseudopolyadenosine, exon-intron splicing sites, repetitive sequences of transposons, and other sequences well characterized as potentially detrimental to gene expression. The GC content of the sequence can be tuned to the average level of a given host cell, calculated using known gene expression levels in the host cell. Possibly, the sequence can be modified to avoid predicted hairpin mRNA secondary structures.

[0048] In the expression cassette or recombinant vector, the expression cassette may additionally contain a 5' leader sequence. This leader sequence can improve transcription efficiency. The leader sequence is known in the art, including but not limited to: parvovirus leader sequences, such as the EMCV leader sequence (5' uncoding region of encephalomyocarditis virus); potato virus group leader sequences, such as the tobacco etch virus (TEV) leader sequence, the maize dwarf mosaic virus (MDMV) leader sequence, and human immunoglobulin heavy chain binding protein (BiP); untranslated leader sequences from alfalfa mosaic virus coating protein mRNA (AMV RNA4); tobacco mosaic virus (TMV) leader sequences; and maize yellow spot virus (MCMV) leader sequences. Other known elements for improving transcription efficiency, such as introns, may also be used.

[0049] The promoter sequence of this invention can be used to initiate transcription of an antisense structure that is at least partially complementary to the messenger RNA (mRNA) of the target nucleic acid sequence. An antisense nucleotide sequence is constructed to hybridize with the corresponding mRNA. The antisense sequence can be modified as long as it is long enough to hybridize with the corresponding mRNA and interfere with its expression. In this way, antisense structures having 80%, preferably 90%, more preferably 95% sequence identity with the corresponding antisense sequence can be used. Furthermore, a portion of the antisense nucleotide sequence can be used to disrupt the expression of the target gene. Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides, or more nucleotides can be used.

[0050] Nucleic acid or nucleic acid sequences operably linked to the promoter of the present invention can encode a target protein. Examples of such nucleic acid or nucleic acid sequences include, but are not limited to, nucleotide sequences encoding resistant polypeptides conferring resistance to abiotic stresses such as drought, temperature, salinity, ozone, and herbicides, or biotic stresses such as pathogen invasion, including insects, viruses, bacteria, fungi, and nematodes, and preventing the development of diseases associated with these organisms.

[0051] In this invention, herbicide resistance proteins can express resistance and / or tolerance to herbicides. These genes include, but are not limited to, hydroxyphenylpyruvate dioxygenase (HPPD) gene, protoporphyrinogen oxidase (PPO) gene, acetyllactate synthase (ALS) gene, 5-enolpyruvate shikimyl-3-phosphate synthase (EPSPS) gene, glyphosate acetyltransferase (PAT) gene, glyphosate oxidoreductase (GOX) gene, and GAT gene.

[0052] In this invention, "insect resistance" refers to a plant's avoidance of symptoms and damage caused by plant-insect interactions. This means preventing insect-induced plant damage, crop damage, plant deformity, and plant diseases, or optionally, minimizing or mitigating insect-induced plant damage, crop damage, plant deformity, and plant diseases. The insects may belong to Lepidoptera (e.g., corn borer), Hemiptera (e.g., stink bug), Coleoptera (e.g., beetles), Orthoptera (e.g., locusts), Homoptera (e.g., aphids), Diptera (e.g., flies), etc. Commonly known in the art, target insect resistance proteins include, but are not limited to, Bacillus toxicity proteins; lectins, including snowdrop lectin, pea lectin, canavalia pea lectin, malt lectin, potato lectin, peanut lectin, etc.; lipoxygenases, including pea lipoxygenase 1 or soybean lipoxygenase; and insect chitosanase, etc.

[0053] Different pests transmit viruses from infected plants to healthy plants in different ways. These viruses include, but are not limited to, rice Dongorubicin virus, tobacco mosaic virus, sweet potato wilt dwarf virus, and sweet potato feather spot virus. Therefore, heterologous nucleotide sequences with antipathogenic activity or that minimize the impact of viral pathogens can be constitutively expressed in plant tissues.

[0054] The promoter sequence and method of this invention can be used to regulate the expression of any target nucleic acid in a plant host to alter the plant's phenotype. Various targeted phenotype alterations include, but are not limited to, changes in the plant's fatty acid composition, amino acid content, and pathogen defense mechanisms. These alterations can be achieved by providing the expression of a heterologous product or increasing the expression of an endogenous product. Alternatively, these alterations can be achieved by reducing the expression of one or more endogenous products in the plant, particularly enzymes or cofactors. These alterations will result in phenotypic changes in the transformed plant.

[0055] Transformation protocols and protocols for introducing nucleotide sequences into plants vary depending on the type of plant or plant cell being transformed, i.e., monocots or dicots. Suitable methods for introducing nucleotide sequences into plant cells and subsequently inserting them into the plant genome include, but are not limited to, Agrobacterium-mediated transformation, microemission bombardment, direct DNA uptake into protoplasts, electroporation, or whisker-based DNA introduction.

[0056] The transformed cells can be grown into plants in a conventional manner. These plants are cultured and pollinated with the same or different transformants to produce hybrids that express the desired identified phenotypic traits. Two or more generations can be cultured to ensure the stable maintenance and inheritance of the desired phenotypic trait, and then seeds that guarantee the expression of the desired phenotypic trait are harvested.

[0057] The host cells of this invention include, but are not limited to, plant cells or bacteria. Bacteria include, but are not limited to, Agrobacterium, Bacillus, Escherichia coli, Salmonella, Pseudomonas, or Rhizobium cells. Plant cells can be non-regenerative or regenerative. Non-regenerative cells are those that cannot be regenerated into a whole plant through in vitro culture. Non-regenerative cells can be in the plant or plant part (e.g., leaf) of this invention. Non-regenerative cells can be cells in a seed or the seed coat of said seed. Mature plant organs (including mature leaves, mature stems, or mature roots) contain at least one non-regenerative cell. On the other hand, plant cells are structurally incomplete living cells, such as plant cells without a nucleus. Specifically, plant cells are sieve tube cells (mature sieve tube cells) without a nucleus. On the other hand, plant cells are reproductive cells, such as ovules or cells that are part of pollen. In one aspect, pollen cells are vegetative (non-reproductive) cells, or sperm cells. On the other hand, plant cells are somatic cells.

[0058] The term "plant" refers to the whole plant, including all plants and plant populations, such as desired and unwanted wild plants or crop plants (including naturally occurring crop plants). Crop plants can be plants obtained through conventional breeding and optimization methods or through biotechnology and recombination methods, or a combination of these methods, including transgenic plants.

[0059] The term "plant part" includes plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can regenerate, plant callus, plant clumps, and intact plant cells in a plant or plant part. Examples of plant parts include embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, etc. It should be understood that parts of transgenic plants within the scope of this invention include, but are not limited to, plant cells, protoplasts, tissues, callus, embryos, and flowers, stems, fruits, leaves, and roots, all of which are derived from transgenic plants or their progeny that have been previously transformed with the DNA molecules of this invention and are therefore at least partially composed of transgenic cells. In one aspect, a plant part is the aforementioned plant cell.

[0060] This invention provides processing of harvested plants or portions containing the constitutive promoters described herein to obtain processed agricultural products. The term "processed agricultural product" refers to any composition or product composed of materials derived from plants, seeds, plant cells, or plant portions containing the constitutive promoters described herein. Specifically, the term "processed agricultural product" includes, but is not limited to, protein concentrates, protein isolates, starch, flour, biomass, and seed oils.

[0061] This invention provides a constitutive promoter and its application, which has the following beneficial effects:

[0062] This invention discloses for the first time a constitutive promoter from Eleusine indica, which exhibits good activity in most tissues and cells of the plant, especially in the roots, stems, leaves, filaments, pollen, grains, and rachis tips of the plant. It can drive the efficient expression of exogenous genes or nucleic acids, providing new tools and options for plant genetic engineering and possessing excellent application potential.

[0063] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0064] Figure 1 is a schematic diagram of the structure of the vector DBNBC-Dual_LUC containing LUC and REN reporter genes of the present invention.

[0065] Figure 2 is a schematic diagram of the structure of the recombinant dual-luciferase expression vector DBN13844 containing the promoter prZmUbi1;

[0066] Figure 3 is a schematic diagram of the recombinant dual-luciferase expression vector DBN14252 containing the promoter prEiUbi2-01.

[0067] Figure 4 is a schematic diagram of the recombinant vector DBN14915 whose PAT gene expression is driven by the constitutive promoter prEiUbi2-01.

[0068] Figure 5 shows the tolerance of transgenic maize with PAT gene driven by the constitutive promoter prEiUbi2-01 to glufosinate herbicide. Detailed Implementation

[0069] The technical solution of the constitutive promoter of the present invention and its application is further illustrated below through specific embodiments.

[0070] Example 1: Obtaining the constitutive promoter prEiUbi2-01

[0071] Using the nucleotide sequence of maize ubiquitin protein as a reference sequence, homologous genes were searched and compared in NCBI (https: / / www.ncbi.nlm.nih.gov / ) to obtain the candidate *Eleusine indica* ubiquitin protein gene (GenBank: QEPD01000483.1). The sequence approximately 3 kb upstream of the *Eleusine indica* ubiquitin protein gene was selected as the target sequence. *Eleusine indica* genomic DNA was used as the PCR amplification template, and primers 1 (SEQ ID NO:2) and 2 (SEQ ID NO:3) were designed for PCR amplification.

[0072] PCR was performed using a 50 μL reaction system, which included 0.5 μL Q5 polymerase, 10 μL reaction buffer, 1 μL 10 mM dNTPs, 2.5 μL each of 10 μM primer 1 and 10 μM primer 2, and genomic DNA <1000 ng. Nuclease-free water was added to bring the total volume to 50 μL.

[0073] The PCR reaction conditions are as follows:

[0074] Among them, Q5 polymerase is from New England. The company's high-fidelity Q5 DNA polymerase has a reaction buffer of... The reaction buffer in the company's High-Fidelity DNA Polymerase kit should be followed according to the specific operating procedures. Company PCR Using Follow the instructions for the High-Fidelity DNA Polymerase (M0491) kit.

[0075] The PCR product was ligated into the blunt-ended Blunt vector (Beijing TransGen Biotech Co., Ltd.). The procedure was performed according to the TransGen Biotech Blunt vector product instructions. The ligation product was then sequenced (Sanger method) to confirm that the constructed Blunt vector contained a promoter fragment of the *Eleusine indica* ubiquitin protein gene with a length of 3075 bp. Its sequence is shown in SEQ ID NO:1 and it was named prEiUbi2-01.

[0076] Example 2: Validation of the effect of the constitutive promoter prEiUbi2-01 in maize callus tissue

[0077] 1. A dual-luciferase expression vector containing the constitutive promoter prEiUbi2-01 was constructed, and a dual-luciferase reporter system containing firefly luciferase (LUC) and rellina luciferase (REN) was introduced. The efficiency of promoter-driven target gene expression was determined by detecting the level of LUC activity. REN was used as an internal control to eliminate inter-group errors caused by factors such as different transformation efficiencies due to Agrobacterium infection in plant tissues.

[0078] The structure of the vector DBNBC-Dual_LUC (vector backbone: pCAMBIA2301 with modified resistance tag, provided by CAMBIA) containing the LUC and REN reporter genes is shown in Figure 1. In the figure, cSpec is the spectinomycin gene, RB is the right boundary, cLUC is the firefly luciferase gene (SEQ ID NO:4), tNos is the Agrobacterium tumefaciens carmine synthase gene terminator (SEQ ID NO:5), prOsUBQ2 is the rice UBQ2 gene promoter (SEQ ID NO:6), cREN is the Renilla luciferase gene (SEQ ID NO:7), tPinII is the potato protease inhibitor II gene terminator (SEQ ID NO:8), prOsAct1 is the rice actin gene promoter (SEQ ID NO:9), cPAT is the phosphinicotinic acid acetyltransferase gene (SEQ ID NO:10), t35S is the cauliflower mosaic virus gene terminator (SEQ ID NO:11), and LB is the left boundary.

[0079] A vector expressing the LUC gene driven by the prEiUbi2-01 promoter was constructed and transformed into maize, with the constitutive promoter prZmUbi1 (SEQ ID NO:12), known in the art, serving as a control. Luciferase activity was measured at different growth stages and in different tissues of maize to compare and analyze the expression pattern and intensity of the prEiUbi2-01 promoter. Constructing the vector using conventional restriction enzyme digestion methods is well-known to those skilled in the art. Specifically, the DBNBC-Dual_LUC vector was linearized using the restriction endonuclease SalⅠ. Primer pairs shown in SEQ ID NO:13 and SEQ ID NO:14, and primer pairs shown in SEQ ID NO:15 and SEQ ID NO:16, were used to amplify the prZmUbi1 element (SEQ ID NO:12) with the adapter sequence and the prEiUbi2-01 element (SEQ ID NO:1) with the adapter sequence, respectively. The linearized DBNBC-Dual_LUC vector was mixed with prZmUbi1 element with adapter sequence and prEiUbi2-01 element with adapter sequence, respectively, for recombination reaction. The operation procedure was performed according to the instructions of Takara In-Fusion Snap Assembly Master Mix kit (Clontech, CA, JPN, CAT: 638949) to construct recombinant dual-luciferase expression vectors DBN13844 and DBN14252, the structures of which are shown in Figures 2 and 3.

[0080] 2. Transformation of Agrobacterium tumefaciens with recombinant dual-luciferase expression vector

[0081] The correctly constructed recombinant dual-luciferase expression vectors DBN13844 and DBN14252 were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA; Cat. No: 18313-015) using liquid nitrogen. The transformation conditions were as follows: 100 μL of Agrobacterium LBA4404 and 3 μL of the recombinant expression vector were placed in liquid nitrogen for 10 min, followed by a 37°C water bath for 10 min. The transformed Agrobacterium LBA4404 was then inoculated into LB tubes and cultured at 28°C and 200 rpm for 2 h. The cultured tubes were then plated onto LB agar plates containing 50 mg / L rifampicin and 50 mg / L spectinomycin until positive single colonies appeared. Single colonies were picked, cultured, and their plasmids were extracted. Sequencing analysis confirmed that the recombinant dual-luciferase expression vectors DBN13844 and DBN14252 had completely correct structures.

[0082] 3. Transient transformation of corn callus tissue

[0083] Maize callus tissue is a highly efficient bioreactor for protein expression. By using Agrobacterium infection to introduce exogenous genes into maize callus tissue, the function of exogenous genes can be preliminarily studied.

[0084] For Agrobacterium-mediated maize transformation, briefly, immature embryos are isolated from maize and contacted with an Agrobacterium suspension, wherein Agrobacterium is capable of transferring T-DNA from the DBN13844 and DBN14252 vectors to at least one cell of one of the embryos (step 1: infection step), in which the embryo is preferably immersed in the Agrobacterium suspension (OD). 660 =0.4-0.6, inoculated in infection medium (MS salt 4.3 g / L, MS vitamins, casein 300 mg / L, sucrose 68.5 g / L, glucose 36 g / L, acetylsuccinone (AS) 40 mg / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg / L, pH 5.3). The embryos are co-cultured with Agrobacterium for a period (3 days) (Step 2: Co-culture step). Preferably, after the infection step, the embryos are cultured on solid medium (MS salt 4.3 g / L, MS vitamins, casein 300 mg / L, sucrose 20 g / L, glucose 10 g / L, AS 100 mg / L, 2,4-D 1 mg / L, agar 8 g / L, pH 5.8). After this co-culture phase, a selective "recovery" step can be performed. In the "recovery" step, the recovery medium (MS salt 4.3 g / L, MS vitamins, casein 300 mg / L, sucrose 30 g / L, 2,4-D 1 mg / L, cephalosporin 250 mg / L, plant gel 3 g / L, pH 5.8) contains at least one known antibiotic that inhibits the growth of Agrobacterium (cephalosporin 150-250 mg / L), without the addition of a selector for plant transformants (Step 3: Recovery Step). The embryos are cultured on a solid medium containing antibiotics but without a selector to eliminate Agrobacterium and provide a recovery period for infected cells, while simultaneously forming callus tissue.

[0085] 4. Detection of LUC / REN dual-luciferase activity in maize callus

[0086] Maize callus tissues transformed into the recombinant dual-luciferase expression vectors DBN13844 and DBN14252 were collected and processed according to the Promega protocol. The Reporter Assay System (E1960) kit was used for testing, and the specific method is as follows:

[0087] Step 1: Dilute 5× Passive lysis buffer (PLB) with 4 times the volume of ultrapure water to obtain 1× PLB; Firefly luciferase reaction solution (LAR II): Dissolve the lyophilized luciferase assay substrate in luciferase assay buffer II (10 mL) and store at -80℃ protected from light; Renidae luciferase reaction solution (Stop&Glo): Dissolve 200 μL of Stop&Glo Substrate (50×) in 10 mL of Stop&Glo buffer to obtain 1× Stop&Glo Reagent solution and store at -80℃ protected from light;

[0088] Step 2: Take the callus tissue after it has been infected and cultured for 5 days, press it on absorbent paper to absorb water, divide the callus tissue transferred into each carrier into 3 parts, place them in 2.0 mL centrifuge tubes with 1 large and 1 small steel ball added, and freeze them in liquid nitrogen; then grind them with a vibratory grinder at 1200 rpm for 1 min.

[0089] Step 3: After grinding, add about 150 μL of 1×PLB to a centrifuge tube, mix well, vortex for 20 seconds, and let stand for 10 minutes to allow for complete lysis; then centrifuge at 12000 rpm for 10 minutes at 4°C, take 100 μL of the supernatant extract, add it to an ELISA plate, and set up 3 replicates.

[0090] Step 4: Add 100 μL of 1× firefly luciferase reaction solution, shake the plate to mix, and use an ELISA reader to detect the activity of firefly luciferase. The detection is completed within 30 minutes. The fluorescence intensity of the detected firefly luciferase is expressed in RLU (relative fluorescence unit).

[0091] Step 5: Add 100 μL of 1× Renina luciferase reaction solution, shake the plate to mix, and use an ELISA reader to detect the activity of Renina luciferase. The detection is completed within 30 minutes. The fluorescence intensity of the detected Renina luciferase is expressed in RLU (relative fluorescence units).

[0092] The BioTek-H1MF microplate reader's detection program is a full-spectrum light intensity reading.

[0093] The results of the detection of firefly luciferase (LUC) and kidney luciferase (REN) activities in transiently transformed maize callus are shown in Table 1. The REN gene was used as an internal control, and the LUC / REN ratio reflected the relative activity intensity of the promoter (LUC / REN ratio = (LUC value of maize callus transformed with recombinant expression vector - LUC value of wild-type maize callus) / (REN value of maize callus transformed with recombinant expression vector - REN value of wild-type maize callus)).

[0094] Table 1. Luciferase activity in transiently transformed maize callus.

[0095] The results in Table 1 show that in transiently transformed maize callus cells, both promoters prEiUbi2-01 and prZmUbi1 can normally drive the expression of the LUC gene, and the transcriptional intensity of prEiUbi2-01 is 1.5 times that of prZmUbi1. Therefore, the promoter prEiUbi2-01 can not only regulate gene expression normally, but also has a stronger transcriptional intensity than commonly used promoters in this field, showing promising application potential.

[0096] Example 3: Validation of the effect of the constitutive promoter prEiUbi2-01 in transgenic maize plants

[0097] 1. Stable transformation of maize using recombinant dual-luciferase vectors DBN13844 and DBN14252

[0098] Transformation was performed using the conventional Agrobacterium infection method. Aseptically cultured maize embryos were co-cultured with the Agrobacterium described in Example 2 to transfer the T-DNA from the constructed recombinant expression vector into the maize chromosome set, thereby generating transgenic maize.

[0099] For Agrobacterium-mediated maize transformation, briefly, immature embryos are isolated from maize and contacted with an Agrobacterium suspension, wherein Agrobacterium is capable of delivering the target nucleotide sequence and the nucleotide sequence of the pat gene to at least one cell of one of the embryos (step 1: infection step), in which the embryos are preferably immersed in an Agrobacterium suspension (OD). 660=0.4-0.6, inoculated in infection medium (MS salt 4.3 g / L, MS vitamins, casein 300 mg / L, sucrose 68.5 g / L, glucose 36 g / L, acetylsuccinone (AS) 40 mg / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg / L, pH 5.3). The embryos are co-cultured with Agrobacterium for a period (3 days) (Step 2: Co-culture step). Preferably, after the infection step, the embryos are cultured on solid medium (MS salt 4.3 g / L, MS vitamins, casein 300 mg / L, sucrose 20 g / L, glucose 10 g / L, AS 100 mg / L, 2,4-D 1 mg / L, agar 8 g / L, pH 5.8). After this co-culture phase, a selective "recovery" step can be performed. In the "recovery" step, the recovery medium (MS salt 4.3 g / L, MS vitamins, casein 300 mg / L, sucrose 30 g / L, 2,4-D 1 mg / L, cephalosporin 250 mg / L, plant gel 3 g / L, pH 5.8) contains at least one known antibiotic that inhibits the growth of Agrobacterium (cephalosporin 150-250 mg / L), without the addition of a selector for plant transformants (Step 3: Recovery Step). Immature embryos are cultured on a solid medium containing antibiotics but without a selector to eliminate Agrobacterium and provide a recovery period for infected cells, while simultaneously forming callus. The callus is then inoculated onto a medium containing a selector (4-[hydroxy(methyl)phosphono]-DL-homoalanine) and the growing transformed callus is selected (Step 4: Selection Step). Preferably, the callus is cultured on a selective solid medium containing a selector (MS salt 4.3 g / L, MS vitamins, casein 300 mg / L, sucrose 30 g / L, cephalosporin 250 mg / L, 4-[hydroxy(methyl)phosphono]-DL-homoalanine 10 mg / L, 2,4-D 1 mg / L, plant gel 3 g / L, pH 5.8), leading to selective growth of transformed cells. The callus then regenerates into a plant (step 5: regeneration step). Preferably, the callus grown on the selective medium is cultured on solid media (MS differentiation medium and MS rooting medium) to regenerate the plant.

[0100] The selected resistant callus tissues were transferred to the MS differentiation medium (MS salt 4.3 g / L, MS vitamins, casein 300 mg / L, sucrose 30 g / L, 6-benzyladenine 2 mg / L, cephalosporin 250 mg / L, 4-[hydroxy(methyl)phosphono]-DL-homoalanine 5 mg / L, plant gel 3 g / L, pH 5.8) and cultured at 25°C for differentiation. The differentiated seedlings were transferred to the MS rooting medium (MS salt 2.15 g / L, MS vitamins, casein 300 mg / L, sucrose 30 g / L, cephalosporin 250 mg / L, indole-3-acetic acid 1 mg / L, plant gel 3 g / L, pH 5.8) and cultured at 25°C until approximately 10 cm tall. They were then transferred to a greenhouse for further cultivation until fruit set. In the greenhouse, the seedlings were cultured at 28°C for 16 hours daily, followed by 8 hours at 20°C.

[0101] 2. Verification of maize plants transformed with recombinant dual-luciferase vectors DBN13844 and DBN14252

[0102] Genomic DNA was extracted using the DNeasy Plant Maxi Kit (Qiagen). The copy number of the screening marker PAT gene in vectors DBN13844 and DBN14252 was detected by Taqman probe real-time PCR. Wild-type maize plants were used as controls. The experiment was repeated three times and the average value was taken.

[0103] The specific method for detecting the PAT gene copy number is as follows:

[0104] Step 1: Take 100 mg of leaves from maize plants that have been transformed with recombinant dual-luciferase vectors DBN13844 and DBN14252 respectively, grind them into a homogenate in a mortar with liquid nitrogen, and take 3 replicates for each sample.

[0105] Step 2: Extract genomic DNA from the above samples. Refer to the instruction manual for specific methods.

[0106] Step 3: Measure the genomic DNA concentration using NanoDrop2000 (Thermo Scientific);

[0107] Step 4: Adjust the genomic DNA concentration to the same value, which is in the range of 80-100 ng / μL;

[0108] Step 5: The copy number of the sample was identified using the Taqman probe-based quantitative real-time PCR method. The sample with a known copy number was used as the standard, and the wild-type maize plant sample was used as the control. Each sample was repeated in triplicate, and the average value was taken. The primers for the quantitative real-time PCR detection of the PAT gene are shown in SEQ ID NO: 17-18, and the probe is shown in SEQ ID NO: 19.

[0109] The PCR reaction system is: JumpStart TM Taq ReadyMix TM 10 μL of (Sigma), 1 μL of 50× primer / probe mixture, 3 μL of genomic DNA, and 6 μL of water (ddH2O). The 50× primer / probe mixture contains 45 μL of each primer at 1 mM concentration, 50 μL of probe at 100 μM concentration, and 860 μL of 1×TE buffer, and is stored in amber tubes at 4°C.

[0110] The PCR reaction conditions are as follows:

[0111] Data were analyzed using SDS2.3 (Applied Biosystems) software. The results showed that the PAT gene had been integrated into the tested maize chromosome as a single copy, resulting in transgenic maize plants containing single-copy promoters prEiUbi2-01 and prZmUbi1.

[0112] 3. Detection of LUC / REN dual-luciferase activity in various tissues of maize plants

[0113] Roots and leaves were sampled from maize T0 plants that had been transfected with promoters prEiUbi2-01 and prZmUbi1 during the seedling stage; stems and leaves were sampled during the tasseling stage; silks and pollen were sampled during the flowering stage; and kernels were sampled during the grain-filling stage. Three replicates were performed for each tissue sample at each stage. The dual-luciferase activity of the samples was detected according to the method described in Example 2 of this application, and the results are shown in Table 2.

[0114] Table 2. Luciferase activity at different stages and parts of stably transformed maize plants.

[0115] Table 2 shows that the promoter prEiUbi2-01 can normally drive constitutive expression of the LUC gene in all tissues during various growth and development stages of maize. In particular, LUC expression driven by prEiUbi2-01 is superior to that driven by prZmUbi1 in leaves, silks, kernels, and the tip of the ear cob. Specifically, in leaves at the seedling and tasseling stages, LUC expression driven by prEiUbi2-01 is 1.4 times and 1.1 times higher than that driven by prZmUbi1, respectively; in silks at the flowering stage, LUC expression driven by prEiUbi2-01 is 2.0 times higher than that driven by prZmUbi1; and in kernels and the tip of the ear cob at the grain-filling stage, LUC expression driven by prEiUbi2-01 is 1.8 times and 2.3 times higher than that driven by prZmUbi1, respectively. It is evident that the promoter prEiUbi2-01 exhibits strong transcriptional activity in maize, and its activity in some tissues is stronger than that of the commonly used promoter prZmUbi1 in this field. The novel constitutive promoter prEiUbi2-01 of this application provides a new tool and option for the expression of exogenous genes in plant species, and has great application potential.

[0116] Example 4: Verification of the effect of the constitutive promoter prEiUbi2-01 on driving PAT gene expression in maize

[0117] 1. Construction of expression vectors containing the constitutive promoter prEiUbi2-01 and the PAT gene

[0118] To further determine the utility of the constitutive promoter prEiUbi2-01, the promoter was operatively linked to a herbicide-tolerant gene, and it was verified whether it could drive the expression of the gene, thereby conferring the herbicide-tolerant trait to the plant.

[0119] Specifically, a plant expression vector for the PAT gene driven by the promoter prEiUbi2-01 was constructed. The backbone vector DBNBC-01 (pCAMBIA2301 with modified resistance tag, available from CAMBIA) was linearized using the restriction endonuclease SalⅠ. The prEiUbi2-01 (SEQ ID NO:1) containing the adapter sequence was amplified using primer pairs SEQ ID NO:20 and SEQ ID NO:21. The cPAT (SEQ ID NO:10), t35S (SEQ ID NO:11), prZmUbi1 (SEQ ID NO:12), cPMI (SEQ ID NO:32), and tNos (SEQ ID NO:5) elements containing the adapter sequence were amplified using primer pairs shown in SEQ ID NO:28 and SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31, SEQ ID NO:22 and SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25, and SEQ ID NO:26 and SEQ ID NO:27 elements.

[0120] The linearized backbone vector DBNBC-01 fragment was mixed with prEiUbi2-01 element with adapter sequence, cPAT element with adapter sequence, t35S element with adapter sequence, prZmUbi1 element with adapter sequence, cPMI element with adapter sequence, and tNos element, respectively, for recombination. The operation procedure was performed according to the instructions of Takara In-Fusion Snap Assembly Master Mix kit (Clontech, CA, JPN, CAT: 638949) to construct the recombinant vector DBN14915 containing the PAT gene, and its structural schematic diagram is shown in Figure 4.

[0121] 2. Recombinant expression vector DBN14915 was transformed into Agrobacterium.

[0122] Referring to Example 2 of this application, the recombinant expression vector DBN14915 was transformed into Agrobacterium LBA4404 using liquid nitrogen, and the plasmid was extracted and sequenced for identification. The results showed that the structure of the recombinant expression vector DBN14915 was completely correct.

[0123] 3. Recombinant expression vector DBN14915 stably transforms maize

[0124] Referring to Example 3 of this application, Agrobacterium tumefaciens transformed with the recombinant expression vector DBN14915 was used to infect maize, and the T-DNA of the recombinant expression vector DBN14915 was inserted into the maize chromosome to obtain the corresponding transgenic maize T0 plants.

[0125] Approximately 100 mg of leaves from transgenic maize T0 plants were collected as a sample. Genomic DNA was extracted using Qiagen's DNeasy Plant Maxi Kit. The copy number of the PAT gene in vector DBN14915 was detected by TaqMan probe-based quantitative real-time PCR. Wild-type maize plants were used as a control. The experiment was performed in triplicate, and the average value was taken. The primers for quantitative real-time PCR detection of the PAT gene are shown in SEQ ID NO:17-18, and the probe is shown in SEQ ID NO:19.

[0126] The experimental results showed that the fragment of the PAT gene driven by prEiUbi2-01 was successfully integrated into the genome of the transgenic maize plants tested, and existed in the form of a single copy.

[0127] 4. Herbicide resistance assessment of genetically modified maize

[0128] Transgenic maize T0 plants incorporating the prEiUbi2-01 promoter and PAT gene, and wild-type maize plants (18 days after sowing), were uniformly sprayed with glufosinate-ammonium at 800 g ai / ha (2 times the field concentration, 2×). Seven days after herbicide application (7DAT), the glufosinate resistance / tolerance level was assessed based on the response of maize leaves and the overall plant. The severity of plant damage was graded as follows: Grade 0: virtually no phytotoxicity; Grade 1: slight burning at the leaf base, ≤10% of the leaf surface area; Grade 2: significant burning at the leaf base, >10% of the leaf surface area, possibly accompanied by slight leaf curling or plant tilting, recovering within 14 days; Grade 3: deformed leaves or tilted growth, not recovering within 14 days; leaves broken at the phytotoxic site; Grade 4: severely deformed plants; wilting, drying, and death of leaves.

[0129] The resistance performance of the transformation event was scored according to the formula X=[∑(N×S) / (T×M)]×100 (X-phytotoxicity score, N-number of affected plants of the same level, S-number of phytotoxicity levels, T-total number of plants, M-highest phytotoxicity level). Resistance was evaluated based on the score: highly resistant (0-15 points), moderately resistant (16-33 points), lowly resistant (34-67 points), and not resistant (68-100 points). The experimental results are shown in Table 3 and Figure 5.

[0130] Table 3. Results of experiments on the tolerance of transgenic maize T0 plants to glufosinate.

[0131] As shown in Table 3, when treated with 2 times the field concentration of glufosinate, compared to wild-type maize, the transgenic maize plants transformed with the PAT gene driven by the constitutive promoter prEiUbi2-01 exhibited less leaf damage and moderate resistance to glufosinate. Wild-type maize plants, on the other hand, showed more severe leaf damage and lacked herbicide resistance. Figure 5 shows that the transgenic maize plants transformed with the PAT gene driven by the constitutive promoter prEiUbi2-01 (the four plants on the right) grew normally with almost no leaf damage, indicating good herbicide tolerance. Wild-type maize (the one on the far left), however, showed leaf withering and death, indicating a lack of herbicide tolerance. In conclusion, the constitutive promoter prEiUbi2-01 of this application can normally drive the expression of the target gene in plants and confer corresponding characteristics.

[0132] Example 5: Construction and activity assay of the truncated form of the constitutive promoter prEiUbi2-01

[0133] 1. Construction of a dual-luciferase expression vector containing a truncated version of the promoter prEiUbi2-01

[0134] Bioinformatics analysis of the constitutive promoter prEiUbi2-01 sequence was performed using online analysis websites such as TSSP. The results showed that the promoter sequence contains cis-acting elements that respond to hormones, cis-acting elements that respond to light signals, and cis-acting elements that participate in hypoxia-specific induction. It also contains multiple cis-acting elements such as CAAT Box related to transcriptional activity and TATA BOX related to transcription initiation.

[0135] To further identify the transcriptional regulatory elements controlling the activity of the constitutive promoter prEiUbi2-01, namely the sequence with the highest promoter-driven target gene expression efficiency and the shortest sequence with transcriptional activity, the full-length promoter prEiUbi2-01 was truncated to different degrees by unidirectional deletion of the 5' end, based on the element distribution in the promoter sequence. The four truncated promoter fragments were 2544bp, 1974bp, 1404bp, and 694bp in size, corresponding to SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively, and were named prEiUbi2-02, prEiUbi2-03, prEiUbi2-04, and prEiUbi2-05.

[0136] Referring to the dual-luciferase expression vector containing the promoter prEiUbi2-01 constructed in Example 2 of this application, the backbone vector DBNBC-Dual_LUC containing LUC and REN dual-luciferase was linearized using the restriction endonuclease SalⅠ. The truncated promoter of the full-length promoter prEiUbi2-01 was amplified using the primer pairs shown in SEQ ID NO:37 and SEQ ID NO:16, SEQ ID NO:38 and SEQ ID NO:16, SEQ ID NO:39 and SEQ ID NO:16, and SEQ ID NO:40 and SEQ ID NO:16, respectively. The linearized backbone vector DBNBC-Dual_LUC fragment was mixed with truncated promoters containing adapter sequences for recombination reactions. The procedure was performed according to the instructions of the Takara In-Fusion Snap Assembly Master Mix kit (Clontech, CA, JPN, CAT: 638949) to obtain dual-luciferase expression vectors DBN14526, DBN14527, DBN14528, and DBN14529 containing truncated promoters prEiUbi2-02, prEiUbi2-03, prEiUbi2-04, and prEiUbi2-05.

[0137] 2. Activity assay of the truncated form of promoter prEiUbi2-01 in maize callus

[0138] Using the method described in Example 2 of this application, vectors DBN14252, DBN14526, DBN14527, DBN14528, and DBN14529 were used to transiently transform maize callus tissue. The corresponding supernatants were prepared for use, and the luciferase activity was detected. The results are shown in Table 4.

[0139] Table 4. Luciferase activity in maize callus tissue transiently transformed by different promoter truncated variants.

[0140] As shown in Table 4, in transiently transformed maize callus cells, the truncated promoters prEiUbi2-02, prEiUbi2-03, and prEiUbi2-04 of the full-length promoter prEiUbi2-01 all exhibited good transcriptional activity. Specifically, LUC expression driven by prEiUbi2-02, prEiUbi2-03, and prEiUbi2-04 was 0.7-fold, 0.7-fold, and 0.4-fold, respectively, higher than that driven by prEiUbi2-01. However, under the influence of prEiUbi2-05, LUC expression was almost nonexistent, indicating that the truncated promoter shown in SEQ ID NO:36 had virtually no transcriptional activity. Therefore, SEQ ID NO:35 is the shortest sequence with promoter activity. Based on the analysis of the active elements and element distribution of the full-length prEiUbi2-01 promoter, those skilled in the art can reasonably expect that when the promoter sequence contains SEQ ID NO:35, it has transcriptional activity and can perform the normal function of the promoter, thereby driving the expression of the target gene.

[0141] In summary, this invention discloses for the first time a constitutive promoter from *Eleusine indica*. The constitutive promoter of this invention shows activity in almost all plant tissues and many cell types, especially in the roots, stems, leaves, filaments, pollen, grains, and rachis tips of plants, and can normally drive the expression of target genes. This provides a new tool and option for plant genetic engineering and has good application prospects.

[0142] While the above descriptions are merely examples of specific embodiments of the present invention, those skilled in the art should understand that these are only illustrative, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, and all such changes or modifications shall fall within the scope of protection of the present invention.

Claims

1. A constitutive promoter, characterized in that, The nucleotide sequence of the constitutive promoter includes SEQ ID NO:

35.

2. The constitutive promoter according to claim 1, characterized in that, The nucleotide sequence of the constitutive promoter includes SEQ ID NO:35, and the constitutive promoter is a truncated sequence of SEQ ID NO:

1.

3. The constitutive promoter according to claim 1 or 2, characterized in that, The nucleotide sequences of the constitutive promoter are shown in SEQ ID NO:1, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:

35.

4. A chimeric gene, characterized in that, The formulation comprising any one of claims 1-3 operably linked to the target nucleic acid; Preferably, the target nucleic acid encodes the target protein.

5. An expression box, characterized in that, It includes the constitutive promoter of any one of claims 1-3 or the chimeric gene of claim 4.

6. A recombinant vector, characterized in that, It comprises a constitutive promoter as described in any one of claims 1-3, a chimeric gene as described in claim 4, or an expression cassette as described in claim 5.

7. A host cell, characterized in that, It comprises the constitutive promoter of any one of claims 1-3, the chimeric gene of claim 4, the expression cassette of claim 5, or the recombinant vector of claim 6.

8. A method for expressing a target nucleic acid in a plant, characterized in that, include: The target nucleic acid, operatively linked to the constitutive promoter of any one of claims 1-3, is stably integrated into plant cells; Preferably, the target nucleic acid is constitutively expressed in plant tissues; Preferably, the target nucleic acid encodes the target protein; More preferably, the target nucleic acid encodes a herbicide-resistant protein or an insect-resistant protein.

9. The method for expressing a target nucleic acid in a plant according to claim 8, characterized in that, The plants mentioned are corn, Arabidopsis thaliana, rapeseed, tobacco, soybean, cotton, chili pepper, beet, pumpkin, eggplant, Chinese cabbage, carrot, tomato, pea, spinach, potato, or peanut.

10. A plant or plant part, characterized in that, It includes the constitutive promoter of any one of claims 1-3, the chimeric gene of claim 4, the expression cassette of claim 5, or the recombinant vector of claim 6.

11. A method for obtaining processed agricultural products, characterized in that, This includes processing the harvest of the plant or part thereof as described in claim 10 to obtain processed agricultural products.

12. Use of the constitutive promoter according to any one of claims 1-3 for constitutive expression of a target nucleic acid in plant tissues.

13. The use of the constitutive promoter according to claim 12 for the constitutive expression of a target nucleic acid in plant tissues, characterized in that, The target nucleic acid encodes the target protein; Preferably, the target nucleic acid encodes a herbicide-resistant protein or an insect-resistant protein.

14. The use of the constitutive promoter according to claim 12 or 13 for constitutively expressing a target nucleic acid in plant tissues, characterized in that, The plants mentioned are corn, Arabidopsis thaliana, rapeseed, tobacco, soybean, cotton, chili pepper, beet, pumpkin, eggplant, Chinese cabbage, carrot, tomato, pea, spinach, potato, or peanut.

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