WO2026020346A1 - Constitutive promoter and use thereof - Google Patents

Abstract

Provided are a constitutive promoter and a use thereof. The nucleotide sequence of the constitutive promoter includes SEQ ID NO: 4, and the constitutive promoter is derived from SEQ ID NO: 1. The constitutive promoter exhibits activity in almost all tissues and multiple types of cells of plants, particularly in the roots, stems, leaves, flowers, pods, and fruits of plants, and has broad prospects for application in plants.

Description

Constitutive promoters and their applications Technical Field

This invention relates to a constitutive promoter and its uses, and more particularly to a constitutive promoter derived from the Ubiquitin gene of rapeseed and its uses.

Background Technology

One of the goals of plant genetic engineering is to produce plants with traits or characteristics desired in agriculture. Commonly desired traits include improved nutritional quality, increased yield, conferring resistance to pests and diseases, enhanced drought and stress tolerance, improved horticultural quality, and conferring herbicide resistance. Current technological advancements have enabled researchers to obtain exogenous polynucleotide molecules (e.g., heterologous or naturally derived genes) and integrate them into the plant genome, where the gene is expressed in plant cells to exhibit the corresponding trait. Importantly, appropriate regulatory signals must be present in suitable structures to achieve expression of the coding sequence of the newly inserted gene in plant cells. These regulatory signals typically include promoter regions, 5' untranslated guide sequences, and 3' transcription terminators/polyadenylation sequences.

Certain promoters can direct RNA synthesis at a certain level of expression in most or all plant tissues and/or during growth and development stages; these promoters are called "constitutive promoters." Constitutive promoters can be classified into strong, moderate, and weak promoters based on their effectiveness in directing RNA synthesis. Constitutive promoters are particularly advantageous in situations where it is necessary to simultaneously express a target gene in different plant tissues to achieve the desired gene function.

Several constitutive promoters functioning in plant cells have been described in existing literature. These include the Gm17gTsf1 promoter for the soybean cell elongation factor gene, 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, isolating more novel constitutive promoters remains a significant interest. These promoters can control the expression of recombinant DNA constructs (or genes) at different levels and can be applied to the expression of multiple genes superimposed in the same transgenic plant.

Summary of the Invention

The purpose of this invention is to provide a novel constitutive promoter and its uses, which enables the efficient expression of heterologous nucleotide sequences in plant tissues.

To achieve the above objectives, the present invention provides a constitutive promoter whose nucleotide sequence includes SEQ ID NO:4, wherein the constitutive promoter is derived from SEQ ID NO:1.

Furthermore, the present invention provides a constitutive promoter whose nucleotide sequence includes SEQ ID NO:4 and is selected from at least a portion of SEQ ID NO:1.

Furthermore, the present invention provides a constitutive promoter with a nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

To achieve the above objectives, the present invention also provides a recombinant DNA construct comprising the above-described constitutive promoter operatively linked to a target heteronucleotide sequence.

Furthermore, the target heteronucleotide sequence encodes the target protein.

To achieve the above objectives, the present invention also provides an expression cassette comprising the above-described recombinant DNA construct.

To achieve the above objectives, the present invention also provides a recombinant vector comprising the above-described expression cassette.

To achieve the above objectives, the present invention also provides a method for expressing a target heteronucleotide sequence in a plant, comprising: stably integrating the target heteronucleotide sequence operatively linked to the above-mentioned constitutive promoter into a plant cell.

Furthermore, the plants mentioned are Arabidopsis thaliana, rapeseed, tobacco, soybean, cotton, chili pepper, beet, pumpkin, eggplant, Chinese cabbage, carrot, tomato, pea, spinach, potato, or peanut.

Preferably, the target heteronucleotide sequence is constitutively expressed in plant tissues.

Furthermore, the target heteronucleotide sequence encodes the target protein.

Preferably, the target heteronucleotide sequence encodes a herbicide-resistant protein.

Preferably, the target heteronucleotide sequence encodes an insect resistance protein.

To achieve the above objectives, the present invention also provides a plant or part thereof comprising the above-described constitutive promoter.

To achieve the above objectives, the present invention also provides a method for obtaining processed agricultural products, comprising processing the above-mentioned plants or parts of the harvested material to obtain processed agricultural products.

To achieve the above objectives, the present invention also provides the use of the above-mentioned constitutive promoter for constitutive expression of a target heteronucleotide sequence in plant tissues.

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

Furthermore, the target heteronucleotide sequence encodes the target protein.

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

In this invention, the term "promoter" refers to a DNA regulatory region, typically containing a TATA box that guides RNA polymerase II to initiate RNA synthesis at a suitable transcription start site within a specific coding sequence. The term "gene" refers to any segment of DNA containing, within a cell, a region of DNA ("transcribed DNA region") that is transcribed into RNA molecules (e.g., mRNA) under the control of a suitable regulatory region (e.g., a plant-expressible promoter region). Thus, a gene 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. Recombinant DNA constructs are any genes not normally found in plant species, or any genes whose promoters are, in their natural state, independent of partially or entirely transcribed DNA regions or at least another regulatory region of the gene.

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 expression. Constitutive promoters are used to operatively link genes, target heterologous nucleotide sequences, or gene editing system guide RNA (gRNA) to 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 tissue is a structural unit in a plant composed of one or more types of cells of the same origin and performing the same function, such as protective tissue, vascular tissue, nutritive tissue, mechanical tissue, and meristematic tissue. 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 term "constitutive expression" refers to the relatively stable and sustained expression of a gene or target heterologous nucleotide sequence 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 at different developmental stages in plants. Constitutive promoters can also ensure the widespread expression of gRNA in host cells, improving the efficiency and accuracy of gene editing.

It has promoter activity and hybridizes with the promoter sequence or fragment thereof of the present invention under stringent conditions. The separated sequences are included in this invention. These sequences are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the sequences of this invention. That is, the range of sequence identity is distributed at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity.

This invention provides a constitutive promoter whose nucleotide sequence includes SEQ ID NO:4 and is selected from at least a portion of SEQ ID NO:1. SEQ ID NO:4 is a fragment of SEQ ID NO:1. Referring to SEQ ID NO:1, the constitutive promoter of this invention can be extended from SEQ ID NO:4 alone to its 5' end or 3' end, or simultaneously to both its 5' and 3' ends, but the length of the nucleotide sequence extended to both ends cannot exceed the 5' or 3' end of SEQ ID NO:1 itself. In other words, constitutive promoters with nucleotide sequences as shown in SEQ ID NO:4, or constitutive promoters obtained by arbitrarily extending from both ends of SEQ ID NO:4 with reference to SEQ ID NO:1, provided that the extension length does not exceed the 5' or 3' end of SEQ ID NO:1 itself, are all within the scope of protection of this invention. Furthermore, for such a constitutive promoter (referring to SEQ ID NO:1, a constitutive promoter obtained by arbitrarily extending both ends of SEQ ID NO:4, with the extension length not exceeding the 5' or 3' end of SEQ ID NO:1 itself), the other nucleotide sequences included in the constitutive promoter besides SEQ ID NO:4 do not affect the activity of the SEQ ID NO:4 sequence itself. The second embodiment of the present invention also demonstrates this conclusion: the prBnUbi14-04 promoter (SEQ ID NO:4) is active, and the prBnUbi14-01 promoter (SEQ ID NO:1), prBnUbi14-02 promoter (SEQ ID NO:2), and prBnUbi14-03 promoter (SEQ ID NO:3) containing SEQ ID NO:4 are all active. Based on the contents of this application, those skilled in the art can reasonably predict that its nucleotide sequence includes SEQ ID NO:4, and that the constitutive promoters selected from at least a portion of SEQ ID NO:1 have the same or similar activities as SEQ ID NO:4.

The promoter sequence and fragments described in this invention, when assembled into a DNA structure to operatively link the promoter sequence with a target heteronucleotide 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 heteronucleotide sequence constitute the recombinant DNA construct provided together in an expression cassette for expression in the target plant. This expression cassette provides numerous restriction sites for inserting the target heteronucleotide sequence, which will be regulated by the promoter sequence of this invention. Transcriptional regulation of the segmental region. 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.

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).

The expression cassette includes the promoter sequence of the present invention transcribed along the 5'-3' direction, a translation initiation region, a target heteronucleotide sequence, and a transcription and translation termination region that functions in plants. The target heteronucleotide sequence can be natural, exogenous to the plant host, or heterologous. Alternatively, the target heteronucleotide sequence can be a natural sequence or a selectively synthesized sequence. "Exogenous" means that the introduced transcription initiation region is not present in the natural plant in which it is introduced. For example, a recombinant DNA construct contains the promoter sequence of the present invention, which is operatively linked to a coding sequence different from the promoter sequence of the present invention.

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).

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.

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.

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.

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.

The promoter sequence of this invention can be used to initiate transcription of an antisense structure that is at least partially complementary to a messenger RNA (mRNA) of a target heteronucleotide sequence. The 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 with 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.

The promoter sequence of this invention is used for the constitutive expression of a target heteronucleotide sequence. A "heteronucleotide sequence" refers to a sequence that is not naturally present with the promoter sequence. Although the nucleotide sequence is heterologous to the promoter sequence, it may be homologous, natural, heterologous, or exogenous to the plant host. A heteronucleotide sequence operatively linked to the promoter of this invention can encode a target protein. Examples of such heteronucleotide sequences include, but are not limited to, nucleotide sequences encoding resistant polypeptides 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.

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.

In this invention, "insect resistance" refers to a plant's avoidance of symptoms and damage caused by plant-insect interactions. Specifically, it refers to preventing insect-induced plant damage, crop damage, plant deformities, and plant diseases. Alternatively, the damage to plants, crops, plant deformities, and plant diseases caused by insects can be minimized or mitigated. The insects may belong to the orders 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. Target insect resistance proteins known in the art include, but are not limited to, Bacillus toxicity proteins; lectins, including snowdrop lectin, pea lectin, canavalia lentin, malt lectin, potato lectin, peanut lectin, etc.; lipoxygenases, including pea lipoxygenase 1 or soybean lipoxygenase; and insect chitinases, etc.

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 dwarf virus, and sweet potato feather spot virus. Therefore, heterologous nucleotide sequences that constitutively express antipathogenic activity or minimize the impact of viral pathogens in plant tissues can be selected.

The promoter sequence and method of this invention can be used to regulate the expression of any desired heteronucleotide sequence in a plant host to alter the plant phenotype. Various desired phenotypic alterations include, but are not limited to, changes in the plant's fatty acid composition, changes in the plant's amino acid content, and changes in plant pathogen defense mechanisms. These alterations can be achieved by providing the expression of a heterologous product or increasing the expression of an endogenous product in the plant. 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.

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.

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.

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.

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 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.

On the one hand, plant parts are plant cells. On the other hand, plant parts are either non-regenerative or regenerative cells. Furthermore, plant cells are somatic cells.

Non-regenerative cells are cells that cannot be regenerated into a whole plant through in vitro culture. Non-regenerative cells can be found 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 reproductive cells, such as ovules or cells that are part of pollen. In another aspect, pollen cells are vegetative (non-reproductive) cells, or sperm cells.

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.

This invention provides a constitutive promoter and its application, which has the following advantages:

1. This invention discloses for the first time a constitutive promoter from the rapeseed Ubiquitin gene, wherein the nucleotide sequence of the constitutive promoter includes SEQ ID NO:4 and is derived from SEQ ID NO:1.

2. The constitutive promoters of this invention exhibit activity in almost all plant tissues and many cell types, particularly in plant roots, stems, leaves, flowers, pods, and fruits.

3. The constitutive promoter of this invention can drive the constitutive expression of exogenous genes in plant tissues.

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

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.

Figure 2 is a schematic diagram of the recombinant expression vector DBN11-C containing the prBnUbi14-01 promoter sequence of the present invention.

Figure 3 is a schematic diagram of the structure of the vector DBNBC-HTG containing the herbicide-resistant gene HTG of the present invention.

Figure 4 is a schematic diagram of the structure of the recombinant expression vector DBN20-C of the present invention.

Detailed Implementation

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

First embodiment: Obtaining the constitutive promoter of the present invention

1. Obtain the prBnUbi14-01 promoter sequence

By searching the public transcriptome database of rapeseed (BnTIR: Brassica napus transcriptome information resource. (hzau.edu.cn)), genes highly expressed in roots, stems, leaves, flowers, seeds, and siliques can be found. The 2030 bp sequence upstream of the gene BnaA08G0137800ZS was selected and named the promoter prBnUbi14-01. Using the genomic DNA sequence of the Brassica napus variety Westar as a PCR amplification template, primers 1 and 2 were designed for PCR amplification.

Primer 1: 5’-aggatcaaagtttttagacaacc-3’, as shown in SEQ ID NO:7 in the sequence listing;

Primer 2: 5’-ctgttaattcacaaaacctaaca-3’, as shown in SEQ ID NO:8 in the sequence listing.

The PCR reaction system is as follows:

The primers consist of 2.5 μL of each primer at a concentration of 10 μM, and the reaction buffer is New England Concentrate. The reaction buffer from the company's High-Fidelity DNA Polymerase kit was added to the above PCR reaction system with nuclease-free water to a final volume of 50 μL. Specific operating procedures were followed according to the New England Journal of Medicine guidelines. Company PCR Using Follow the instructions in the High-Fidelity DNA Polymerase (M0491) kit manual.

The PCR reaction conditions are as follows:

The PCR amplification product was ligated with a blunt-ended Blunt vector (TransGen cloning vector, Beijing). The procedure was performed according to the TransGen Blunt vector product instructions. The ligation product was then sequenced (Sanger sequencing) to confirm the prBnUbi14-01 promoter sequence, as shown in SEQ ID NO:1 in the sequence listing.

2. Obtain the promoter sequences prBnUbi14-02, prBnUbi14-03, prBnUbi14-04, prBnUbi14-05, and prBnUbi14-06.

Using the prBnUbi14-01 gene sequence as a PCR amplification template, the following primer pairs were designed: primer 2 (SEQ ID NO:8) and primer 3 (SEQ ID NO:9), primer 2 (SEQ ID NO:8) and primer 4 (SEQ ID NO:10), primer 2 (SEQ ID NO:8) and primer 5 (SEQ ID NO:11), primer 2 (SEQ ID NO:8) and primer 6 (SEQ ID NO:12), primer 2 (SEQ ID NO:8) and primer 7 (SEQ ID NO:11). 3) Following the method described above for obtaining the prBnUbi14-01 promoter sequence, PCR amplification reactions were performed using the primer pairs described above, and the prBnUbi14-02 promoter sequence (SEQ ID NO:2), prBnUbi14-03 promoter sequence (SEQ ID NO:3), prBnUbi14-04 promoter sequence (SEQ ID NO:4), prBnUbi14-05 promoter sequence (SEQ ID NO:5) and prBnUbi14-06 promoter sequence (SEQ ID NO:6) were obtained sequentially.

3. Synthesize the above-mentioned prBnUbi14-01 to prBnUbi14-06 promoter sequences.

The 5' and 3' ends of the above-mentioned prBnUbi14-01 promoter sequence, prBnUbi14-02 promoter sequence, prBnUbi14-03 promoter sequence, prBnUbi14-04 promoter sequence, prBnUbi14-05 promoter sequence, and prBnUbi14-06 promoter sequence, as well as the prGm17gTsf1 control promoter sequence (SEQ ID NO:14), pr35S control promoter sequence (SEQ ID NO:15), and prAtH4A748:lTEV chimeric control promoter sequence (SEQ ID NO:16) were separated. Do not connect universal connector primer 1:

5’ Universal Connector Primer 1: 5’-ctaaaaccaaaatccagtggactagt-3’, as shown in SEQ ID NO:17 in the sequence listing;

The 3’ universal adapter primer 1: 5’-atgtttttggcgtcttccat-3’, as shown in SEQ ID NO:18 in the sequence listing.

Second embodiment: Verification of the effect of promoter elements driving LUC reporter gene expression in transgenic tobacco.

1. A dual-luciferase reporter system was introduced, and recombinant expression vectors containing the prBnUbi14-01 promoter sequence, the prBnUbi14-02 promoter sequence, the prBnUbi14-03 promoter sequence, the prBnUbi14-04 promoter sequence, the prBnUbi14-05 promoter sequence, and the prBnUbi14-06 promoter sequence were constructed respectively.

Constructing vectors using conventional enzyme digestion methods is well known to those skilled in the art. A schematic diagram of the structure of the vector DBNBC-Dual_LUC (vector backbone: modified pCAMBIA2301 with resistance tag (available from CAMBIA)) containing the LUC and REN reporter genes is shown in Figure 1 (Spec: spectinomycin gene; RB: right border; prAtAct2: Arabidopsis thaliana Act2 gene promoter (SEQ ID NO: 19); REN: Renilla luciferase gene (SEQ ID NO: 20); t35s: cauliflower virus 35S terminator (SEQ ID NO: 21); SpeI: restriction endonuclease SpeI recognition site; LUC: Firefly luciferase gene (SEQ ID NO:22); tPsE9: terminator of pea RbcS gene (SEQ ID NO:23); prAtUbi10: promoter of Arabidopsis ubiquitin 10 gene (SEQ ID NO:24); spAtCTP2: Arabidopsis chloroplast transport peptide (SEQ ID NO:25); cEPSPS: 5-enolpyruvate-shikimate-3-phosphate synthase gene (SEQ ID NO:26); tNos: terminator of carmine synthase gene (SEQ ID NO:27); LB: left border).

The above-mentioned vector DBNBC-Dual_LUC was linearized by digesting it with the restriction endonuclease SpeI. The digestion product was purified to obtain the linearized DBNBC-Dual_LUC expression vector. The prBnUbi14-01 promoter sequence ligated with the universal adapter primer 1 was then used to carry out a recombination reaction with the linearized DBNBC-Dual_LUC expression vector. The operation was performed according to the instructions of the Takara In-Fusion Snap Assembly Master Mix kit (Clontech, CA, JPN, CAT: 638949) to construct the recombinant expression vector DBN11-C, the structural schematic diagram of which is shown in Figure 2.

The recombinant expression vector DBN11-C was transformed into *E. coli* DH5α competent cells using a heat shock method. The heat shock conditions were as follows: 100 μL of *E. coli* DH5α competent cells and 20 μL of recombinant plasmid DNA (recombinant expression vector DBN11-C) were gently mixed and then heat-shocked in a 42°C water bath for 30 s, followed immediately by placing on ice for 2 min. Then, 250 μL of antibiotic-free LB broth (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, pH adjusted to 7.5 with NaOH) was added, and the cells were cultured at 37°C with shaking (200 rpm/min) for 1 h. The cells were then incubated upside down on LB agar plates containing 50 mg/L spectinomycin at 37°C for 12 h. Positive colonies were picked and cultured overnight in LB broth containing 50 mg/L spectinomycin at 37°C with shaking (200 rpm/min). Plasmid extraction was performed using the alkaline lysis method: The bacterial culture was centrifuged at 12,000 rpm for 1 min, the supernatant was discarded, and the precipitated bacterial cells were resuspended in 100 μL of ice-cold solution I (25 mM Tris-HCl, 10 mM EDTA (ethylenediaminetetraacetic acid), 50 mM glucose, pH = 8.0); 200 μL of freshly prepared solution II (0.2 M NaOH, 1% SDS (sodium dodecyl sulfate)) was added, the tube was inverted 4 times to mix, and the mixture was placed on ice for 3-5 min; 150 μL of ice-cold solution III (3 M potassium acetate, 5 M acetic acid) was added, and the mixture was immediately and thoroughly mixed, and the mixture was placed on ice for 5-10 min. The sample was centrifuged at 12000 rpm for 5 min at 4℃. The supernatant was transferred to a new 2 mL centrifuge tube, and two volumes of anhydrous ethanol were added. After mixing, the mixture was incubated at room temperature for 5 min. The sample was then centrifuged again at 12000 rpm for 5 min at 4℃. The supernatant was discarded, and the precipitate was washed with 70% ethanol (V/V) and air-dried. The precipitate was dissolved in 30 μL of TE (10 mM Tris-HCl, 1 mM EDTA, pH = 8.0) containing RNase (20 μg/mL). The RNA was digested by incubating in a water bath at 37℃ for 30 min. The sample was then stored at -20℃ for later use. The extracted plasmid was sequenced and identified. The results showed that the recombinant expression vector DBN11-C contained the nucleotide sequence shown in SEQ ID NO:1 in the sequence listing, which is the prBnUbi14-01 promoter sequence.

Following the method described above for constructing the recombinant expression vector DBN11-C containing the prBnUbi14-01 promoter sequence, the following sequences are connected: the prBnUbi14-02 promoter sequence linked to universal adapter primer 1, the prBnUbi14-03 promoter sequence linked to universal adapter primer 1, the prBnUbi14-04 promoter sequence linked to universal adapter primer 1, the prBnUbi14-05 promoter sequence linked to universal adapter primer 1, and the [other sequences are missing from the original text]. The prBnUbi14-06 promoter sequence, the prGm17gTsf1 control promoter sequence connected to the universal adapter primer 1, the pr35S control promoter sequence connected to the universal adapter primer 1, and the prAtH4A748:lTEV chimeric control promoter sequence connected to the universal adapter primer 1 were respectively subjected to recombination reactions with the linearized DBNBC-Dual_LUC expression vector to obtain recombinant expression vectors DBN12-C to DBN19-C. Sequencing was used to verify the recombination expression. The above nucleotide sequences were correctly inserted into vectors DBN12-C to DBN19-C.

2. Transformation of Agrobacterium with recombinant expression vector

The correctly constructed recombinant expression vectors DBN11-C to DBN19-C were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA; Cat. No: 18313-015) using liquid nitrogen. The transformation conditions were as follows: 100 μL Agrobacterium LBA4404, 3 μL plasmid DNA (recombinant expression vector); incubation in liquid nitrogen for 10 min, followed by a 37°C water bath for 10 min; the transformed Agrobacterium LBA4404 was inoculated into LB tubes and cultured at 28°C and 200 rpm for 2 h; then plated onto LB agar plates containing 50 mg/L rifampicin and 50 mg/L spectinomycin until positive single colonies grew. Single colonies were picked, cultured, and their plasmids were extracted. The extracted plasmids were sequenced and identified, and the results showed that the recombinant expression vectors DBN11-C to DBN19-C had completely correct structures.

3. Instantaneous transformation of tobacco leaves

Tobacco leaves are efficient bioreactors for protein expression. Exogenous genes were introduced into tobacco leaves for expression using the Agrobacterium injection permeation method, and the effectiveness of the constitutive promoter of this invention was verified by high-throughput protein expression.

The methods for converting tobacco leaves are as follows:

Step 1: Plant tobacco. Cultivate the tobacco under conditions of 14 hours of light/10 hours of darkness, 25°C, and 70% relative humidity for 4-5 weeks, and then harvest the tobacco leaves.

Step 2: Select the following Agrobacterium strains transformed with recombinant expression vectors DBN11-C, DBN12-C, DBN13-C, DBN14-C, DBN15-C, DBN16-C, DBN17-C, DBN18-C, and DBN19-C respectively from Part 2 of this embodiment, and clone them into 1 mL of LB liquid medium containing antibiotics (tryptone 10 g/L, yeast extract 10 g/L, NaCl). 5 g/L of antibiotics (50 mg/mL rifampicin, 50 mg/mL spectinomycin, 10 mg/mL tetracycline) were added to the culture medium and cultured at 28°C with shaking (200 rpm) until the logarithmic growth phase of Agrobacterium (OD ₆₀₀ = 0.5-0.6). 1 mL of the logarithmic-phase Agrobacterium culture was then transferred to 20 mL of LB liquid medium containing antibiotics (10 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, 50 mg/mL rifampicin, 50 mg/mL spectinomycin, 10 mg/mL tetracycline) and cultured at 28°C with shaking (200 rpm) until... Agrobacterium was centrifuged at 5000 rpm for 10 min at room temperature during the logarithmic phase (OD ₆₀₀ = 0.5-0.6). The cells were collected and resuspended in a staining buffer (containing 10 mM _MgCl2 , 10 mM MES, 150 μM acetylsylcholine, pH = 5.6) until the OD ₆₀₀ reached 0.8. The cells were then allowed to stand at room temperature for 2-3 h to obtain Agrobacterium cultures transformed with recombinant expression vectors DBN11-C to DBN19-C for injection.

Step 3: Using a 1mL needle, gently make a small incision on the back of the tobacco leaf obtained in Step 1 of this embodiment (be careful not to puncture). Then, using a syringe without the needle, draw up the Agrobacterium tumefaciens solution transformed with recombinant expression vectors DBN11-C to DBN19-C in Step 2 of this embodiment and inject it into the tobacco leaf through the small incision. This will allow the T-DNA (including the prAtAct2 promoter sequence, REN gene sequence, t35s terminator sequence, and sequences selected from prBnUbi14-01, prBnUbi14-02, and prBn...) from the recombinant expression vectors DBN11-C to DBN19-C to be injected into the tobacco leaf. The following promoter sequences were introduced into tobacco leaves: Ubi14-03, prBnUbi14-04, prBnUbi14-05, prBnUbi14-06, prGm17gTsf1 control promoter, pr35S control promoter, and prAtH4A748:lTEV chimeric control promoter; LUC gene sequence; tPsE9 terminator sequence; prAtUbi10 promoter sequence; spAtCTP2 nucleotide sequence; cEPSPS gene sequence; and tNos terminator sequence. Wild-type tobacco leaves (CK1) were used as a control. Water-stained areas on the tobacco leaves were marked with a marker.

Step 4: Place the tobacco leaves injected in Step 3 of this embodiment and wild-type tobacco leaves in the dark for 12 hours, and then incubate them in a constant temperature incubator at 21°C for 2 days. Cut off the labeled areas of the tobacco leaves, and take three equal mass portions of leaves with labeled areas transferred to recombinant expression vectors DBN11-C to DBN19-C and wild-type tobacco leaves as biological replicates. Freeze-mill them in liquid nitrogen, and then add 1×Passive lysis buffer (PLB) buffer to each. Centrifuge at 12000 rpm for 10 min at 4°C, and collect the supernatant for later use.

4. Detection of the effect of the constitutive promoter of the present invention on driving LUC reporter gene expression in tobacco leaves.

Step 5: Take 100 μL of the supernatant from Step 4 above, add it to an ELISA plate, and set up 3 replicates. Add 100 μL of 1× firefly luciferase reaction solution LAR II (dissolve the lyophilized luciferase assay substrate in luciferase assay buffer II (Promega)). The luciferase activity of fireflies was obtained from the Reporter Assay System (E1960) kit and stored at -80°C protected from light. After shaking to mix, the luciferase activity was measured using a BioTek-H1MF microplate reader. The assay was completed within 30 minutes. The unit for the measured luciferase activity is... RLU (Relative Light Unit).

Step 6: Add 100 μL of 1× Reninax luciferase reaction solution Stop&Glo (obtained by dissolving 200 μL of Stop&Glo Substrate (50×) in 10 mL of Stop&Glo buffer and storing at -80℃ protected from light), shake the plate to mix, and use a BioTek-H1MF microplate reader to detect the Reninax luciferase activity value. The detection is completed within 30 min, and the unit of the detected Reninax luciferase (REN) activity value is RLU (relative light units).

To eliminate inter-group errors caused by factors such as different transformation efficiencies due to Agrobacterium infection in plant tissues, the REN gene was used as an internal reference. The LUC/REN ratio reflects the relative activity intensity of the promoter (LUC/REN ratio = (LUC value of tobacco leaves transformed with different recombinant expression vectors - LUC value of wild-type tobacco leaves) / (REN value of tobacco leaves transformed with different recombinant expression vectors - REN value of wild-type tobacco leaves)). The experimental results of LUC and REN enzyme activity detection in transiently transformed tobacco leaves are shown in Table 1.

Table 1. Enzyme activities of LUC and REN and the LUC/REN ratio in tobacco leaves after transient transformation.

The results in Table 1 show that: (1) the promoters prBnUbi14-01, prBnUbi14-02, prBnUbi14-03 and prBnUbi14-04 of the present invention are generally active and can drive LUC gene expression in tobacco leaves; the LUC/REN values of prBnUbi14-05 and prBnUbi14-06 are 0, indicating that prBnUbi14-05 and prBnUbi14-06 have basically no promoter activity; (2) in tobacco leaves, compared with the control promoters pr35S and prAtH4A748:lTEV, prBnUbi14-02 has a higher activity in driving LUC gene expression.

Third embodiment: Verification of the effect of promoter elements driving LUC reporter gene expression in transgenic Arabidopsis thaliana.

1. Transformation of Agrobacterium tumefaciens with recombinant expression vector

The recombinant expression vectors DBN11-C, DBN17-C to DBN19-C, which were correctly constructed in part 1 of the second embodiment above, were transformed into Agrobacterium GV3101 using liquid nitrogen. The transformation conditions were as follows: 100 μL Agrobacterium GV3101, 3 μL plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 min, then in a 37°C water bath for 10 min; the transformed Agrobacterium GV3101 was inoculated into LB tubes and cultured at 28°C and 200 rpm for 2 h; then plated onto LB agar plates containing 50 mg/L rifampicin and 50 mg/L spectinomycin until positive single clones grew. Single clones were picked, cultured, and their plasmids were extracted. The extracted plasmids were sequenced and identified. The results showed that the recombinant expression vectors DBN11-C, DBN17-C to DBN19-C had completely correct structures.

2. Obtaining transgenic Arabidopsis plants

Wild-type Arabidopsis seeds were suspended in a 0.1% (w/v) agarose solution. The suspended seeds were stored at 4°C for 2 days to complete the necessary dormancy to ensure synchronous germination. A mixture of vermiculite and horse manure was irrigated from the bottom with water until moist, and the soil mixture was drained for 24 hours. The pretreated seeds were planted on the soil mixture and covered with a moisture-retaining cover for 7 days. The seeds were then germinated and cultivated in a greenhouse under long-day conditions (16 hours light/8 hours dark) with constant temperature (22°C), constant humidity (40-50%), and light intensity of 120-150 μmol/ ^m² s⁻¹. The plants were initially irrigated with Hogland's solution, followed by deionized water, keeping the soil moist but not saturated.

Arabidopsis thaliana was transformed using the flower immersion method. Selected Agrobacterium colonies were inoculated with one or more 15-30 mL aliquots of LB medium containing spectinomycin (50 mg/L) and rifampin (10 mg/L) (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH adjusted to 7.5 with NaOH). The pre-cultures were incubated overnight at 28°C with constant shaking at 220 rpm. Each pre-culture was used to inoculate two 500 mL aliquots of the aforementioned LB medium containing spectinomycin (50 mg/L) and rifampin (10 mg/L), and the cultures were incubated overnight at 28°C with continuous shaking. Cells were pelleted by centrifugation at approximately 4000 rpm for 20 min at room temperature, and the supernatant was discarded. The cell pellet was gently resuspended in 500 mL of osmotic medium containing 1/2 × MS salt/vitamin B5, 10% (w/v) sucrose, 0.044 μM benzylaminopurine (10 μL/L (stock solution in 1 mg/mL DMSO)) and 300 μL/L Silwet L-77. Approximately one-month-old Arabidopsis plants were immersed in the resuspended cell medium for 5 min, ensuring that the newest inflorescences were submerged. The Arabidopsis plants were then laid flat on their sides and covered, kept moist in the dark for 24 hours, and then cultured normally at 22°C with a photocycle of 16 hours of light and 8 hours of darkness. Seeds were harvested after about 4 weeks.

Newly harvested _T1 seeds (prBnUbi14-01 promoter sequence, prGm17gTsf1 control promoter sequence, pr35S control promoter sequence, prAtH4A748:lTEV chimeric control promoter sequence) were dried at room temperature for 7 days. The seeds were then sown in 26.5cm × 51cm germination trays, with each tray receiving 200mg of _T1 seeds (approximately 10,000 seeds). These seeds had been pre-suspended in distilled water and stored at 4°C for 2 days to complete the necessary dormancy and ensure synchronous germination.

Mix vermiculite with horse manure and irrigate the bottom of the soil with water until moist, then drain by gravity. Using a pipette, evenly sow the pretreated seeds onto the soil mixture and cover with a moisture-retaining cover for 4-5 days. Remove the cover one day before initial transformant selection using a glyphosate spray (selecting the co-transformed EPSPS gene) after germination.

Seven days after planting (DAP) and again at 11 DAP, _T1 plants (cotyledon stage and 2-4 leaf stage, respectively) were sprayed with a 0.5% solution of Roundup herbicide (356 g ae/L glyphosate) at a spray volume of 10 mL/tray (703 L/ha) using a DeVilbiss compressed air nozzle, providing an effective dose of 420 g ae/ha of glyphosate per application. Surviving plants (actively growing plants) were identified 4-7 days after the final spray and transplanted into 7 cm × 7 cm square pots (2-4 plants per pot) prepared with horse manure and vermiculite. The transplanted plants were covered with a moisture-retaining cover for 3-4 days and placed in a 22°C incubator as before or directly transferred to a greenhouse. Then, the cover was removed and the plants were planted in a greenhouse (temperature 22±5℃, 50±30%RH, 14h light: 10h dark, minimum 500μE/m2s-1 natural + supplemental light) for at least 1 day before testing the effect of promoter element driving LUC reporter gene.

3. Detection of the effect of the constitutive promoter of the present invention on driving LUC reporter gene expression in various tissues of Arabidopsis thaliana.

The _T1 transformant was selected from untransformed seed background using a glyphosate selection scheme. Arabidopsis _T1 plants transformed with the prBnUbi14-01 promoter sequence, Arabidopsis _T1 plants transformed with the prGm17gTsf1 control promoter sequence, Arabidopsis _T1 plants transformed with the pr35S control promoter sequence, and Arabidopsis _T1 plants transformed with the prAtH4A748:lTEV chimeric control promoter sequence were obtained from part 2 of this embodiment. Samples were taken from different parts of the above-mentioned Arabidopsis _T1 plants at different stages as test samples.

Samples were taken from three parts during the rosette stage as test samples: roots, stems, and leaves;

Samples were taken from four parts during the bolting stage as test samples: roots, stems, leaves, and flowers;

Samples were taken from four parts during the maturity stage as test samples: roots, stems, leaves, and pods.

Wild-type Arabidopsis thaliana (CK2) samples from the same part of the plant at the same growth stage were used as negative control samples.

Three identical samples of test samples from different time periods and parts of the plant, which were respectively transformed into recombinant expression vectors DBN11-C, DBN17-C to DBN19-C, and negative control samples from the same time period and part of wild-type Arabidopsis thaliana plants, were taken as biological replicates. The samples were cryogenically ground in liquid nitrogen, and then 1×PLB buffer was added to each sample. The samples were centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was collected for later use.

Following steps 5 and 6 in Example 4 of the second embodiment, dual-luciferase detection was performed on the test samples and negative control samples, using the REN gene as an internal reference. The LUC/REN ratio reflected the relative activity intensity of the promoter (LUC/REN ratio = (LUC value of test samples transformed into different recombinant expression vectors at different times and in different parts - LUC value of wild-type plants at the same time and in the same part) / (REN value of test samples transformed into different recombinant expression vectors at different times and in different parts - REN value of wild-type plants at the same time and in the same part)). The LUC/REN ratios of stably transformed Arabidopsis thaliana at different times and in different parts are shown in Table 2.

Table 2. Ratio of LUC/REN in different parts of Arabidopsis thaliana at different stages of stable transformation.

The results in Table 2 show that: (1) the promoter prBnUbi14-01 of the present invention is active and is expressed in the roots, stems, leaves, flowers and pods of Arabidopsis thaliana plants, indicating that the promoter prBnUbi14-01 can drive the constitutive expression of the target heterologous gene in the plant.

(2) In the leaves of Arabidopsis plants at the rosette, bolting and maturity stages, as well as in the mature pods, prBnUbi14-01 showed higher activity in driving LUC gene expression compared with the control promoters prGm17gTsf1, pr35S and prAtH4A748:lTEV.

(3) In the stems of Arabidopsis plants during the rosette and bolting stages, as well as in the roots during the rosette stage, prBnUbi14-01 showed higher activity in driving LUC gene expression compared to the control promoters pr35S and prAtH4A748:lTEV.

(4) In the flowers of Arabidopsis thaliana during the bolting stage, the prBnUbi14-01 promoter showed higher activity in driving LUC gene expression compared with the prAtH4A748:lTEV control promoter.

Fourth Example: Validation of the effect of promoter elements driving LUC reporter gene expression in transgenic soybeans

1. Transformation of Agrobacterium tumefaciens with recombinant expression vector

The correctly constructed recombinant expression vectors DBN11-C, DBN17-C, DBN18-C, and DBN19-C were transformed into Agrobacterium EHA101 using the liquid nitrogen method. The transformation conditions were as follows: 100 μL Agrobacterium EHA101, 3 μL plasmid DNA (recombinant expression vector); incubation in liquid nitrogen for 10 min, followed by a 37°C water bath for 10 min; the transformed Agrobacterium EHA101 was inoculated into LB tubes and cultured at 28°C and 200 rpm for 2 h; then plated onto LB agar plates containing 50 mg/L rifampicin and 50 mg/L spectinomycin until positive single colonies grew. Single colonies were picked, cultured, and their plasmids were extracted. The extracted plasmids were sequenced and identified. The results showed that the recombinant expression vectors DBN11-C, DBN17-C, DBN18-C, and DBN19-C had completely correct structures.

2. Obtaining transgenic soybean plants

Following the conventional Agrobacterium infection method, cotyledonary node tissues of aseptically cultured soybean variety SY2043C were co-cultured with the Agrobacterium described in Part 1 of this embodiment to infect the T-DNA (including the prAtAct2 promoter sequence, REN gene sequence, t35S terminator sequence, one selected from the prBnUbi14-01 promoter sequence, prGm17gTsf1 control promoter sequence, pr35S control promoter sequence, and prAtH4A748:lTEV chimeric control promoter sequence, LUC gene sequence, tPsE9 terminator sequence, prAtUbi10 promoter sequence, and spAtCTP2 nucleotide sequence) in the recombinant expression vectors DBN11-C, DBN17-C, DBN18-C, and DBN19-C. The cEPSPS gene sequence and tNos terminator sequence were transferred into the soybean chromosome, resulting in soybean plants with the prBnUbi14-01 promoter sequence, soybean plants with the prGm17gTsf1 control promoter sequence, soybean plants with the pr35S control promoter sequence, and soybean plants with the prAtH4A748:lTEV chimeric control promoter sequence.

For Agrobacterium-mediated soybean transformation, mature soybean seeds were germinated in soybean germination medium (3.1 g/L B5 salt, B5 vitamin, 20 g/L sucrose, 8 g/L agar, pH 5.6). Seeds were inoculated onto the germination medium and cultured under the following conditions: temperature 25 ± 1℃; photoperiod (light/dark) 16/8 h. One day after germination, a cotyledon and the first true leaf were removed and inoculated onto a pretreated medium containing cytokinins (MS salt 4.3 g/L, vitamin B5, sucrose 20 g/L, agar 8 g/L, 2-morpholinoethanesulfonic acid (MES) 4 g/L, zeatin (ZT) 2 mg/L, 6-benzyladenine (6-BAP) 1 mg/L, acetylsyringone (AS) 40 mg/L, pH = 5.3). Three days after inoculation on the pretreated medium, the cotyledonary node was wounded with the back of a scalpel, and Agrobacterium suspension was applied to the wounded cotyledonary node tissue. Agrobacterium can deliver the prBnUbi14-01 promoter sequence, prGm17gTsf1 control promoter sequence, pr35S control promoter sequence, and prAtH4A748:lTEV chimeric control promoter sequence to the wounded cotyledonary node tissue (Step 1: Infection Step). In this step, the cotyledonary segment tissue is preferably immersed in Agrobacterium suspension (OD ₆₆₀ = 0.5-0.8, infection medium (MS salt 2.15 g/L, vitamin B5, sucrose 20 g/L, glucose 10 g/L, acetylsuccinone (AS) 40 mg/L, 2-morpholinoethanesulfonic acid (MES) 4 g/L, zeatin (ZT) 2 mg/L, pH 5.3) to initiate inoculation. The cotyledonary segment tissue is co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-culture step). Preferably, after the infection step, the cotyledonary segment tissue is placed in solid medium (MS salt 4.3 g/L, vitamin B5, sucrose 20 g/L, glucose 10 g/L, MES 4 g/L, ZT) Cultured on a medium containing 2 mg/L B5 salt, 8 g/L agar, pH 5.6. Following this co-culture phase, a selective "recovery" step can be performed. In the "recovery" step, the medium is restored (B5 salt 3.1 g/L, B5 vitamin, MES 1 g/L, sucrose 30 g/L, ZT...). The culture medium contains at least one known antibiotic that inhibits the growth of Agrobacterium (cephalosporin 150-250 mg/L), along with 2 mg/L of agar, 8 g/L of cephalosporin, 150 mg/L of glutamic acid, 100 mg/L of aspartic acid, and pH 5.6, without the addition of a plant transformant (Step 3: Recovery Step). Preferably, the cotyledonary regenerated tissue blocks are cultured on a solid medium containing antibiotics but without a selectant to eliminate Agrobacterium and provide a recovery period for infected cells. Next, the cotyledonary regenerated tissue blocks are cultured on a medium containing a selectant (glyphosate) and the growing transformed callus is selected (Step 4: Selection Step). Preferably, the cotyledonary regenerated tissue blocks are cultured on a screening solid medium containing a selectant (B5 salt 3.1 g/L, pH 5.6). The cells are cultured on a medium containing B5 vitamins, MES 1 g/L, sucrose 30 g/L, 6-benzyladenine (6-BAP) 1 mg/L, agar 8 g/L, cephalosporin 150 mg/L, glutamate 100 mg/L, aspartic acid 100 mg/L, and N-(phosphonocarboxymethyl)glycine 0.25 mol/L (pH 5.6), resulting in selective growth of the transformed cells. The transformed cells then regenerate into plants (step 5: regeneration step). Preferably, tissue blocks regenerated from cotyledonary nodes grown on a medium containing the selective agent are cultured on solid media (B5 differentiation medium and B5 rooting medium) to regenerate plants.

The selected resistant tissue blocks were transferred to the B5 differentiation medium (B5 salt 3.1 g/L, B5 vitamin, MES 1 g/L, sucrose 30 g/L, ZT 1 mg/L, agar 8 g/L, cephalosporin 150 mg/L, glutamic acid 50 mg/L, aspartic acid 50 mg/L, gibberellin 1 mg/L, auxin 1 mg/L, N-(phosphocarboxymethyl)glycine 0.25 mol/L, pH 5.6) and cultured at 25°C for differentiation. The differentiated seedlings were transferred to the B5 rooting medium (B5 salt 3.1 g/L, B5 vitamin, MES 1 g/L, sucrose 30 g/L, agar 8 g/L, cephalosporin 150 mg/L, indole-3-butyric acid (IBA) 1 mg/L) and cultured at 25°C until approximately 10 cm tall, then transferred to a greenhouse for further cultivation until fruit set. In the greenhouse, the plants were cultured at 26°C for 16 hours each day, followed by 8 hours at 20°C.

3. Verify transgenic soybean plants using TaqMan

Approximately 100 mg of leaves were collected from soybean plants transformed with the prBnUbi14-01 promoter sequence, the prGm17gTsf1 control promoter sequence, the pr35S control promoter sequence, and the prAtH4A748:lTEV chimeric control promoter sequence, respectively. Genomic DNA was extracted using Qiagen's DNeasy Plant Maxi Kit. The copy number of the EPSPS gene was determined by TaqMan probe-based quantitative PCR to identify the copy numbers of the prBnUbi14-01, prGm17gTsf1, pr35S, and prAtH4A748:lTEV genes. Wild-type soybean plants were used as controls, and the analysis was performed according to the following method. The experiment was repeated in triplicate, and the average value was used.

The specific method for detecting the EPSPS gene copy number is as follows:

Step 6: Take 100 mg of leaves from soybean plants transformed with the prBnUbi14-01 promoter sequence, soybean plants transformed with the prGm17gTsf1 control promoter sequence, soybean plants transformed with the pr35S control promoter sequence, soybean plants transformed with the prAtH4A748:lTEV chimeric control promoter sequence, and wild-type soybean plants, respectively. Grind each sample into a homogenate in a mortar using liquid nitrogen. Take 3 replicates for each sample.

Step 7: Use Qiagen's DNeasy Plant Mini Kit to extract genomic DNA from the above samples. Refer to the product manual for specific methods.

Step 8: Determine the genomic DNA concentration of the above samples using NanoDrop 2000 (Thermo Scientific);

Step 9: Adjust the genomic DNA concentration of the above samples to the same concentration value, wherein the concentration value ranges from 80-100 ng/μL;

Step 10: The copy number of the samples was identified using TaqMan probe-based quantitative real-time PCR. Samples with known copy numbers were used as standards, and wild-type soybean plant samples were used as controls. Each sample was tested in triplicate, and the average value was taken. The primer and probe sequences for quantitative real-time PCR were as follows:

The following primers and probes are used to detect the EPSPS gene sequence:

Primer 1: ggtgtgcaggtgaagtctgaag is shown in SEQ ID NO:28 in the sequence listing;

Primer 2: gtctttggtccacgcaaggt is shown in SEQ ID NO:29 in the sequence listing;

Probe 1: cggtgatcgtcttccagt is shown in SEQ ID NO:30 in the sequence listing;

The PCR reaction system is as follows:

The 50× primer/probe mixture contains 45 μL of each primer at a concentration of 1 mM, 50 μL of the probe at a concentration of 100 μM, and 860 μL of 1×TE buffer, and is stored in amber tubes at 4°C.

The PCR reaction conditions are as follows:

The data was analyzed using SDS2.3 software (Applied Biosystems).

Analysis of the EPSPS gene copy number results confirmed that the prBnUbi14-01 promoter sequence, the prGm17gTsf1 control promoter sequence, the pr35S control promoter sequence, and the prAtH4A748:lTEV chimeric control promoter sequence had all been integrated into the chromosomes of the soybean plants tested. Furthermore, soybean plants transformed with the prBnUbi14-01 promoter sequence and those transformed with... Soybean plants with the prGm17gTsf1 control promoter sequence, soybean plants with the pr35S control promoter sequence, and soybean plants with the prAtH4A748:lTEV chimeric control promoter sequence all produced single-copy transgenic soybean plants.

4. Detection of the effect of the constitutive promoter of the present invention on driving LUC reporter gene expression in various soybean tissues.

In this embodiment, soybean plants transformed with the prBnUbi14-01 promoter sequence, soybean plants transformed with the prGm17gTsf1 control promoter sequence, soybean plants transformed with the pr35S control promoter sequence, and soybean plants transformed with the prAtH4A748:lTEV chimeric control promoter sequence were obtained. Samples were taken from different parts of the above transgenic soybean plants at different stages as test samples.

Samples were taken from three parts during the vegetative growth stage (V3 stage) as test samples: roots, stems, and leaves;

Samples were taken from six parts during the reproductive growth period as test samples: roots, stems, leaves, flowers, pods, and fruits;

Wild-type soybean plants (CK3) at the same growth stage and in the same part were sampled as negative control samples.

Test samples from different periods and parts of the plant and negative control samples from wild-type soybean plants (CK3) from the same period and part of the plant (three lines were sampled at each period, and three replicates of the same mass were sampled from each part of each line) were cryogenically ground in liquid nitrogen, and then 1×PLB buffer was added to each sample. The samples were centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected for later use.

The test samples and negative control samples were subjected to dual-luciferase detection according to steps 5 and 6 in the second embodiment 4, with the REN gene as an internal reference. The LUC/REN ratio reflects the relative activity intensity of the promoter (the definition of the LUC/REN ratio is the same as that in Table 2 of the third embodiment above). The LUC/REN ratios of different parts of soybeans at different stages of stable transformation are shown in Table 3.

Table 3. LUC/REN ratios at different stages of stable transformation in different parts of soybean.

The results in Table 3 show that: (1) the promoter prBnUbi14-01 of this invention is expressed in the roots, stems, leaves, flowers, pods and fruits of soybean plants, indicating that the promoter prBnUbi14-01 can drive the constitutive expression of the target heterologous gene in the plant; (2) in the leaves of soybean during the vegetative growth stage, compared with the prAtH4A748:lTEV control promoter, prBnUbi14-01 has a higher activity in driving the expression of the LUC gene; (3) in the leaves, roots, flowers and fruits of soybean during the reproductive growth stage, compared with the prAtH4A748:lTEV control promoter, prBnUbi14-01 has a higher activity in driving the expression of the LUC gene.

Fifth Example: Detection of Herbicide Resistance in Transgenic Arabidopsis Plants

1. Construct a recombinant expression vector that drives the herbicide-tolerant gene HTG using prBnUbi14-01.

The 5' and 3' ends of the prBnUbi14-01 promoter sequence, as well as the prGm17gTsf1 control promoter sequence, the pr35S control promoter sequence, and the prAtH4A748:lTEV chimeric control promoter sequence, were ligated to universal adapter primer 2, respectively.

5’ Universal Connector Primer 2: 5’-cacgtgaccctagtcacttaaagcttggcgcgcc-3’, as shown in SEQ ID NO:31 in the sequence listing;

3’ Universal Connector Primer 2: 5’-cagtagctggtgttggaggcat-3’, as shown in SEQ ID NO:32 in the sequence listing.

Constructing vectors using conventional enzyme digestion methods is well known to those skilled in the art. Figure 3 shows a schematic diagram of the structure of the vector DBNBC-HTG containing the herbicide resistance gene HTG (vector backbone: pCAMBIA2301 modified with resistance tag (available from CAMBIA)). (Spec: spectinomycin gene; RB: right border; AscI: restriction endonuclease AscI recognition site; HTG: hydroxyphenylpyruvate dioxygenase gene (SEQ ID NO:33); t35s: cauliflower virus 35s terminator (SEQ ID NO:21); prAtUbi10: promoter of Arabidopsis ubiquitin 10 gene (SEQ ID NO:24); spAtCTP2: Arabidopsis chloroplast transport peptide (SEQ ID NO:25); cEPSPS: 5-enolpyruvate shikimate-3-phosphate synthase gene (SEQ ID NO:26); tNos: terminator of carmine synthase gene (SEQ ID NO:27); LB: left border).

The above-mentioned vector DBNBC-HTG was linearized by digestion with the restriction endonuclease AscI. The digestion product was purified to obtain the linearized DBNBC-HTG expression vector. The prBnUbi14-01 promoter sequence ligated with the universal adapter primer 2 was then used to carry out a recombination reaction with the linearized DBNBC-HTG expression vector. The operation was performed according to the instructions of the Takara In-Fusion Snap Assembly Master Mix kit (Clontech, CA, JPN, CAT: 638949) to construct the recombinant expression vector DBN20-C, the structural schematic diagram of which is shown in Figure 4.

The recombinant expression vector DBN20-C was transformed into *E. coli* DH5α competent cells using a heat shock method. The heat shock conditions were as follows: 100 μL of *E. coli* DH5α competent cells and 20 μL of recombinant plasmid DNA (recombinant expression vector DBN20-C) were gently mixed and then heat-shocked in a 42°C water bath for 30 s, followed immediately by placing on ice for 2 min. Then, 250 μL of antibiotic-free LB broth (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, pH adjusted to 7.5 with NaOH) was added, and the cells were cultured at 37°C with shaking (200 rpm/min) for 1 h. The cells were then incubated upside down on LB agar plates containing 50 mg/L spectinomycin at 37°C for 12 h. Positive colonies were picked and cultured overnight in LB broth containing 50 mg/L spectinomycin at 37°C with shaking (200 rpm/min). Plasmid extraction was performed using the alkaline lysis method: The bacterial culture was centrifuged at 12,000 rpm for 1 min, the supernatant was discarded, and the precipitated bacterial cells were resuspended in 100 μL of ice-cold solution I (25 mM Tris-HCl, 10 mM EDTA (ethylenediaminetetraacetic acid), 50 mM glucose, pH = 8.0); 200 μL of freshly prepared solution II (0.2 M NaOH, 1% SDS (sodium dodecyl sulfate)) was added, the tube was inverted 4 times to mix, and the mixture was placed on ice for 3-5 min; 150 μL of ice-cold solution III (3 M potassium acetate, 5 M acetic acid) was added, and the mixture was immediately and thoroughly mixed, and the mixture was placed on ice for 5-10 min. The sample was centrifuged at 12000 rpm for 5 min at 4℃. The supernatant was transferred to a new 2 mL centrifuge tube, and two volumes of anhydrous ethanol were added. After mixing, the mixture was incubated at room temperature for 5 min. The sample was then centrifuged again at 12000 rpm for 5 min at 4℃. The supernatant was discarded, and the precipitate was washed with 70% ethanol (V/V) and air-dried. The precipitate was dissolved in 30 μL of TE (10 mM Tris-HCl, 1 mM EDTA, pH = 8.0) containing RNase (20 μg/mL). The RNA was digested by incubating in a water bath at 37℃ for 30 min. The sample was then stored at -20℃ for later use. The extracted plasmid was sequenced and identified. The results showed that the recombinant expression vector DBN20-C contained the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing, which is the prBnUbi14-01 promoter sequence.

The recombinant expression vector DBN20-C containing the prBnUbi14-01 promoter sequence was constructed according to the method described above, and the prGm17gTsf1 control promoter sequence and the pr35S control promoter sequence connected to the universal adapter primer 2 were used as well as the method for constructing the recombinant expression vector DBN20-C containing the prBnUbi14-01 promoter sequence and ... The prAtH4A748:lTEV chimeric control promoter sequence of the universal adapter primer 2 was used to perform recombination reactions with the linearized DBNBC-HTG expression vector to obtain recombinant expression vectors DBN21-C to DBN23-C in sequence. Sequencing verified that the above nucleotide sequences were correctly inserted in recombinant expression vectors DBN21-C to DBN23-C.

2. Transformation of Agrobacterium with recombinant expression vector

Following the method for transforming Agrobacterium with recombinant expression vectors in Part 1 of the third embodiment described above, the correctly constructed recombinant expression vectors DBN20-C to DBN23-C were transformed into Agrobacterium GV3101 using liquid nitrogen. Sequencing verification results showed that the structures of recombinant expression vectors DBN20-C to DBN23-C were completely correct.

3. Detection of the herbicide resistance effect of the promoter-driven herbicide-tolerant gene HTG in transgenic Arabidopsis plants.

Following the method described in Part 2 of the third embodiment above, Arabidopsis inflorescences were immersed in the Agrobacterium tumefaciens culture described in Part 2 of this embodiment to transfer the T-DNA from the recombinant expression vectors DBN20-C to DBN23-C constructed in Part 2 of this embodiment into the Arabidopsis chromosome, thereby obtaining the corresponding transgenic Arabidopsis plants, namely, Arabidopsis _T1 plants transformed with the prBnUbi14-01 promoter sequence, Arabidopsis T1 plants transformed with the prGm17gTsf1 control promoter sequence, Arabidopsis _T1 plants transformed with the pr35S control promoter sequence _, and Arabidopsis _T1 plants transformed with the prAtH4A748:lTEV chimeric control promoter sequence.

The _T1 transformant was selected from untransformed seed background using a glyphosate selection scheme. Arabidopsis _T1 plants transformed with the prBnUbi14-01 promoter sequence, Arabidopsis _T1 plants transformed with the prGm17gTsf1 control promoter sequence, Arabidopsis _T1 plants transformed with the pr35S control promoter sequence, Arabidopsis _T1 plants transformed with the prAtH4A748:lTEV chimeric control promoter sequence, and wild-type Arabidopsis plants (CK4) (18 days after sowing) were sprayed with 4 times the field concentration (100 g ai/ha) of bensulfuron-methyl to test herbicide tolerance in Arabidopsis. Seven days after spraying, the degree of damage to each plant from the herbicide was determined based on the proportion of leaf whitening area (leaf whitening area ratio = leaf whitening area / total leaf area × 100%): level 0 was basically no whitening phenotype, level 1 was leaf whitening area ratio less than 50%, level 2 was leaf whitening area ratio greater than 50%, and level 3 was leaf whitening area ratio of 100%.

The resistance performance of each recombinant expression vector in the transformation event was scored according to the formula X＝[Σ(N×S)/(T×M)]×100 (X-phytotoxicity score, N-number of plants with the same level of damage, S-number of phytotoxicity levels, T-total number of plants, M-highest phytotoxicity level). Resistance was evaluated based on the score: highly resistant plants (0-15 points). Moderately resistant plants (16-33 points), weakly resistant plants (34-67 points), and non-resistant plants (68-100 points). The experimental results are shown in Table 4.

Table 4. Results of tolerance experiment of transgenic Arabidopsis thaliana _T1 plants to benzyladenine.

For Arabidopsis, 4 times the field concentration of benzimidone is the effective dose for high-stress treatment. The results in Table 4 show that: (1) Compared with CK4, Arabidopsis plants transformed with the prBnUbi14-01 promoter sequence were tolerant to benzimidone at 4 times the field concentration. It can be seen that the constitutive promoter prBnUbi14-01 of this invention can drive the expression of the target heterologous gene in plants. (2) Compared with the pr35S and prAtH4A748:lTEV control promoters, prBnUbi14-01 has a better effect on driving the herbicide resistance gene HTG in transgenic Arabidopsis plants.

In summary, this invention discloses for the first time a constitutive promoter from the Ubiquitin gene of rapeseed. The constitutive promoter of this invention shows activity in almost all tissues and many types of cells in plants, especially in the roots, stems, leaves, flowers, pods, and fruits of plants, and has broad application prospects in plants.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (18)

A constitutive promoter, characterized in that its nucleotide sequence includes SEQ ID NO:4, said constitutive promoter being derived from SEQ ID NO:1. The constitutive promoter according to claim 1 is characterized in that its nucleotide sequence includes SEQ ID NO:4 and is selected from at least a portion of SEQ ID NO:1. The constitutive promoter according to claim 1 or 2 is characterized in that its nucleotide sequence is as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. A recombinant DNA construct comprising a constitutive promoter according to any one of claims 1-3 operably linked to a target heteronucleotide sequence. The recombinant DNA construct according to claim 4 is characterized in that the target heteronucleotide sequence encodes the target protein. An expression cassette comprising the recombinant DNA construct of claim 4 or 5. A recombinant vector comprising the expression cassette of claim 6. A method for expressing a target heteronucleotide sequence in a plant, characterized by comprising: stably integrating the target heteronucleotide sequence operatively linked to a constitutive promoter according to any one of claims 1-3 into a plant cell. The method for expressing a target heteronucleotide sequence in a plant according to claim 8, wherein the plant is Arabidopsis thaliana, rapeseed, tobacco, soybean, cotton, pepper, beet, pumpkin, eggplant, Chinese cabbage, carrot, tomato, pea, spinach, potato or peanut. The method for expressing a target heteronucleotide sequence in a plant according to claim 8 is characterized in that the target heteronucleotide sequence is constitutively expressed in plant tissues. The method for expressing a target heteronucleotide sequence in a plant according to claim 8, wherein the target heteronucleotide sequence encodes a target protein. The method for expressing a target heteronucleotide sequence in a plant according to claim 11, wherein the target heteronucleotide sequence encodes a herbicide-resistant protein. The method for expressing a target heteronucleotide sequence in a plant according to claim 11, wherein the target heteronucleotide sequence encodes an insect resistance protein. A plant or part thereof, characterized in that it comprises a constitutive promoter as described in any one of claims 1-3. A method for obtaining processed agricultural products, characterized by comprising: [the following steps are described in claim 14] The plant or part of the harvested material is processed to obtain processed agricultural products. Use of a constitutive promoter according to any one of claims 1-3 for constitutive expression of a target heteronucleotide sequence in plant tissues. The use of the constitutive promoter according to claim 16 for constitutive expression of a target heteronucleotide sequence in plant tissues, characterized in that the plant is Arabidopsis thaliana, rapeseed, tobacco, soybean, cotton, pepper, beet, pumpkin, eggplant, Chinese cabbage, carrot, tomato, pea, spinach, potato or peanut. The use of the constitutive promoter according to claim 16 for constitutive expression of a target heteronucleotide sequence in plant tissues, characterized in that the target heteronucleotide sequence encodes a target protein.