Constitutive promoter and use thereof

Abstract

The invention relates to a constitutive promoter and application thereof. The nucleotide sequence of the constitutive promoter comprises SEQ ID NO: 35. The constitutive promoter disclosed by the invention almost shows activity in most tissues and many types of cells of a plant, especially in roots, stems, leaves, filaments, pollen, grains and spike-stalk apexes of the plant, and has a wide application prospect in the plant.

Description

Technical Field

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

[0002] One of the goals of plant genetic engineering is to modify plants according to people's needs, so as to produce plants with desired characteristics or traits. Commonly desired traits include improving nutritional value, increasing yield, imparting resistance to pests and diseases, improving drought and stress tolerance, improving horticultural quality, and imparting herbicide resistance. Currently, researchers have been able to obtain exogenous genes (such as genes from heterologous or natural sources) and integrate the genes into the plant genome to express them to show the corresponding traits.

[0003] When expressing target genes in plants, it is very important to select appropriate regulatory signals. Typical regulatory signals include promoter regions, 5' untranslated guide sequences, and 3' transcription terminator / polyadenylation sequences. Among them, the promoter is a DNA sequence located upstream of the 5' end of the structural gene that can be recognized, bound, and initiated by RNA polymerase to initiate gene transcription. In plant genetic engineering, promoters are often divided into three categories according to their mode of action and function: constitutive promoters, specific promoters, and inducible promoters. Constitutive promoters are a type of promoter that guides RNA synthesis at a certain expression level in most or all tissues and / or growth and development stages of plants, and can be divided into strong, medium, and weak promoters according to their effectiveness in guiding RNA synthesis. Since in many cases it is necessary to simultaneously express the target gene (or chimeric gene) in different tissues of the plant to obtain the desired gene function, constitutive promoters are particularly advantageous in the above cases.

[0004] Some constitutive promoters that work in plant cells have been described in the existing literature, including the promoters Gm1 7gTsf1 of the soybean cell elongation factor gene, the nopaline (nos) promoter carried on the tumor-inducing plasmid of Agrobacterium tumefaciens, the octopine synthase (ocS) promoter, and the caulimovirus promoter, such as the cauliflower mosaic virus (CaMV) 19S or 35S promoter, the CaMV 35S promoter with a repeat enhancer, and the figwort mosaic virus (FMV) 35S promoter, which have been used in transgenic expression plant constructs. Although some constitutive promoters have been obtained, it is still a hot topic in the art to explore and isolate more sources and constitutive promoters that are safe and efficient, and can control the expression of target genes (or chimeric genes) at different levels or be used for the superposition expression of multiple genes in the same transgenic plant. Summary of the invention

[0005] The purpose of the present invention is to provide a new constitutive promoter and its use. The promoter is derived from Eleusine indica L. and can regulate the efficient expression of target nucleic acid in tissues such as roots, stems, leaves, filaments, pollen, grains, and cob tops of plants, providing new tools and options for plant genetic engineering and having very broad application prospects.

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

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

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

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

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

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

[0012] Further preferably, the nucleotide sequence of the constitutive promoter is as shown in SEQ ID NO: 1, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35.

[0013] In a second aspect, the present invention also provides a chimeric gene comprising the above constitutive promoter operably linked to a target nucleic acid.

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

[0015] In a third aspect, the present invention further provides an expression cassette, comprising the constitutive promoter or chimeric gene of the present invention.

[0016] In a fourth aspect, the present invention further provides a recombinant vector comprising the constitutive promoter or chimeric gene or expression cassette of the present invention.

[0017] In a fifth aspect, the present invention further provides a host cell, which comprises 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 operably linked to the above-mentioned constitutive promoter into a plant cell.

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

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

[0021] More preferably, the nucleic acid of interest encodes a herbicide tolerance protein.

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

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

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

[0025] In an eighth aspect, the present invention also provides a method for obtaining processed agricultural products, comprising processing the harvested material of the above-mentioned plants or plant parts to obtain processed agricultural products.

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

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

[0028] More preferably, the nucleic acid of interest encodes a herbicide tolerance protein.

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

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

[0031] The term "promoter" in the present invention refers to a DNA regulatory region, usually containing a TATA box that can guide RNA polymerase II to initiate RNA synthesis at a suitable transcription start site of a specific coding sequence.

[0032] The term "gene" or "nucleic acid" as used herein 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 an appropriate regulatory region (e.g., a plant expressible promoter region). A gene or nucleic acid 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 a partial or complete transcribed DNA region or with at least another regulatory region of the gene under natural conditions.

[0033] The term "target gene" or "target nucleic acid" in the present invention may be an endogenous gene or nucleic acid, or an exogenous or heterologous gene or nucleic acid. An endogenous gene or nucleic acid refers to a gene naturally found or present in a plant species or plant host, and an exogenous or heterologous gene or nucleic acid refers to any gene that does not belong to or does not exist in a plant species or plant host. Alternatively, the target gene or target nucleic acid may be a natural sequence or a selectively synthesized sequence.

[0034] The target gene or target nucleic acid of the present invention can be homologous to the promoter of the present application, for example, the target gene or target nucleic acid and the promoter are derived from the same plant species or are naturally connected together. The target gene or target nucleic acid of the present invention can also be exogenous or heterologous to the promoter of the present application, that is, the target gene or target nucleic acid and the promoter are derived from different plant species or are non-naturally connected together or exist together. For example, a chimeric gene comprises a promoter sequence of the present invention, and the promoter sequence of the present invention is operably connected to a coding sequence different from the promoter sequence of the present invention. Although the target gene or target nucleic acid sequence is heterologous to the promoter sequence, it may be homologous or natural or heterologous or exogenous to the plant host.

[0035] The term "constitutive promoter" in the present invention 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 show a certain degree of gene / nucleic acid expression. The constitutive promoter is used to operably connect the target gene, target nucleic acid or gene editing system guide RNA (gRNA) so that it is expressed in most cells of the organism, and the initiated expression has a certain persistence. It is understandable that for the term "constitutive promoter", there may be some changes in the absolute level of expression or activity between different tissues and developmental stages of an organism. The tissue is a structural unit in a 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, and several different tissues are organically coordinated and closely connected to form different organs (organs), and 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. In the present invention, the terms "constitutive expression" and "constitutively expressed" 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 the plant. For example, the cauliflower mosaic virus CaMV35S promoter can initiate high-intensity expression of exogenous genes in most organs and different developmental stages in plants; constitutive promoters can also ensure the widespread expression of gRNA in host cells, thereby improving the efficiency and accuracy of gene editing. The promoter of the present invention can be homologous, exogenous or heterologous to the plant species or plant host. Homologous means that the promoter is derived from the plant species or plant host into which the transcription initiation region is introduced, and exogenous or heterologous means that the promoter or transcription initiation region does not exist in the natural plant or plant host into which the transcription initiation region is introduced.

[0036] The present 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 technical solution of the present invention, the nucleotide sequence of the constitutive promoter includes SEQ ID NO: 35 and is derived from SEQ ID NO: 1. Further, the nucleotide sequence of the promoter of the present invention includes SEQ ID NO: 35 and is at least a part of SEQ ID NO: 1 or is 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 truncated sequence of the 5' end (N end) of SEQ ID NO: 1, and the truncation 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, and is also the remaining fragment (truncated fragment) after truncating 1671 bp from the 5' end of SEQ ID NO: 1. The constitutive promoter of the present invention can be extended to its 5' end or 3' end on the basis of SEQ ID NO: 35. In a preferred technical solution, the constitutive promoter of the present invention can refer to SEQ ID NO: 1 and extend to its 5' end on the basis 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 the present application is a truncated nucleotide fragment obtained by truncating or removing nucleotides with a length of less than or equal to 1671 bp from the 5' end of SEQ ID NO: 1, and the truncated fragment includes SEQ ID NO: 35. The constitutive promoters obtained as described above are all within the protection scope of the present invention.

[0038] For the above promoter, the other nucleotides included in its nucleotide sequence except SEQ ID NO: 35 do not affect the activity of the promoter itself. Example 5 of the present invention also proves this conclusion: the prEiUbi2-04 promoter (SEQ ID NO: 35) is active, and the promoters prEiUbi2-02 (SEQ ID NO: 33), prEiUbi2-03 (SEQ ID NO: 34), and prEiUbi2-01 (SEQ ID NO: 1) containing SEQ ID NO: 35 all have good activity. Those skilled in the art can reasonably expect based on the contents recorded in this application that SEQ ID NO: 35 is the shortest fragment (minimum unit) that exerts the function of the promoter, and when the nucleotide sequence of the promoter includes SEQ ID NO: 35, it has the function and activity of the promoter. Similarly, when the nucleotide sequence of the promoter includes SEQ ID NO: 35 and is selected from at least a portion of SEQ ID NO: 1 or a truncated sequence of SEQ ID NO: 1, a person skilled in the art can also 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 having promoter activity and being 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, i.e., the range of sequence identity is distributed in at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0040] Isolated sequences having promoter activity and hybridizing to the promoter sequence of the present invention or fragments thereof 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 distributed at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0041] The promoter sequence of the present invention and its fragment, when assembled into DNA structure so that the promoter sequence is operably connected with the target nucleic acid sequence, are used for genetic manipulation of any plant. The "operably connected" refers to the functional connection between the promoter sequence of the present invention and the second sequence, wherein the promoter sequence starts and regulates the transcription of the DNA sequence corresponding to the second sequence. Generally, operably connected means that the nucleic acid sequence connected is continuous, adjacently combines two protein coding regions when necessary, and is in the same reading frame. In this way, the promoter nucleotide sequence and the target nucleotide sequence constitute the chimeric gene and are provided together in an expression cassette to express in the target plant. This expression cassette provides a large number of restriction sites to insert the target nucleotide sequence, and the target nucleotide sequence will be subject to transcriptional regulation of the regulatory region comprising the promoter sequence of the present invention. The expression cassette may additionally contain at least one additional gene that will be co-transformed into the organism. Alternatively, additional genes can be provided on multiple expression cassettes.

[0042] The expression cassette may additionally contain a selectable marker gene. Generally, the expression cassette will contain a selectable marker gene for selecting transformed cells. The selectable marker gene is used to select transformed cells or tissues. The selectable marker gene includes, but is 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, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D) resistance genes.

[0043] The expression cassette comprises a promoter sequence of the present invention transcribed in the 5'-3' direction, a translation initiation region, a heterologous nucleotide sequence of interest, and transcriptional and translational termination regions functional in plants.

[0044] The termination region can be derived from the promoter sequence of the present invention, can also be derived from the heterologous nucleotide sequence of interest that can be operably linked, or can be derived from another source. Traditional termination regions can be obtained from the Ti plasmid of Agrobacterium tumefaciens, such as the carnitine synthase and nopaline synthase (NOS) termination regions.

[0045] In the preparation of expression cassettes, the various DNA fragments may be manipulated to provide the DNA sequence in the proper orientation and, where appropriate, the proper reading frame. Thus, acceptors or linkers may be used to join the DNA fragments, or other manipulations may be performed to provide convenient restriction sites, remove excess DNA, remove restriction sites, etc. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitution, such as conversion and transformation, may be involved.

[0046] Where appropriate, the heterologous nucleotide sequence of interest can be optimized to increase expression in transformed plants, i.e., the gene can be synthesized using plant-preferred codons to improve expression.

[0047] As is well known in the art, other sequence modifications can improve the gene expression level in the cell host. These include, but are not limited to, removal of the repetitive sequences encoding false polyadenylation signals, exon-intron splice site signals, transposons, and other fully characterized sequences that may be detrimental to gene expression. The GC content of the sequence can be adjusted to the average level of the specified host cell, citing the known gene expression level in the host cell for calculation. Possibly, the modified sequence is to avoid the predicted hairpin mRNA secondary structure.

[0048] In the expression cassette or recombinant vector, the expression cassette may additionally contain a 5' leader sequence. The leader sequence may serve to improve transcription efficiency. The leader sequence is known in the art and includes, but is not limited to, picornavirus leaders, such as the EMCV leader (encephalomyocarditis virus 5' non-coding region); Potatovirus leaders, such as the tobacco etch virus (TEV) leader, the maize dwarf mosaic virus (MDMV) leader, and the human immunoglobulin heavy chain binding protein (BiP); the non-translated leader from the alfalfa mosaic virus coat protein mRNA (AMV RNA4); the tobacco mosaic virus (TMV) leader; and the maize chlorosis mottle virus (MCMV) leader. Other known elements that improve transcription efficiency, such as introns, etc., may also be used.

[0049] The promoter sequence of the present invention can be used to initiate the transcription of an antisense structure that is at least partially complementary to the messenger RNA (mRNA) of the target nucleic acid sequence. Antisense nucleotide sequences are constructed to hybridize with the corresponding mRNA. As long as the antisense sequence is long enough to hybridize with the corresponding mRNA and interfere with its expression, the antisense sequence can be modified. In this way, antisense structures having 80%, preferably 90%, and more preferably 95% sequence identity with the corresponding antisense sequence can be used. In addition, 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] The nucleic acid or nucleic acid sequence operably linked to the promoter of the present invention can encode a protein of interest. Examples of such nucleic acids or nucleic acid sequences include, but are not limited to, nucleotide sequences encoding polypeptides that confer resistance to abiotic stresses such as drought, temperature, salinity, ozone, and herbicides, or biotic stresses such as pathogen attack, including insects, viruses, bacteria, fungi, and nematodes, and prevent the production of diseases associated with these organisms.

[0051] The herbicide tolerance protein of the present invention can express resistance and / or tolerance to herbicides. These genes include, but are not limited to, hydroxyphenylpyruvate dioxygenase (HPPD) gene, protoporphyrinogen oxidase (PPO) gene, acetolactate synthase (ALS) gene, 5-enolpyruvyl-3-phosphate synthase (EPSPS) gene, glufosinate acetyltransferase (PAT) gene, glyphosate oxidoreductase (GOX) gene, GAT gene, etc.

[0052] In the present invention, "insect resistance" refers to the symptoms and damage caused by plant-insect interactions that plants avoid. That is, plant damage, crop damage, plant shape and plant diseases caused by insects are prevented, or alternatively, plant damage, crop damage, plant shape and plant diseases caused by insects are reduced to a minimum or alleviated. The insects may belong to Lepidoptera (such as corn borers), Hemiptera (such as stink bugs), Coleoptera (such as beetles), Orthoptera (such as locusts), Homoptera (such as aphids), Diptera (such as flies), etc. As is well known in the art, target insect resistance proteins include, but are not limited to, Bacillus toxic proteins; lectins, wherein lectins include snowdrop lectin, pea lectin, jack bean lectin, malt lectin, potato lectin, peanut lectin, etc.; lipoxygenases, wherein lipoxygenases include pea lipoxygenase 1 or soybean lipoxygenase; insect chitinase, etc.

[0053] Different pests transfer viruses from infected plants to healthy plants in different ways. Such viruses include, but are not limited to, rice tungro baculovirus, tobacco mosaic virus, sweet potato chlorotic stunt virus, and sweet potato feathery mottle virus, etc. Therefore, heterologous nucleotide sequences that constitutively express anti-pathogenic activity or minimize the effects of viral pathogens can be selected in plant tissues.

[0054] The promoter sequence and method of the present invention can be used for the expression regulation of any target nucleic acid in a plant host to change the phenotype of the plant. Various target phenotype changes include, but are not limited to, changing the fatty acid composition of the plant, changing the amino acid content of the plant, changing the defense mechanism of plant pathogens, etc. The above changes can be obtained by providing the expression of a heterologous product or increasing the expression of a plant endogenous product. Alternatively, the above changes can be obtained by reducing the expression of one or more endogenous products in the plant, particularly enzymes or cofactors. The above changes will result in a phenotype change 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, microbead bombardment, direct DNA uptake into protoplasts, electroporation, or silicon whisker-mediated DNA introduction.

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

[0057] Host cells of the present invention include but are not limited to plant cells or bacteria. Bacteria include but are not limited to Agrobacterium, Bacillus, Escherichia, Salmonella, Pseudomonas or Rhizobium cells. Plant cells are non-renewable cells or renewable cells. Non-renewable cells refer to cells that cannot be regenerated into whole plants by in vitro culture. Non-renewable cells can be in plants or plant parts (e.g., leaves) of the present invention. Non-renewable cells can be cells in seeds or the seed coat of the seeds. Mature plant organs (including mature leaves, mature stems or mature roots) contain at least one non-renewable cell. On the other hand, plant cells are living cells with incomplete structures, such as plant cells without nuclei. Specifically, plant cells are sieve tube cells (mature sieve tube cells) without nuclei. On the other hand, plant cells are reproductive cells, such as ovules or cells as 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 whole plants, including whole plants and plant populations, such as desired and undesired wild plants or crop plants (including naturally occurring crop plants). Crop plants can be plants obtained by traditional breeding and optimization methods or by biotechnology and recombinant 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 be regenerated, plant calli, plant clumps, and intact plant cells in plants or plant parts. The plant parts are, for example, embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stalks, roots, root tips, anthers, etc. It should be understood that the parts of transgenic plants within the scope of the present invention include, but are not limited to, plant cells, protoplasts, tissues, calli, embryos, and flowers, stems, fruits, leaves and roots, and the above plant parts are derived from transgenic plants or their progeny that have been transformed with the DNA molecules of the present invention in advance and are therefore at least partially composed of transgenic cells. In one aspect, the plant part is the above-mentioned plant cell.

[0060] The present invention provides a process for processing a harvested product of a plant or part thereof containing a constitutive promoter of the present invention to obtain a processed agricultural product. The term "processed agricultural product" refers to any composition or product consisting of materials derived from a plant, seed, plant cell or plant part containing a constitutive promoter of the present invention. Specifically, the term "processed agricultural product" includes, but is not limited to, protein concentrates, protein isolates, starch, flour, biomass and seed oils.

[0061] The present invention provides a constitutive promoter and its use, which has the following beneficial effects:

[0062] The present invention discloses for the first time a constitutive promoter from Eleutherodactylus truncatula, which shows good activity in most tissues and cells of plants, especially in tissues such as roots, stems, leaves, filaments, pollen, grains, and cob tips of plants, and can drive the efficient expression of exogenous genes or nucleic acids, providing new tools and options for plant genetic engineering, and having very excellent application potential.

[0063] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The schematic diagram of the structure of the vector DBNBC-Dual LUC containing LUC and REN reporter genes of the present invention;

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

[0066] Figure 3 Schematic diagram of the structure of the recombinant dual-luciferase expression vector DBN14252 containing the promoter prEiUbi2-01;

[0067] Figure 4 Schematic diagram of the structure of the recombinant vector DBN14915 driven by the constitutive promoter prEiUbi2-01 to express the PAT gene;

[0068] Figure 5 This is a diagram showing the tolerance of transgenic corn with the PAT gene driven by the constitutive promoter prEiUbi2-01 to the glufosinate herbicide. DETAILED DESCRIPTION

[0069] The technical scheme of the constitutive promoter and its use of the present invention is further illustrated by specific examples below.

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

[0071] The nucleotide sequence of maize ubiquitin protein was used as the reference sequence in NCBI

[0072] (https: / / www.ncbi.nlm.nih.gov / ) was searched and compared with homologous genes, and a candidate G. elegans ubiquitin protein gene (GenBank: QEPD01000483.1) was obtained. The sequence about 3 kb upstream of the G. elegans ubiquitin protein gene was selected as the target sequence. G. elegans genomic DNA was used as a PCR amplification template, and primer 1 (SEQ ID NO: 2) and primer 2 (SEQ ID NO: 3) were designed for PCR amplification.

[0073] PCR used a 50 μL reaction system, including 0.5 μL of Q5 polymerase, 10 μL of reaction buffer, 1 μL of 10 mM dNTPs, 2.5 μL each of 10 μM primer 1 and 10 μM primer 2, and genomic DNA <1000 ng, supplemented with nuclease-free water to 50 μL.

[0074] The PCR reaction conditions were:

[0075]

[0076] Among them, Q5 polymerase is New England The company's high-fidelity Q5DNA polymerase, the reaction buffer is The reaction buffer in the company's High-Fidelity DNA Polymerase kit, the specific operation steps are as follows Company PCR Using The procedure was performed according to the instructions of High-Fidelity DNA Polymerase (M0491) kit.

[0077] The above PCR product was connected to a blunt-end Blunt vector (Beijing Quanshijin Company) according to the operating steps of the Blunt vector product manual of Quanshijin Company, and then the ligation product was sequenced (Sanger method) to confirm that the constructed Blunt vector contained a promoter fragment of the ubiquitin protein gene of Glechoma longituba with a length of 3075 bp, the sequence of which is shown in SEQ ID NO: 1 and named prEiUbi2-01.

[0078] Example 2: Verification of the effect of the constitutive promoter prEiUbi2-01 in corn callus

[0079] 1. Construct a dual-luciferase expression vector containing the constitutive promoter prEiUbi2-01 and introduce a dual-luciferase reporter system (Dual-Luciferase Reporter) containing firefly luciferase (LUC) and Rellina luciferase (REN). The efficiency of promoter-driven target gene expression is determined by detecting the activity of LUC. REN is used as an internal reference to eliminate inter-group errors caused by factors such as different transformation efficiencies of plant tissues due to Agrobacterium infection.

[0080] The structure of the vector DBNBC-Dual LUC (vector backbone: pCAMBIA2301 with modified resistance tags, provided by CAMBIA) containing LUC and REN reporter genes is as follows: Figure 1 As shown. Among them, cSpec is the spectinomycin gene, RB is the right border, cLUC is the firefly luciferase gene (SEQ ID NO: 4), tNos is the Agrobacterium tumefaciens nopaline 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 proteinase inhibitor II gene terminator (SEQ ID NO: 8), prOsAct1 is the rice actin gene promoter (SEQ ID NO: 9), cPAT is the phosphinothricin acetyltransferase gene (SEQ ID NO: 10), t35S is the cauliflower mosaic virus gene terminator (SEQ ID NO: 11), and LB is the left border.

[0081] A vector for LUC gene expression driven by prEiUbi2-01 promoter was constructed and transformed into corn, and the constitutive promoter prZmUbil (SEQ ID NO: 12) known in the art was used as a control. Luciferase activity was measured in each growth period and each tissue cell of corn to compare and analyze the expression pattern and expression intensity of the promoter prEiUbi2-01. It is well known to those skilled in the art to construct a vector using conventional enzyme digestion methods. Specifically, the DBNBC-Dual_LUC vector was linearized using the restriction endonuclease SalI, and the primer pairs shown in SEQ ID NO: 13 and SEQ ID NO: 14, and the primer pairs shown in SEQ ID NO: 15 and SEQ ID NO: 16 were used to amplify the prZmUbil element with a linker sequence (SEQ ID NO: 12) and the prEiUbi2-01 element with a linker sequence (SEQ ID NO: 1). The linearized DBNBC-Dual_LUC vector was mixed with the prZmUbi1 element with a linker sequence and the prEiUbi2-01 element with a linker sequence for recombination reaction. The operation steps were carried out according to the instructions of the In-Fusion Snap Assembly Master Mix kit of Takara Company (C1ontech, CA, JPN, CAT: 638949), and the recombinant dual luciferase expression vectors DBN13844 and DBN14252 were constructed. The structures are shown in Figure 2 and Figure 3 shown.

[0082] 2. Transformation of Agrobacterium with recombinant dual luciferase expression vector

[0083] The correctly constructed recombinant dual luciferase expression vectors DBN13844 and DBN14252 were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA; Cat. No: 18313-015) by liquid nitrogen method. 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, then incubated in 37°C warm water for 10 min, the transformed Agrobacterium LBA4404 was inoculated into an LB test tube and cultured at 28°C and 200 rpm for 2 h, and then spread on an LB solid plate containing 50 mg / L of Rifampicin and 50 mg / L of Spectinomycin until a positive single clone grew, a single clone was picked for culture and its plasmid was extracted, and the extracted plasmid was sequenced and identified, and the results showed that the structure of the recombinant dual luciferase expression vectors DBN13844 and DBN14252 was completely correct.

[0084] 3. Transient transformation of corn callus

[0085] Corn callus is a biological cell reactor for efficient protein expression. The exogenous genes are introduced into corn callus by Agrobacterium infection, so as to conduct preliminary research on the functions of the exogenous genes.

[0086] For Agrobacterium-mediated transformation of maize, briefly, immature embryos are isolated from maize and contacted with an Agrobacterium suspension, wherein the Agrobacterium is capable of transferring the T-DNA in the DBN13844 and DBN14252 vectors to at least one cell of one of the embryos (step 1: infection step). In this step, the embryos are preferably immersed in the Agrobacterium suspension (OD 660 =0.4-0.6, infection medium (MS salts 4.3 g / L, MS vitamins, casein 300 mg / L, sucrose 68.5 g / L, glucose 36 g / L, acetosyringone (AS) 40 mg / L, 2,4-di-chlorophenoxyacetic acid (2,4-D) 1 mg / L, pH 5.3)) to start the inoculation. The young embryos are co-cultivated with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step). Preferably, the young embryos are cultured on solid medium (MS salts 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 the infection step. After this co-cultivation stage, there can be an optional "recovery" step. In the "recovery" step, at least one antibiotic known to inhibit the growth of Agrobacterium (cephalosporin 150-250 mg / L) is present in the recovery medium (MS salts 4.3 g / L, MS vitamins, casein 300 mg / L, sucrose 30 g / L, 2,4-D 1 mg / L, cephalosporin 250 mg / L, phytagel 3 g / L, pH 5.8), and no selective agent for plant transformants is added (step 3: recovery step). The young embryos are cultured on solid medium with antibiotics but no selective agent to eliminate Agrobacterium and provide a recovery period for infected cells while callus is formed.

[0087] 4. Detection of LUC / REN dual luciferase activity in corn callus

[0088] The corn callus tissues transferred with the above-mentioned recombinant dual-luciferase expression vectors DBN13844 and DBN14252 were respectively taken and the recombinant dual-luciferase expression vectors DBN13844 and DBN14252 were respectively transferred with the recombinant dual-luciferase expression vectors DBN13844 and DBN14252. Reporter Assay System (E1960) kit was used for detection. The specific method is as follows:

[0089] 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 freeze-dried powder luciferase assay substrate with luciferase assay buffer II (10 mL), and store at -80°C in the dark; Renilla 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°C in the dark;

[0090] Step 2: Take the callus tissue after infection and culture for 5 days, press it on absorbent paper to absorb water, divide the callus tissue transferred with each vector into 3 parts, place it in a 2.0mL centrifuge tube with 1 large and 1 small steel ball, and freeze it in liquid nitrogen; then grind it with a vibration grinder at 1200rpm for 1min;

[0091] Step 3: After grinding, add about 150 μL 1×PLB to the centrifuge tube, mix well, and vortex for 20 seconds. Let it stand for 10 minutes to fully lyse. Then centrifuge at 4°C, 12,000 rpm for 10 minutes, take 100 μL of the supernatant extract, add it to the ELISA plate, and set up 3 replicates.

[0092] Step 4: Add 100 μL of 1× firefly luciferase reaction solution, shake the plate to mix, and detect the activity of firefly luciferase with a microplate reader. The detection is completed within 30 minutes, and the fluorescence intensity of the detected firefly luciferase is expressed in RLU (relative fluorescence unit);

[0093] Step 5: Add 100 μL of 1× Renilla luciferase reaction solution, shake the plate to mix, and detect the activity of Renilla luciferase with a microplate reader. The detection is completed within 30 minutes, and the fluorescence intensity of the detected Renilla luciferase is expressed in RLU (relative fluorescence unit).

[0094] Among them, the detection program of the microplate reader (BioTek-H1MF) is full spectrum light intensity reading.

[0095] The results of the activity detection of firefly luciferase (LUC) and Renilla luciferase (REN) in the transiently transformed maize callus are shown in Table 1. Among them, the REN gene is an internal reference, and the ratio of LUC / REN reflects the relative activity strength of the promoter (LUC / REN ratio = (LUC value of maize callus transformed with the recombinant expression vector-LUC value of wild-type maize callus) / (REN value of maize callus transformed with the recombinant expression vector-REN value of wild-type maize callus)).

[0096] Table 1 Luciferase activity in transiently transformed maize callus

[0097]

[0098] 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 transcription intensity of prEiUbi2-01 is 1.5 times that of prZmUbi1. It can be seen that the promoter prEiUbi2-01 can not only normally regulate gene expression, but also has a stronger transcription intensity than the commonly used promoters in this field, and has good application prospects.

[0099] Example 3: Verification of the effect of the constitutive promoter prEiUbi2-01 in transgenic corn plants

[0100] 1. Stable transformation of maize with recombinant dual-luciferase vectors DBN13844 and DBN14252

[0101] Conventional Agrobacterium infection method was used for transformation. The sterile cultured maize embryos were co-cultured with the Agrobacterium described in Example 2 to transfer the T-DNA in the constructed recombinant expression vector into the maize chromosome set to produce transgenic maize events.

[0102] For Agrobacterium-mediated transformation of maize, briefly, immature embryos are separated from maize and contacted with an Agrobacterium suspension, wherein the Agrobacterium is capable of transmitting 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 this step, the embryos are preferably immersed in the Agrobacterium suspension (OD 660=0.4-0.6, infection medium (MS salts 4.3 g / L, MS vitamins, casein 300 mg / L, sucrose 68.5 g / L, glucose 36 g / L, acetosyringone (AS) 40 mg / L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1 mg / L, pH 5.3)) to start the inoculation. The young embryos are co-cultivated with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step). Preferably, the young embryos are cultured on solid medium (MS salts 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 the infection step. After this co-cultivation stage, there can be an optional "recovery" step. In the "recovery" step, at least one antibiotic known to inhibit the growth of Agrobacterium (cephalosporin 150-250 mg / L) is present in the recovery medium (MS salts 4.3 g / L, MS vitamins, casein 300 mg / L, sucrose 30 g / L, 2,4-D 1 mg / L, cephalosporin 250 mg / L, phytagel 3 g / L, pH 5.8), and no selection agent for plant transformants is added (step 3: recovery step). The young embryos are cultured on solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for infected cells while callus is formed. Next, the callus is inoculated on a medium containing a selection agent (4-[hydroxy(methyl)phosphonyl]-DL-homoalanine) and selected for growing transformed callus (step 4: selection step). Preferably, callus is cultured on a screening solid medium with a selective agent (MS salts 4.3 g / L, MS vitamins, casein 300 mg / L, sucrose 30 g / L, cephalosporin 250 mg / L, 4-[hydroxy(methyl)phosphonyl]-DL-homoalanine 10 mg / L, 2,4-D 1 mg / L, phytagel 3 g / L, pH 5.8), resulting in selective growth of transformed cells. Then, callus is regenerated into plants (step 5: regeneration step), preferably, callus grown on a medium containing a selective agent is cultured on a solid medium (MS differentiation medium and MS rooting medium) to regenerate plants.

[0103] The resistant callus obtained by screening was transferred to the MS differentiation medium (MS salt 4.3g / L, MS vitamin, casein 300mg / L, sucrose 30g / L, 6-benzyladenine 2mg / L, cephalosporin 250mg / L, 4-[hydroxy(methyl)phosphonyl]-DL-homoalanine 5mg / L, plant gel 3g / L, pH 5.8), and cultured and differentiated at 25°C. The differentiated seedlings were transferred to the MS rooting medium (MS salt 2.15g / L, MS vitamin, casein 300mg / L, sucrose 30g / L, cephalosporin 250mg / L, indole-3-acetic acid 1mg / L, plant gel 3g / L, pH 5.8), cultured at 25°C to a height of about 10cm, and moved to a greenhouse for culture until fruiting. In the greenhouse, culture was carried out at 28°C for 16h every day and then at 20°C for 8h.

[0104] 2. Verification of corn plants transformed with recombinant dual-luciferase vectors DBN13844 and DBN14252

[0105] Genomic DNA was extracted using DNeasy Plant Maxi Kit (Qiagen), and the copy number of the screening marker PAT gene in vectors DBN13844 and DBN14252 was detected by Taqman probe fluorescent quantitative PCR method. Wild-type corn plants were used as controls. The experiment was repeated three times and the average value was taken.

[0106] The specific method for detecting the copy number of PAT gene is as follows:

[0107] Step 1: Take 100 mg of leaves from corn plants transformed with the recombinant dual-luciferase vectors DBN13844 and DBN14252, grind them into homogenate in a mortar with liquid nitrogen, and take 3 replicates for each sample;

[0108] Step 2: Extract genomic DNA from the above samples. For specific methods, refer to the instructions;

[0109] Step 3: Determine the genomic DNA concentration using NanoDrop2000 (Thermo Scientific);

[0110] Step 4: adjusting the genomic DNA concentration to the same concentration value, wherein the concentration value ranges from 80-100 ng / μL;

[0111] Step 5: The Taqman probe fluorescence quantitative PCR method was used to identify the copy number of the sample, with the identified sample with a known copy number as the standard, and the sample of the wild-type corn plant as the control, with 3 replicates for each sample, and the average value was taken; the fluorescence quantitative PCR primers for detecting the PAT gene are shown in SEQ ID NO: 17-18, and the probe is shown in SEQ ID NO: 19.

[0112] PCR reaction system: JumpStart TM Taq ReadyMix TM (Sigma) 10 μL, 50× primer / probe mixture 1 μL, genomic DNA 3 μL, water (ddH2O) 6 μL. 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 an amber tube at 4°C.

[0113] The PCR reaction conditions were:

[0114]

[0115] The data were analyzed using SDS2.3 software (Applied Biosystems). The experimental results showed that the PAT gene had been integrated into the chromosome of the tested maize in the form of a single copy, thereby obtaining transgenic maize plants containing a single copy of the promoter prEiUbi2-01 and the promoter prZmUbi1.

[0116] 3. Detection of LUC / REN dual luciferase activity in various tissues of corn plants

[0117] Roots and leaves were sampled at the seedling stage, stems and leaves were sampled at the trumpet stage, silks and pollens were sampled at the flowering stage, and grains were sampled at the grain filling stage of corn T0 plants into which promoters prEiUbi2-01 and prZmUbil were transferred, and 3 replicates were taken for each tissue sample in each period. The samples were tested for dual luciferase activity according to the method in Example 2 above, and the test results are shown in Table 2.

[0118] Table 2 Luciferase activity at different stages and in different parts of stably transformed maize plants

[0119]

[0120] The results in Table 2 show that: in each growth and development stage of corn, the promoter prEiUbi2-01 can normally drive the constitutive expression of the LUC gene in each tissue, especially in leaves, filaments, grains and cob tops, the expression of LUC driven by prEiUbi2-01 is better than that driven by prZmUbi1. Specifically, in leaves at the seedling stage and the trumpet stage, the expression of LUC driven by prEiUbi2-01 is 1.4 times and 1.1 times that driven by prZmUbil, respectively; in filaments at the flowering stage, the expression of LUC driven by prEiUbi2-01 is 2.0 times that driven by prZmUbi1; in grains and cob tops at the filling stage, the expression of LUC driven by prEiUbi2-01 is 1.8 times and 2.3 times that driven by prZmUbi1, respectively. It can be seen that the promoter prEiUbi2-01 has strong transcriptional activity in corn, and its activity in some tissues is stronger than that of the promoter prZmUbi1 commonly used in the art. The novel constitutive promoter prEiUbi2-01 of the present application provides a new tool and option for the expression of exogenous genes in plant species, and has great application prospects.

[0121] Example 4: Verification of the effect of constitutive promoter prEiUbi2-01 in driving PAT gene expression in corn

[0122] 1. Construction of expression vector containing constitutive promoter prEiUbi2-01 and PAT gene

[0123] In order to further confirm the utility of the constitutive promoter prEiUbi2-01, the promoter was operably linked to a herbicide-tolerant gene and verified whether it could drive the expression of the gene, thereby conferring the herbicide-tolerant trait to the plant.

[0124] Specifically, a plant expression vector in which the PAT gene was driven by the promoter prEiUbi2-01 was constructed. The backbone vector DBNBC-01 (pCAMBIA2301 with modified resistance tag, available from CAMBIA) was linearized using restriction endonuclease SalI, and the primer pair of SEQ ID NO: 20 and SEQ ID NO: 21 was used to amplify prEiUbi2-01 (SEQ ID NO: 1) with a linker sequence, and the primer pair shown in SEQ ID NO: 28 and SEQ ID NO: 29, the primer pair shown in SEQ ID NO: 30 and SEQ ID NO: 31, the primer pair shown in SEQ ID NO: 22 and SEQ ID NO: 23, the primer pair shown in SEQ ID NO: 24 and SEQ ID NO: 25, and the primer pair shown in SEQ ID NO: 26 and SEQ ID NO: 27 were used to amplify cPAT (SEQ ID NO: 10) element with a linker sequence, t35S (SEQ ID NO: 11) element, prZmUbi1 (SEQ ID NO: 12) element, cPMI (SEQ ID NO: 32) element and tNos (SEQ ID NO: 5) element.

[0125] The linearized backbone vector DBNBC-01 fragment was mixed with the prEiUbi2-01 element with a linker sequence, the cPAT element with a linker sequence, the t35S element with a linker sequence, the prZmUbil element with a linker sequence, the cPMI element with a linker sequence, and the tNos element for recombination reaction. The operation steps were carried out according to the instructions of the In-Fusion SnapAssembly Master Mix kit of Takara Company (Clontech, CA, JPN, CAT: 638949), and the recombinant vector DBN14915 containing the PAT gene was constructed. The schematic diagram of its structure is shown in FIG. Figure 4 shown.

[0126] 2. Transformation of Agrobacterium with recombinant expression vector DBN14915

[0127] Referring to Example 2 of the present application, the recombinant expression vector DBN14915 was transformed into Agrobacterium LBA4404 using the liquid nitrogen method, and the plasmid was extracted for sequencing identification. The results showed that the structure of the recombinant expression vector DBN14915 was completely correct.

[0128] 3. Stable transformation of corn with recombinant expression vector DBN14915

[0129] Referring to Example 3 of the present application, Agrobacterium transformed with the recombinant expression vector DBN14915 was used to infect corn, and the T-DNA of the recombinant expression vector DBN14915 was inserted into the corn chromosome to obtain the corresponding transgenic corn T0 plant.

[0130] About 100 mg of leaves of transgenic corn T0 plants were taken as samples, and their genomic DNA was extracted using Qiagen's DNeasy Plant MaxiKit. The copy number of the PAT gene of vector DBN14915 was detected by TaqMan probe fluorescence quantitative PCR method, and wild-type corn plants were used as controls. The experiment was repeated 3 times and the average value was taken. The fluorescence quantitative PCR primers for detecting the PAT gene are shown in SEQ ID NO: 17-18, and the probe is shown in SEQ ID NO: 19.

[0131] The experimental results showed that the fragment of the PAT gene driven by prEiUbi2-01 had been successfully integrated into the genome of the transgenic corn plants tested and existed in the form of a single copy.

[0132] 4. Evaluation of herbicide resistance in transgenic corn

[0133] Transgenic maize T0 plants and wild-type maize plants (18 days after sowing) with the prEiUbi2-01 promoter and PAT gene were uniformly sprayed with 800 g ai / ha (2 times the field concentration, 2×) of glufosinate. Seven days after spraying the herbicide (7DAT), the resistance / tolerance level of maize leaves and the overall plant was evaluated. The damage level of the plants was divided into the following levels: basically no phenotype of drug damage as level 0; slight burns at the base of the leaves, with a burn area of ​​≤10% as level 1; obvious burns at the base of the leaves, with a burn area of ​​>10%. There may be slight curling of the leaves or tilting of the plants, which can be recovered within 14 days to level 2; deformed leaves of the plants, or tilted growth of the plants, which have not recovered after 14 days; broken leaves from the drug-damaged parts as level 3; severe deformed plants; wilting leaves, dryness and death as level 4.

[0134] The resistance performance of the transformation event was scored according to the formula X = [∑ (N × S) / (T × M)] × 100 (X-drug damage score, N-number of plants damaged at the same level, S-number of pesticide damage levels, T-total number of plants, M-highest pesticide damage level), and resistance was evaluated according to the scores: high resistance (0-15 points), medium resistance (16-33 points), low resistance (34-67 points), and no resistance (68-100 points). The experimental results are shown in Table 3 and Figure 5 shown.

[0135] Table 3 Results of the tolerance experiment of transgenic corn T0 plants to glufosinate

[0136]

[0137] The results in Table 3 show that when treated with glufosinate at twice the field concentration, the transgenic corn plant that has been transformed with the PAT gene driven by the constitutive promoter prEiUbi2-01 has less leaf damage than the wild-type corn plant and has a moderate resistance to glufosinate, while the wild-type corn plant has more severe leaf damage and has no herbicide resistance. Figure 5 It can be seen that the transgenic corn (four plants on the right) into which the PAT gene driven by the constitutive promoter prEiUbi2-01 was transferred can grow normally with almost no leaf damage, indicating that it has good tolerance to herbicides; while the leaves of the wild-type corn (the one on the far left) dried up and died, that is, it has no herbicide tolerance. In summary, the constitutive prEiUbi2-01 of the present application can normally drive the expression of the target gene in plant species and give the plant corresponding characteristics.

[0138] Example 5. Construction of truncated version of constitutive promoter prEiUbi2-01 and activity detection

[0139] 1. Construction of dual luciferase expression vector containing promoter prEiUbi2-01 truncated form

[0140] The sequence of the constitutive promoter prEiUbi2-01 was analyzed by bioinformatics using online analysis websites such as TSSP, and it was determined that the promoter sequence contained cis-acting elements for hormone response, cis-acting elements for light signal response, cis-acting elements involved in hypoxia-specific induction, etc. It also had multiple cis-acting elements such as CAAT Box related to transcriptional activity and TATA BOX related to transcription initiation.

[0141] In order to further identify the transcriptional regulatory elements that control the activity of the constitutive promoter prEiUbi2-01, that is, the sequence with the highest efficiency of the promoter driving the expression of the target gene and the shortest sequence with transcriptional activity, the full-length promoter prEiUbi2-01 was subjected to different degrees of 5' end unidirectional fragment deletion and truncation according to the distribution of elements in the promoter sequence. The sizes of the four truncated promoter fragments were 2544bp, 1974bp, 1404bp, and 694bp, respectively, corresponding to SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, and were named prEiUbi2-02, prEiUbi2-03, prEiUbi2-04, and prEiUbi2-05.

[0142] Referring to the dual luciferase expression vector containing the promoter prEiUbi2-01 constructed in Example 2 of the present application, the backbone vector DBNBC-Dual_LUC containing the LUC and REN dual luciferins was linearized using the restriction endonuclease SalI, and the primer pairs shown in SEQ ID NO: 37 and SEQ ID NO: 16, the primer pairs shown in SEQ ID NO: 38 and SEQ ID NO: 16, the primer pairs shown in SEQ ID NO: 39 and SEQ ID NO: 16, and the primer pairs shown in SEQ ID NO: 40 and SEQ ID NO: 16 were used to amplify the truncated promoter of the full-length promoter prEiUbi2-01, respectively. The linearized backbone vector DBNBC-Dual LUC fragment was mixed with the truncated promoter with a linker sequence for recombination reaction. The operation steps were carried out according to the instructions of Takara's 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.

[0143] 2. Activity detection of truncated promoter prEiUbi2-01 in maize callus

[0144] The vectors DBN14252, DBN14526, DBN14527, DBN14528 and DBN14529 were transiently transformed into corn callus using the method in Example 2 of the present application, and the corresponding supernatants were prepared for use and the luciferase activity was detected. The results are shown in Table 4.

[0145] Table 4 Luciferase activity in maize callus transiently transformed with different promoter truncations

[0146]

[0147] As shown in the data in Table 4, in the transiently transformed maize callus cells, the truncated promoters prEiUbi2-02, prEiUbi2-03, and prEiUbi2-04 of the full-length promoter prEiUbi2-01 all have good transcriptional activity. Specifically, the LUC expression driven by prEiUbi2-02, prEiUbi2-03, and prEiUbi2-04 is 0.7 times, 0.7 times, and 0.4 times that driven by prEiUbi2-01, respectively. Under the drive of prEiUbi2-05, LUC is almost not expressed, indicating that the truncated promoter shown in SEQ ID NO: 36 has almost no transcriptional activity. It can be seen that SEQ ID NO: 35 is the shortest sequence with promoter activity. Combined with 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 function as a promoter normally, thereby driving the expression of the target gene.

[0148] In summary, the present invention discloses for the first time a constitutive promoter from Eleutherodactylus truncatula. The constitutive promoter of the present invention shows activity in almost most tissues and many types of cells of plants, especially in the roots, stems, leaves, filaments, pollen, grains, and cob tips of plants, and can normally drive the expression of target genes, providing a new tool and option for plant genetic engineering, and has good application prospects.

[0149] Although only specific implementation examples of the present invention are described above, those skilled in the art should understand that these are only examples, and the protection scope of the present invention is defined by the appended claims. Those skilled in the art may make various changes or modifications to these implementations without departing from the principles and essence of the present invention, but these changes or modifications all fall within the protection scope 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 A constitutive promoter comprising any one of claims 1 to 3 operably linked to a nucleic acid of interest; Preferably, the target nucleic acid encodes a target protein.

5. An expression cassette, characterized in that It comprises the constitutive promoter according to any one of claims 1 to 3 or the chimeric gene according to claim 4.

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

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

8. A method for expressing a target nucleic acid in a plant, characterized in that: include: Stably integrating a nucleic acid of interest operably linked to a constitutive promoter according to any one of claims 1 to 3 into a plant cell; Preferably, the nucleic acid of interest is constitutively expressed in plant tissue; Preferably, the target nucleic acid encodes a target protein; More preferably, the nucleic acid of interest encodes a herbicide tolerance protein or an insect resistance protein.

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

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

11. A method for obtaining processed agricultural products, characterized in that: The method comprises processing the harvested product of the plant or part thereof according to claim 10 to obtain a processed agricultural product.

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

13. Use of the constitutive promoter according to claim 12 for constitutively expressing a target nucleic acid in plant tissue, characterized in that: The target nucleic acid encodes a target protein; Preferably, the nucleic acid of interest encodes a herbicide tolerance protein or an insect resistance protein.

14. Use of the constitutive promoter according to claim 12 or 13 for constitutively expressing a target nucleic acid in plant tissue, characterized in that: The plant is corn, Arabidopsis, rapeseed, tobacco, soybean, cotton, pepper, beet, pumpkin, eggplant, Chinese cabbage, carrot, tomato, pea, spinach, potato or peanut.

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