CN118853674A - Constitutive promoters and uses thereof - Google Patents

Abstract

The invention relates to a constitutive promoter and application thereof, wherein the nucleotide sequence of the constitutive promoter comprises SEQ ID NO. 3, and the constitutive promoter is derived from SEQ ID NO. 1. The constitutive promoter of the invention shows activity in most tissues and cells of many types of plants, especially in roots, stems, leaves, flowers, pod skin and fruits of plants, and has wide application prospect on plants.

Description

Constitutive promoters and uses thereof

Technical Field

The invention relates to a constitutive promoter and application thereof, in particular to a constitutive promoter from rape Histone genes and application thereof.

Background

One of the goals of plant genetic engineering is to produce plants having agriculturally desirable characteristics or traits, often desirable traits including improved nutritional quality, increased yield, conferring pest resistance, improved drought and stress tolerance, improved horticultural quality, conferring herbicide resistance, and the like. Current technological advances have enabled researchers to acquire exogenous polynucleotide molecules (e.g., heterologous or naturally derived genes) and integrate the polynucleotide molecules into the plant genome, which genes are expressed in plant cells to exhibit the corresponding traits. It is important that the appropriate regulatory signals must be present in the appropriate structure to obtain expression of the coding sequence of the newly inserted gene in the plant cell. These regulatory signals typically include a promoter region, a5 'untranslated leader sequence, and a 3' transcription terminator/polyadenylation sequence.

Certain promoters are capable of directing RNA synthesis at a level of expression in most or all tissues and/or growth stages of a plant and are referred to as "constitutive promoters". Constitutive promoters can be classified into strong, medium and weak promoters according to their effect on directing RNA synthesis. Constitutive promoters are particularly advantageous in these situations, since in many cases it is necessary to express the gene of interest simultaneously in different tissues of the plant in order to obtain the desired gene function.

Several constitutive promoters have been described in the prior art, which are active in plant cells, including the promoter Gm17gTsf1 of the soybean cell elongation factor gene, the nopaline synthase (nos) promoter carried on the Agrobacterium tumefaciens (Agrobacterium tumefaciens) tumor-inducing plasmid, the octopine synthase (ocs) promoter and the cauliflower mosaic virus (Caulimovirus) promoter, such as the cauliflower mosaic virus (CaMV) 19S or 35S promoter, the CaMV 35S promoter with repeated enhancers and the Figwort Mosaic Virus (FMV) 35S promoter, which have been used in plant constructs for transgenic expression. Although some constitutive promoters are currently available, there is still a great interest in isolating more novel constitutive promoters which are capable of controlling expression of recombinant DNA constructs (or genes) at different levels, as well as for the expression of multiple genes superimposed on the same transgenic plant.

Disclosure of Invention

The invention aims to provide a novel constitutive promoter and application thereof, wherein the promoter can enable a heterologous nucleotide sequence to be expressed in plant tissues with high efficiency.

To achieve the above object, the present invention provides a constitutive promoter, the nucleotide sequence of which comprises SEQ ID NO:3, said constitutive promoter is derived from SEQ ID NO:1.

Further, the present invention provides a constitutive promoter, the nucleotide sequence of which comprises SEQ ID NO:3 and is selected from SEQ ID NO: 1.

Further, the invention provides a constitutive promoter, the nucleotide sequence of which is shown in SEQ ID NO:1. SEQ ID NO:2 or SEQ ID NO: 3.

To achieve the above object, the present invention also provides a recombinant DNA construct comprising the above constitutive promoter operably linked to a heterologous nucleotide sequence of interest.

Further, the heterologous nucleotide sequence of interest encodes a protein of interest.

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

In order to achieve the above object, the present invention also provides a recombinant vector comprising the above expression cassette.

To achieve the above object, the present invention also provides a method for expressing a heterologous nucleotide sequence of interest in a plant, comprising: the heterologous nucleotide sequence of interest operably linked to the constitutive promoter described above is stably integrated into the plant cell.

Further, the plant is arabidopsis thaliana, rape, tobacco, soybean, cotton, capsicum, beet, pumpkin, eggplant, chinese cabbage, carrot, tomato, pea, spinach, potato or peanut.

Preferably, the heterologous nucleotide sequence of interest is constitutively expressed in plant tissue.

Further, the heterologous nucleotide sequence of interest encodes a protein of interest.

Preferably, the heterologous nucleotide sequence of interest encodes a herbicide tolerance protein.

Preferably, the heterologous nucleotide sequence of interest encodes an insect-resistant protein.

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

To achieve the above object, the present invention also provides a method for obtaining processed agricultural products, comprising treating a harvest of the above plants or parts to obtain processed agricultural products.

In order to achieve the above object, the present invention also provides a use of the above constitutive promoter for constitutive expression of a heterologous nucleotide sequence of interest in plant tissue.

Preferably, the plant is arabidopsis thaliana, canola, tobacco, soybean, cotton, capsicum, beet, pumpkin, eggplant, chinese cabbage, carrot, tomato, pea, spinach, potato, or peanut.

Further, the heterologous nucleotide sequence of interest encodes a protein of interest.

The terms "comprising," including, "and" comprising "in this disclosure mean" including but not limited to.

The term "promoter" as used herein refers to a DNA regulatory region, typically comprising a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at a suitable transcription initiation site for a particular coding sequence. The term "gene" in the present invention refers to any DNA fragment containing a DNA region (the "transcribed DNA region") transcribed into an RNA molecule (e.g. mRNA) in a cell under the control of a suitable regulatory region (e.g. a plant expressible promoter region). Thus, a gene may contain several operably linked DNA fragments, such as a promoter, a 5 'untranslated leader sequence, a coding region, and a 3' untranslated region containing a polyadenylation site. Endogenous plant genes are genes found naturally in plant species. A recombinant DNA construct is any gene that is not normally found in a plant species, or any gene whose promoter is naturally unrelated to a partially or fully transcribed DNA region or to at least another regulatory region of the gene.

The term "constitutive promoter" in the present invention refers to a special class of gene regulatory sequences. Under the control of such promoters, most or all tissues and/or stages of growth of an organism exhibit a degree of gene expression. Constitutive promoters are used for expression of an operably linked gene, a heterologous nucleotide sequence of interest, or a gene editing system guide RNA (gRNA) in most cells of an organism, with some persistence of expression being initiated. It will be appreciated 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 formed by gathering one or more types of cells with the same source and performing the same function in the plant body, such as a protective tissue, a guide tissue, a nutrition tissue, a mechanical tissue and a meristematic tissue, and different tissues are organically matched and closely connected to form different organs (organs), and the different organs are mutually matched to more effectively complete the whole life activity process of the organism. The growth and development stages can be classified into embryo stage, seedling stage, maturation stage and senescence stage according to the difference of plant morphology and function.

The term "constitutive expression" in the present invention means that the gene or heterologous nucleotide sequence of interest exhibits relatively stable and sustained expression in most or all tissues and/or growth and development phases of the plant, for example, the cauliflower mosaic virus CaMV35S promoter, which is capable of initiating high-strength expression of the exogenous gene in the plant in most organs and at various developmental stages; the constitutive promoter can ensure the wide expression of gRNA in host cell and raise the efficiency and accuracy of gene editing.

Isolated sequences that have promoter activity and hybridize under stringent conditions to the promoter sequences of the present invention or fragments thereof are 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., sequence identity ranges from at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity.

The application provides a constitutive promoter, the nucleotide sequence of which comprises SEQ ID NO:3 and is selected from SEQ ID NO: 1. SEQ ID NO:3 is SEQ ID NO:1, with reference to SEQ ID NO:1, a constitutive promoter of the application can be represented in SEQ ID NO:3 to its 5 'end or 3' end alone or to both its 5 'and 3' ends, but the length of the nucleotide sequence extending to both ends thereof cannot exceed the length of the nucleotide sequence of SEQ ID NO:1 itself, or a 3' end. In other words, the nucleotide sequence is as set forth in SEQ ID NO:3, or a constitutive promoter as set forth in SEQ ID NO:1, by the sequence set forth in SEQ ID NO:3 and the extension length thereof is not more than the extension length of SEQ ID NO:1 or 3' end of the host cell is within the scope of the present application. Further, with respect to such a constitutive promoter (see SEQ ID NO:1, a constitutive promoter obtained by arbitrarily extending both ends of SEQ ID NO:3 and having an extension length not exceeding the 5 'end or 3' end of SEQ ID NO:1 itself), the constitutive promoter includes a sequence other than SEQ ID NO:3 does not affect the sequence of SEQ ID NO:3 activity of the sequence itself. This conclusion is also demonstrated by the second embodiment of the present application: prBnH4 the 4-03 promoter (SEQ ID NO: 3) is active and comprises the sequence of SEQ ID NO:3 (SEQ ID NO: 1) and the prBnH4-02 promoter (SEQ ID NO: 2). One skilled in the art can reasonably predict, based on the disclosure herein, a nucleotide sequence comprising SEQ ID NO:3 and is selected from SEQ ID NO:1, each having a constitutive promoter sequence identical to SEQ ID NO:3, the same or similar activity.

The promoter sequences and fragments thereof of the present invention are useful for genetic manipulation of any plant when assembled into a DNA structure such that the promoter sequence is operably linked to a heterologous nucleotide sequence of interest. The term "operably linked" refers to a functional linkage between a promoter sequence of the present invention and a second sequence, wherein the promoter sequence initiates and regulates transcription of a DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, if necessary, joined together in adjacent, in reading frame with the two protein coding regions. In this way, the promoter nucleotide sequence is provided together with the heterologous nucleotide sequence of interest constituting the recombinant DNA construct in an expression cassette for expression in a plant of interest. Such an expression cassette provides a large number of restriction sites for insertion of a heterologous nucleotide sequence of interest that will be transcriptionally regulated by a regulatory region comprising the promoter sequence of the present invention. The expression cassette may additionally contain at least one additional gene to be co-transformed into the organism. Alternatively, additional genes may be provided on multiple expression cassettes.

The expression cassette may additionally contain a selectable marker gene. Typically, the expression cassette will comprise a selectable marker gene for selection of transformed cells. The selectable marker gene is used to select for transformed cells or tissues. Such selectable marker genes include, but are not limited to, genes encoding antibiotic resistance (e.g., 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.

The expression cassette comprises the promoter sequence of the invention transcribed in the 5'-3' direction, a translation initiation region, a heterologous nucleotide sequence of interest and transcription and translation termination regions which function in plants. The heterologous nucleotide sequence of interest may be native or foreign or heterologous to the plant host. Alternatively, the heterologous nucleotide sequence of interest may be a natural sequence or a selectively synthetic sequence. By "exogenous" is meant that the introduced transcription initiation region is absent from the native plant into which it was introduced. For example, a recombinant DNA construct comprises a promoter sequence of the invention operably linked to a coding sequence that differs from the promoter sequence of the invention.

The termination region may be derived from the promoter sequences of the present invention, may be derived from operably linked heterologous nucleotide sequences of interest, or may be derived from another source. Conventional termination regions are available from the Ti plasmid of Agrobacterium tumefaciens, such as the carnitine synthase and nopaline synthase (NOS) termination regions.

In the preparation of the expression cassette, the different DNA fragments can be manipulated to provide DNA sequences in the appropriate orientation and, where appropriate, reading frames. Therefore, acceptors or linkers can be used to bind the DNA fragments, or other manipulations can be performed to provide convenient restriction sites, remove excess DNA, remove restriction sites, and the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, re-substitution, such as transformation and conversion, may be involved.

Where appropriate, the heterologous nucleotide sequence of interest may be optimized to increase the amount of expression in the transformed plant. That is, plant preferred codons may be used to synthesize genes to improve expression.

Additional sequence modifications are known in the art to increase the level of gene expression in a cellular host. These include, but are not limited to, removal of repetitive sequences encoding pseudo-polyadenylation signals, exon-intron splice site signals, transposons, and other sequences that are well characterized as potentially detrimental to gene expression. The G-C content of the sequences can be adjusted to the average level of the indicated host cell and calculated by reference to the known gene expression levels in the host cell. Possibly, the sequence is 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. The leader sequence may function to improve transcription efficiency. Such leader sequences are known in the art and include, but are not limited to, picornaviral leader sequences, such as EMCV leader sequences (5' non-coding region of encephalomyocarditis virus); potexvirus leader sequences, such as Tobacco Etch Virus (TEV) leader sequence, maize Dwarf Mosaic Virus (MDMV) leader sequence, and human immunoglobulin heavy chain binding protein (BiP); an untranslated leader sequence from alfalfa mosaic virus-coated protein mRNA (AMV RNA 4); tobacco Mosaic Virus (TMV) leader sequence; and a maize chlorosis spot virus (MCMV) leader sequence. Other known elements that improve transcription efficiency, such as introns, etc., may also be used.

The promoter sequences of the present invention may be used to initiate transcription of antisense constructs at least partially complementary to messenger RNA (mRNA) of a heterologous nucleotide sequence of interest. The antisense nucleotide sequence was constructed to hybridize with the corresponding mRNA. Modification of the antisense sequence can be performed as long as the antisense sequence hybridizes to the corresponding mRNA and interferes with its expression. In this way, antisense constructs having 80%, preferably 90%, more preferably 95% sequence identity to the corresponding antisense sequences can be used. In addition, a portion of the antisense nucleotide sequence may be used to disrupt expression of the target gene. Generally, sequences of at least 50 nucleotides, 100 nucleotides, 200 nucleotides or more may be used.

The promoter sequences of the invention are used for constitutive expression of the heterologous nucleotide sequence of interest. "heterologous nucleotide sequence" refers to a sequence that does not naturally occur with the promoter sequence. Although the nucleotide sequence is heterologous to the promoter sequence, it may be homologous or native or heterologous or exogenous to the plant host. The heterologous nucleotide sequence operably linked to the promoter of the present invention may encode a protein of interest. Examples of such heterologous nucleotide sequences include, but are not limited to, nucleotide sequences encoding polypeptides conferring 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 the prevention of diseases associated with the production of these organisms.

The herbicide tolerance protein of the present invention may express resistance and/or tolerance to herbicides. These genes include, but are not limited to, hydroxyphenylpyruvate dioxygenase (HPPD) genes, protoporphyrinogen oxidase (PPO) genes, acetolactate synthase (ALS) genes, 5-enolpyruvylshikimyl-3-phosphate synthase (EPSPS) genes, oxamidophosphorylase (PAT) genes, glyphosate Oxidoreductase (GOX) genes, GAT genes, and the like.

By "insect resistance" is meant herein that the plant is protected from symptoms and damage caused by plant-insect interactions. I.e., to prevent, or alternatively to minimize or reduce insect-induced plant damage, crop damage, plant damage, and plant disease. The insect may be of the order lepidoptera (e.g., corn borer), hemiptera (e.g., stink bugs), coleoptera (e.g., beetles), orthoptera (e.g., migratory locust), homoptera (e.g., aphids), diptera (e.g., flies), and the like. Insect resistance proteins of interest, as known in the art, include, but are not limited to, bacillus toxic proteins; lectins, wherein the lectin comprises galanthamine lectin, pea lectin, jack bean lectin, malt lectin, potato lectin, peanut lectin, etc.; lipoxygenase, wherein the lipoxygenase comprises pea lipoxygenase 1 or soybean lipoxygenase; insect chitinase, and the like.

The manner in which different pests transmit viruses from infected plants to healthy plants is different. Such viruses include, but are not limited to, rice Douglas baculovirus, tobacco mosaic virus, sweet potato chlorosis virus, sweet potato pekoe virus, and the like. Thus, heterologous nucleotide sequences may be selected that constitutively express in plant tissue have anti-pathogenic activity or minimize the effects of viral pathogens.

The promoter sequences and methods of the present invention can be used for expression regulation of any heterologous nucleotide sequence of interest in a plant host to alter the phenotype of the plant. The various types of purpose phenotype alterations include, but are not limited to, altering the fatty acid composition of the plant, altering the amino acid content of the plant, altering plant pathogen defense mechanisms, and the like. Such alterations may be obtained by providing for expression of heterologous products or by increasing expression of endogenous products in the plant. Alternatively, the alteration may be obtained by reducing the expression of one or more endogenous products in the plant, in particular enzymes or cofactors. Such changes will result in a change in the phenotype of 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., monocotyledonous or dicotyledonous plants. Suitable methods for introducing nucleotide sequences into plant cells and subsequent insertion into plant genomes include, but are not limited to, agrobacterium-mediated transformation, microprojectile bombardment, direct DNA uptake into protoplasts, electroporation or whisker-silicon-mediated DNA introduction.

The cells that have been transformed can be grown into plants in a conventional manner. These plants are grown and pollinated with the same transformant or different transformants to obtain the identified phenotype characteristics required for expression of the hybrid. Two or more generations may be grown to ensure stable maintenance and inheritance of the expression of the desired phenotypic trait, and then seeds may be harvested to ensure expression of the desired phenotypic trait.

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 may be plants obtained by conventional breeding and optimization methods or by biotechnology and recombinant methods, or combinations 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 be regenerated, plant calli, clumps (plant clumps) and whole plant cells in plants or plant parts. Such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, and the like. It is to be understood that 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, which are derived from transgenic plants or their progeny which have been previously transformed with the DNA molecules of the present invention and thus at least partially consist of the transgenic cells.

In one aspect, the plant part is a plant cell. In yet another aspect, the plant part is a non-regenerable or regenerable cell. In another aspect, the plant cell is a somatic cell.

Non-regenerable cells refer to cells that cannot be regenerated into whole plants by in vitro culture. Non-regenerable cells can be in plants or plant parts (e.g., leaves) of the present invention. The non-regenerable cells may be cells in the seed or seed coat of the seed. Mature plant organs (including mature leaves, mature stems or mature roots) contain at least one non-regenerable cell.

In another aspect, the plant cell is a germ cell, such as an ovule or a cell that is part of pollen. In one aspect, the pollen cell is a vegetative (non-germ) cell, or a sperm cell.

The present invention provides for the treatment of a harvest of plants or parts comprising the constitutive promoter of the invention to obtain processed agricultural products. The term "processed agricultural product" refers to any composition or product consisting of material derived from a plant, seed, plant cell or plant part comprising a constitutive promoter of the invention. In particular, the term "processed agricultural product" includes, but is not limited to, protein concentrates, protein isolates, starches, flours, biomass, and seed oils.

The invention provides a constitutive promoter and application thereof, and the constitutive promoter has the following advantages:

1. The invention discloses a constitutive promoter from rape Histone gene for the first time, wherein the nucleotide sequence of the constitutive promoter comprises SEQ ID NO:3 and is derived from SEQ ID NO:1.

2. The constitutive promoters of the invention show activity in almost most tissues and many types of cells of plants, in particular in roots, stems, leaves, flowers, pod skin, fruits of plants.

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

The technical scheme of the invention is further described in detail through the drawings and the embodiments.

Drawings

FIG. 1 is a schematic diagram of the structure of a vector DBNBC-Dual_LUC containing LUC and REN reporter genes of the present invention;

FIG. 2 is a schematic diagram showing the structure of a recombinant expression vector DBN11-D containing the prBnH-01 promoter sequence according to the present invention;

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

FIG. 4 is a schematic diagram showing the structure of the recombinant expression vector DBN18-D of the present invention.

Detailed Description

The constitutive promoter of the present invention and its technical scheme of use are further described below by specific examples.

First example, obtaining the constitutive promoter of the present invention

1. Obtaining prBnH a 4-01 promoter sequence

Genes that are expressed in high abundance in tissues such as roots, stems, leaves, flowers, seeds, and fruits can be retrieved by querying the public transcriptome database of canola (BnTIR: brassica napus transcriptome information resource. (hzau. Edu. Cn)). A925 bp sequence upstream of gene BnaC G0011300ZS was selected and designated promoter prBnH-01. Taking the cabbage type rape Westar genome DNA sequence as a PCR amplification template, designing a primer 1 and a primer 2 for PCR amplification:

primer 1:5'-ctgctccgctagtttctgtcctt-3' as set forth in SEQ ID NO:5 is shown in the figure;

Primer 2:5'-ctgaataccatcaaaacaatacc-3' as set forth in SEQ ID NO: shown at 6.

The PCR reaction system is as follows:

The primers contained 2.5. Mu.L of each primer at a concentration of 10. Mu.M, the reaction buffer was NEW ENGLAND The PCR reaction system was supplemented with nuclease-free water to 50. Mu.L in the reaction buffer of the High-FIDELITY DNA Polymerase kit, incorporated herein by reference. The specific operation steps are according to NEW ENGLAND Company PCR UsingHigh-Fidelity DNAPolymerase (M0491) kit instructions.

The PCR reaction conditions were:

the PCR amplified product is connected with a Blunt end Blunt vector (full-scale gold company cloning vector, beijing), the operation steps are carried out according to the specification of the full-scale gold company product Blunt vector, then the connected product is sequenced (Sanger method sequencing), and the prBnH-01 promoter sequence is confirmed, as shown in SEQ ID NO: 1.

2. PrBnH4-02, prBnH4-03 and prBnH4-04 promoter sequences were obtained

The prBnH4-01 gene sequence is used as a PCR amplification template, and the following primer pairs are respectively designed: primer 2 (SEQ ID NO: 6) and primer 3 (SEQ ID NO: 7), primer 2 (SEQ ID NO: 6) and primer 4 (SEQ ID NO: 8), primer 2 (SEQ ID NO: 6) and primer 5 (SEQ ID NO: 9), PCR amplification was performed using the above-described primer pairs, respectively, according to the method of obtaining the prBnH4-01 promoter sequence, to obtain the prBnH4-02 promoter sequence (SEQ ID NO: 2), the prBnH4-03 promoter sequence (SEQ ID NO: 3) and the prBnH4-04 promoter sequence (SEQ ID NO: 4) in this order.

3. PrBnH4 a 4-01 to prBnH4-04 promoter sequence was ligated to the primer

The prBnH-01 promoter sequence, prBnH-02 promoter sequence, prBnH4-03 promoter sequence and prBnH-04 promoter sequence described above, and prGm17gTsf1 control promoter sequence (SEQ ID NO: 10), pr35S control promoter sequence (SEQ ID NO: 11) and prAtH A748 were used: the 5 'and 3' ends of the 1TEV chimeric control promoter sequence (SEQ ID NO: 12) were ligated to universal adaptor primer 1:

5' -terminal universal adaptor primer 1:5'-ctaaaaccaaaatccagtggactagt-3' as set forth in SEQ ID NO: 13;

3' -terminal universal adaptor primer 1:5'-atgtttttggcgtcttccat-3' as set forth in SEQ ID NO: 14.

Second example, verification of the Effect of promoter element driving the expression of LUC reporter Gene in transgenic tobacco

1. A Dual luciferase (Dual-Luciferase Reporter) report system was introduced to construct a recombinant expression vector containing prBnH4-01 promoter sequence, a recombinant expression vector containing prBnH4-02 promoter sequence, a recombinant expression vector containing prBnH4-03 promoter sequence and a recombinant expression vector containing prBnH4-04 promoter sequence, respectively.

The construction of vectors using conventional methods of cleavage is well known to those skilled in the art. The structure of vector DBNBC-Dual_LUC (vector backbone: pCAMBIA2301 (available from CAMBIA Co.) with modified resistance tag) containing LUC and REN reporter genes is shown in FIG. 1 (Spec: spectinomycin resistance gene; RB: right border; prAtAct: arabidopsis Act2 gene promoter (SEQ ID NO: 15), REN: renilla luciferase gene (SEQ ID NO: 16), t35s: cauliflower virus 35s terminator (SEQ ID NO: 17), speI: restriction enzyme SpeI recognition site; LUC: firefly luciferase gene (SEQ ID NO: 18), tPsE: terminator of RbcS gene (SEQ ID NO: 19), prAtUbi: promoter of Arabidopsis Ubiquitin (Ubiqui) 10 gene (SEQ ID NO: 20), spAtCTP: arabidopsis thaliana transit peptide (SEQ ID NO: 21), cEPSPS: 5-enolpyruvylshikimate-3-phosphate synthase gene (SEQ ID NO: 16), and R1: 4. Ipomace gene (SEQ ID NO: 498).

The vector DBNBC-Dual_LUC was digested with restriction enzyme SpeI, thereby linearizing the vector DBNBC-Dual_LUC, purifying the digested product to obtain a linearized DBNBC-Dual_LUC expression vector, and recombining the prBnH-01 promoter sequence linked to the universal adaptor primer 1 with the linearized DBNBC-Dual_LUC expression vector, wherein the procedure was performed according to the instructions of Takara In-Fusion Snap Assembly Master Mix kit (Clontech, CA, JPN, CAT: 638949) to construct a recombinant expression vector DBN11-D, the structure of which is schematically shown In FIG. 2.

The recombinant expression vector DBN11-D is used for transforming competent cells of escherichia coli DH5 alpha by a heat shock method, and the heat shock conditions are as follows: 100 mu L of escherichia coli DH5 alpha competent cells, 20 mu L of recombinant plasmid DNA (recombinant expression vector DBN 11-D), carrying out light flick, uniformly mixing, carrying out water bath heat shock at 42 ℃ for 30s, and immediately placing on ice for 2min; mu.L of antibiotic-free LB liquid medium (tryptone 10g/L, yeast extract 5g/L, naCl _10g/L, pH adjusted to 7.5 with NaOH) was added and incubated with shaking (200 rpm) at 37℃for 1 h. Then, the positive clone colonies were picked up by culturing in an inverted manner on the LB solid plate containing 50mg/L of spectinomycin (Spectinomycin) at 37℃for 12 hours, and cultured overnight in LB liquid medium containing 50mg/L of spectinomycin at 37℃with shaking (200 rpm). Extracting the plasmid by an alkaline lysis method: centrifuging the bacterial liquid at 12000rpm for 1min, removing supernatant, and suspending the precipitated bacterial body with 100uL of ice-precooled solution I (25 mM Tris-HCl, 10mM EDTA (ethylenediamine tetraacetic acid), 50mM glucose, pH=8.0); 200. Mu.L of freshly prepared solution II (0.2M NaOH, 1% SDS (sodium dodecyl sulfate)) was added, the tube was inverted 4 times, mixed, and placed on ice for 3-5min; adding 150 μl ice-cold solution III (3M potassium acetate, 5M acetic acid), immediately mixing, and standing on ice for 5-10min; centrifuging at 4deg.C and 12000rpm for 5min, transferring supernatant to new 2mL centrifuge tube, adding 2 times volume of absolute ethanol, mixing, and standing at room temperature for 5min; centrifuging at 4deg.C and 12000rpm for 5min, removing supernatant, washing precipitate with 70% ethanol (V/V), and air drying; 30. Mu.L of TE (10 mM Tris-HCl, 1mM EDTA, pH=8.0) containing RNase (20. Mu.g/mL) was added to dissolve the precipitate; digesting RNA in a water bath at 37 ℃ for 30 min; preserving at-20deg.C. Sequencing and identifying the extracted plasmid, and the result shows that the recombinant expression vector DBN11-D contains SEQ ID NO:1, i.e., the prBnH-01 promoter sequence.

According to the above method for constructing recombinant expression vector DBN11-D comprising prBnH4-01 promoter sequence, said prBnH-02 promoter sequence linked to said universal adaptor primer 1, said prBnH-03 promoter sequence linked to said universal adaptor primer 1, said prBnH4-04 promoter sequence linked to said universal adaptor primer 1, said prGm17gTsf1 control promoter sequence linked to said universal adaptor primer 1, said pr35S control promoter sequence linked to said universal adaptor primer 1 and said prAtH A748 linked to said universal adaptor primer 1 are described above: and (3) respectively carrying out recombination reaction on the 1TEV chimeric control promoter sequences and the linearized DBNBC-dual_LUC expression vector to sequentially obtain recombinant expression vectors DBN12-D to DBN17-D, and carrying out sequencing to verify that the nucleotide sequences in the recombinant expression vectors DBN12-D to DBN17-D are correctly inserted.

2. Recombinant expression vector transformation of agrobacterium

The correctly constructed recombinant expression vectors DBN11-D to DBN17-D were transformed into Agrobacterium LBA4404 (Invitrogen, chicago, USA; cat. No. 18313-015) by liquid nitrogen method, respectively, under the following transformation conditions: 100. Mu.L of Agrobacterium LBA4404, 3. Mu.L of plasmid DNA (recombinant expression vector); placing in liquid nitrogen for 10min, and warm water bath at 37deg.C for 10min; the transformed agrobacterium LBA4404 is inoculated in an LB test tube and cultured for 2 hours at the temperature of 28 ℃ and the rotating speed of 200rpm, and is coated on an LB solid plate containing 50mg/L of Rifampicin (RIFAMPICIN) and 50mg/L of spectinomycin until positive monoclonal grows out, the monoclonal culture is selected, plasmids are extracted, sequencing identification is carried out on the extracted plasmids, and the result shows that the recombinant expression vectors DBN11-D to DBN17-D have completely correct structures.

3. Transient transformation of tobacco lamina

The tobacco leaf is a biological cell reactor for high-efficiency protein expression, exogenous genes are introduced into the tobacco leaf for expression by using an agrobacterium injection infiltration method, and the effect of the constitutive promoter is verified by using high-flux protein expression.

The tobacco leaf transformation method comprises the following steps:

Step1, planting tobacco, namely culturing the tobacco for 4-5 weeks under the conditions of 14h illumination/10 h darkness, 25 ℃ and 70% relative humidity, and collecting tobacco leaves;

Step 2, sequentially picking the Agrobacterium strain transformed with the recombinant expression vector DBN11-D, the Agrobacterium strain transformed with the recombinant expression vector DBN12-D, the Agrobacterium strain transformed with the recombinant expression vector DBN13-D, the Agrobacterium strain transformed with the recombinant expression vector DBN14-D, the Agrobacterium strain transformed with the recombinant expression vector DBN15-D, the Agrobacterium strain transformed with the recombinant expression vector DBN16-D, the Agrobacterium strain transformed with the recombinant expression vector DBN17-D of the present example, cloning them into 1mL of LB liquid medium (tryptone 10g/L, yeast extract 10g/L, naCl 5g/L, rifampicin (RIFAMPICIN) 50mg/mL, spectinomycin 50mg/mL, tetracycline 10 mg/mL) containing antibiotics, respectively, culturing at 28deg.C with shaking (200 rpm) to logarithmic phase of Agrobacterium growth (OD ₆₀₀ =0.5-0.6), taking 1mL of Agrobacterium solution of logarithmic phase and transferring to 20mL of LB liquid medium containing antibiotics (tryptone 10g/L, yeast extract 10g/L, naCl 5g/L, rifampicin (RIFAMPICIN) 50mg/mL, spectinomycin 50mg/mL, tetracycline 10 mg/mL), culturing at 28deg.C with shaking (200 rpm) to logarithmic phase of Agrobacterium (OD ₆₀₀ =0.5-0.6), centrifuging at 5000rpm at room temperature for 10min, collecting thallus, suspending Agrobacterium with infection solution (containing 10mMMgCl ₂, 10mMMES, 150 μm acetosyringone, pH=5.6) to OD ₆₀₀ =0.8, standing at room temperature for 2-3h, respectively obtaining agrobacterium tumefaciens bacteria liquid which is used for injection and is transformed with recombinant expression vectors DBN11-D to DBN 17-D;

Step 3, lightly pointing a small opening (note not to be pierced) on the back of the tobacco leaf obtained in step 1 of the present embodiment by using a 1mL needle, then respectively sucking the agrobacterium solution transformed with the recombinant expression vectors DBN11-D to DBN17-D in step 2 of the present embodiment by using a needle tube with the needle removed, injecting the agrobacterium solution into the tobacco leaf from the small opening of the leaf to transfer the T-DNA (comprising prAtAct promoter sequence, REN gene sequence, T35S terminator sequence, prBnH-01 promoter sequence, prBnH-02 promoter sequence, prBnH4-03 promoter sequence, prBnH-04 promoter sequence, prGm17gTsf1 control promoter sequence, pr35S control promoter sequence and prAtH A748:1TEV chimeric control promoter sequence, LUC gene sequence, tPsE terminator sequence, prAtUbi promoter sequence, spAtCTP nucleotide sequence, cEPSPS gene sequence and tNos promoter sequence) in the recombinant expression vectors DBN11-D to the tobacco leaf; wild-type tobacco leaf (CK 1) was also used as a control. Marking the water-stained area of the tobacco leaves by using a marker;

and 4, placing the tobacco leaves and the wild tobacco leaves injected in the step 3 of the embodiment in darkness for 12 hours, culturing in a constant temperature incubator for 2 days at the temperature of 21 ℃, cutting off the tobacco leaf marking area, taking three parts of the tobacco leaves respectively transferred into the marking areas of the recombinant expression vectors DBN11-D to DBN17-D and the wild tobacco leaves with the same mass as biological repetition, freezing and grinding by liquid nitrogen, then respectively adding 1X PASSIVE LVSIS Buffer (PLB) and centrifuging at 12000rpm for 10 minutes at the temperature of 4 ℃, and respectively sucking the supernatant for standby.

4. Effect detection of constitutive promoter of the present invention to drive LUC reporter gene expression in tobacco leaf

Step 5, 100. Mu.L of the supernatant obtained in the above step 4 was added to the ELISA plate, and 3 repetitions were set, and 100. Mu.L of 1 Xfirefly luciferase reaction solution LAR II (dissolving lyophilized powder luciferase assay substrate in luciferase assay buffer II (Promega Co., dual-Reporter ASSAY SYSTEM (E1960) kit), storing in a dark place at-80 ℃), shaking plates, mixing uniformly, detecting the activity value of firefly luciferase by using a microplate reader BioTek-H1MF, and finishing the detection within 30min, wherein the unit of the detected activity value of firefly luciferase is RLU (relative light unit).

Step 6, 100. Mu.L of 1 XRenilla luciferase reaction solution Stop & Glo (200. Mu.L of Stop & Glo Substrate (50X) is dissolved in 10mL of Stop & Glo buffer, stored at-80 ℃ in a dark place), and the shaking plates are uniformly mixed, and the Renilla luciferase activity value is detected by using a microplate reader Bi _o Tek-H1MF, and the detection is completed within 30min, wherein the detected Renilla luciferase (REN) activity value unit is RLU (relative light unit).

In order to eliminate the inter-group errors of plant tissues caused by different factors such as agrobacterium infection conversion efficiency, with REN gene as an internal reference, the ratio of LUC/REN reflects the relative activity intensity of the promoter (LUC/REN ratio= (LUC value of tobacco leaf transformed into different recombinant expression vectors-LUC value of wild type tobacco leaf)/(REN value of tobacco leaf transformed into different recombinant expression vectors-REN value of wild type tobacco leaf), and the test results of the enzyme activity detection of transient transformed tobacco leaf LUC and REN are shown in table 1.

TABLE 1 enzyme Activity values for LUC and REN in transient transformed tobacco leaves and LUC/REN ratio

Promoters	LUC enzyme activity value/RLU	REN enzyme activity value/RLU	LUC/REN value
				prBnH4-0l	42601.0±1836.1	85947.0±3016.7	0.5±0.0
prBnH4-02	92849.1±3259.0	327658.0±14122.1	0.3±0.0
				prBnH4-03	78485.8±2449.5	455354.7±11725.4	0.2±0.0
prBnH4-04	7645.9±196.9	162159.7±5061.0	0.0±0.0
				prGm17gTsf1	419375.0±135474.9	322980.0±10757.2	1.3±0.2
pr35S	138048.5±3824.8	276097.1±8707.3	0.5±0.0
				prAtH4A748：1TEV	1509.3±39.9	38091.3±1078.1	0.0±0.0
CK1	87.2±7.6	177.2±16.7	/

The results in table 1 show that: (1) The promoters prBnH, prBnH, 4-02 and prBnH, 4-03 of the invention are universally active and can drive the LUC gene expression in tobacco leaves; the LUC/REN value of prBnH4-04 was 0, indicating that prBnH-04 had substantially no promoter activity; (2) in tobacco lamina, and prAtH a748: the 1TEV control promoter has higher activity of prBnH4-01, prBnH4-02, and prBnH4-03 driving LUC gene expression.

Third example, verification of the Effect of promoter element driving the expression of LUC reporter Gene in transgenic Arabidopsis

1. Recombinant expression vector transformation of agrobacterium

The recombinant expression vectors DBN11-D, DBN-D to DBN17-D, which had been constructed correctly in part 1 of the second example described above, were transformed into Agrobacterium GV3101 by liquid nitrogen method, respectively, under the following conditions: 100. Mu.L of Agrobacterium GV3101, 3. Mu.L of plasmid DNA (recombinant expression vector); placing in liquid nitrogen for 10min, and warm water bath at 37deg.C for 10min; the transformed agrobacterium GV3101 is inoculated in an LB test tube and cultured for 2 hours at the temperature of 28 ℃ and the rotating speed of 200rpm, and the agrobacterium is coated on an LB solid plate containing 50mg/L of Rifampicin (RIFAMPICIN) and 50mg/L of spectinomycin until positive monoclone grows out, the monoclone is selected for culture, plasmids are extracted, and sequencing identification is carried out on the extracted plasmids, so that the result shows that the recombinant expression vectors DBN11-D, DBN-D to DBN17-D are completely correct in structure.

2. Obtaining transgenic Arabidopsis plants

Wild-type Arabidopsis seeds were suspended in 0.1% (w/v) agarose solution. The suspended seeds were kept at 4 ℃ for 2 days to fulfill the need for dormancy to ensure synchronized germination of the seeds. The soil mixture was drained for 24 hours by mixing the horsemanure with vermiculite and irrigating with water bottom until wet. The pretreated seeds were planted on the soil mixture and covered with a moisture-retaining cover for 7 days. Seeds were germinated and plants were grown in a greenhouse under long-day conditions (16 h light/8 h dark) with a constant temperature (22 ℃) and constant humidity (40-50%) and a light intensity of 120-150. Mu. Mol/m ²s^-1. Plants were initially irrigated with Hoagland's nutrient solution followed by deionized water to keep the soil moist but not wet.

Arabidopsis thaliana was transformed using the floral dip method. One or more 15-30mL of precultures of LB medium (tryptone 10g/L, yeast extract 5g/L, naCl g/L, pH adjusted to 7.5 with NaOH) containing spectinomycin (50 mg/L) and rifampicin (10 mg/L) were inoculated with the selected Agrobacterium colonies. The preculture was incubated overnight at a constant shaking speed at a temperature of 28℃at 220 rpm. Each preculture was used to inoculate two 500ml cultures of the LB medium containing spectinomycin (50 mg/L) and rifampicin (10 mg/L) and the cultures were incubated at 28℃with continuous shaking overnight. The cells were pelleted by centrifugation at about 4000rpm for 20min at room temperature and the resulting supernatant was discarded. The cell pellet was gently resuspended in 500mL of an osmotic medium containing 1/2 XMS salt/B5 vitamin, 10% (w/v) sucrose, 0.044. Mu.M benzylaminopurine (10. Mu.L/L (stock solution in 1mg/mL DMSO)) and 300. Mu.L/L Silwet L-77. Arabidopsis plants of about 1 month of age were soaked in the permeate medium containing the resuspended cells for 5min, ensuring that the latest inflorescences were submerged. The sides of the Arabidopsis plants were then laid down and covered, moisturized for 24 hours in a dark environment, and Arabidopsis plants were normally cultivated at a temperature of 22℃with a 16h light/8 h dark photoperiod. Seeds were harvested after about 4 weeks.

Freshly harvested (prBnH 4-01 promoter sequence, prGm17gTsf control promoter sequence, pr35S control promoter sequence, prAtH A748: lTEV chimeric control promoter sequence) T ₁ seeds were dried at room temperature for 7 days. Seeds were planted in 26.5cm x 51cm germination trays, each tray receiving 200mg of T ₁ seeds (about 10000 seeds) which had been previously suspended in distilled water and stored at a temperature of 4 ℃ for 2 days to fulfill the need for dormancy to ensure synchronized germination of the seeds.

Mixing Ma Fentu with vermiculite and irrigating with water bottom until wet, draining with gravity. The pretreated seeds were planted uniformly on the soil mixture with a pipette and covered with a moisturizing cap for 4-5 days. The cover was removed 1 day prior to the initial transformant selection using post-emergence glyphosate spray (selection for co-transformed EPSPS gene).

After 7 days of planting (DAP) and again using a DeVilbiss compressed air nozzle at 11DAP, T ₁ plants (cotyledonary stage and 2-4 foliar stage, respectively) were sprayed with a 0.5% solution of Roundup herbicide (356 gae/L glyphosate) at a spray volume of 10 mL/tray (703L/ha) to provide an effective amount of glyphosate per application of 420 gae/ha. Surviving plants (actively growing plants) were identified 4-7 days after the final spraying and transplanted into 7cm x 7cm square pots (2-4 plants per tray) prepared with struvite and vermiculite, respectively. The transplanted plants were covered with a moisture-retaining cover for 3-4 days and placed in a culture chamber at a temperature of 22℃as before or directly transferred into a greenhouse. The cover is then removed and the plants are grown in a greenhouse (22.+ -. 5 ℃ C., 50.+ -. 30% RH,14h light: 10h darkness, minimum 500. Mu.E/m ²s^-1 natural + supplemental light) at least 1 day before testing the effect of the promoter element to drive the LUC reporter gene.

3. Effect detection of constitutive promoter of the present invention for driving LUC reporter gene expression in Arabidopsis thaliana tissues

The T ₁ transformants were selected from the untransformed seed background using the glyphosate selection protocol. The Arabidopsis T ₁ plant into which the prBnH-01 promoter sequence was transferred, the Arabidopsis T ₁ plant into which the prGm17gTsf1 control promoter sequence was transferred, the Arabidopsis T ₁ plant into which the pr35S control promoter sequence was transferred, and the Arabidopsis T ₁ into which the prAtH A748 was transferred in part 2 of this example were obtained: the arabidopsis T ₁ plant lTEV chimeric to the control promoter sequence was sampled as test samples at different positions of the arabidopsis T ₁ plant at different periods, respectively:

The rosette period 3 sites were sampled as test samples: roots, stems and leaves;

sampling 4 parts in the bolting period to serve as test samples: root, stem, leaf, flower;

samples were taken from 4 sites at maturity as test samples: root, stem, leaf, pod.

Wild-type Arabidopsis thaliana (CK 2) at the same site in the same growth period was sampled as a negative control sample.

Taking three test samples of the same mass and respectively transferred into different parts of recombinant expression vectors DBN11-D, DBN-D to DBN17-D in different periods and negative control samples of the same parts of wild arabidopsis plants in the same period as biological repetition, freezing and grinding by liquid nitrogen, respectively adding 1 XPLB buffer solution, centrifuging at 12000rpm for 10min at 4 ℃, and respectively sucking supernatant for later use.

The test samples and the negative control samples were tested according to the methods of step 5 and step 6 in the second example 4, and the relative activity intensity of the promoter was reflected by the level of the LUC/REN ratio (LUC/REN ratio= (LUC value of the test sample at different sites in different periods when different recombinant expression vectors were transferred-LUC value of the same site in the same period of the wild type plant)/(REN value of the test sample at different sites in different periods when different recombinant expression vectors were transferred-REN value of the same site in the same period of the wild type plant)) using REN gene as an internal reference. The LUC/REN ratios at different sites of the stably transformed Arabidopsis at different times are shown in Table 2.

TABLE 2 LUC/REN ratio at different sites in stably transformed Arabidopsis thaliana at different periods

The results in Table 2 show that: (1) The promoter prBnH-01 of the invention has activity, and is expressed in roots, stems, leaves, flowers and pods of arabidopsis plants, which indicates that the promoter prBnH-01 can drive the constitutive expression of a target heterologous gene in the plants. (2) In the rosette, bolting and maturing leaves of Arabidopsis plants, the method is characterized by comprising the following steps of prAtH A748: prBnH4-01 has higher activity in driving LUC gene expression than the 1TEV control promoter. (3) In the rosette and bolting stage stems, roots and mature stage pods of arabidopsis plants, the plant rosettes and rosettes are matched with pr35S and prAtH A748: prBnH4-01 has higher activity in driving LUC gene expression than the 1TEV control promoter. (4) In the flowers of arabidopsis plants in the bolting stage, the plants are combined with prAtH A748: prBnH4-01 has higher activity in driving LUC gene expression than the 1TEV control promoter.

Fourth example, verification of the Effect of promoter element driving expression of LUC reporter Gene in transgenic Soybean

1. Recombinant expression vector transformation of agrobacterium

The correctly constructed recombinant expression vectors DBN11-D, DBN-D to DBN17-D are respectively transformed into agrobacterium EHA101 by a liquid nitrogen method, and the transformation conditions are as follows: 100. Mu.L of Agrobacterium EHA101, 3. Mu.L of plasmid DNA (recombinant expression vector); placing in liquid nitrogen for 10min, and warm water bath at 37deg.C for 10min; inoculating the transformed agrobacterium EHA101 into an LB test tube, culturing for 2 hours at the temperature of 28 ℃ and the rotating speed of 200rpm, coating the LB solid plate containing 50mg/L of Rifampicin (RIFAMPICIN) and 50mg/L of spectinomycin until positive monoclonal grows out, picking up the monoclonal, culturing and extracting plasmids, and sequencing and identifying the extracted plasmids, wherein the result shows that the recombinant expression vectors DBN11-D, DBN15-D to DBN17-D are completely correct in structure.

2. Obtaining transgenic soybean plants

The cotyledonary node tissue of the soybean variety SY2043C aseptically cultured was co-cultured with the agrobacterium described in part 1 of this example in accordance with the conventionally employed agrobacterium infection method to transfer the above recombinant expression vector DBN11-D, DBN-D to T-DNA in DBN17-D (including prAtAct promoter sequence, REN gene sequence, T35S terminator sequence, one of the control promoter sequences selected from prBnH-01, prGm gTsf1, pr35S control promoter sequence and prAtH a748: lTEV chimeric control promoter sequence, LUC gene sequence, tPsE terminator sequence, prAtUbi10 promoter sequence, spAtCTP nucleotide sequence, cEPSPS gene sequence and tNos terminator sequence) into the soybean chromosome group to obtain soybean plants transferred into prBnH-01 promoter sequence, soybean plants transferred into prGm gTsf1 control promoter sequence, soybean plants transferred into 35S control promoter sequence and prAtH a748 of prAtH S control promoter sequence: lTEV soybean plants chimeric to the control promoter sequence.

For Agrobacterium-mediated transformation of soybean, briefly, mature soybean seeds were germinated in soybean germination medium (B5 salt 3.1g/L, B vitamin, sucrose 20g/L, agar 8g/L, pH 5.6), seed inoculated onto germination medium, and cultured under the following conditions: the temperature is 25+/-1 ℃; the photoperiod (light/dark) was 16/8h. 1 day after germination, one cotyledon and the first true leaf were removed, inoculated onto a pretreatment medium containing cytokinin (MS salt 4.3g/L, B vitamin, sucrose 20g/L, agar 8g/L, 2-morpholinoethanesulfonic acid (MES) 4g/L, zeatin (ZT) 2mg/L, 6-benzyladenine (6-BAP) 1mg/L, acetosyringone (AS) 40mg/L, pH=5.3), and after inoculation on the pretreatment medium for 3 days, wounded at cotyledon node with the back of a scalpel, the wounded cotyledon node tissue was contacted with an Agrobacterium suspension, wherein Agrobacterium was able to sequence the prBnH-01 promoter sequence, prGm17gTsf1 control promoter sequence, pr35S control promoter sequence and prAtH a748: lTEV chimeric control promoter sequence was delivered to wounded cotyledonary node tissue (step 1: invasion step). In this step, cotyledonary node tissue is preferably immersed in an agrobacterium suspension (OD ₆₆₀ = 0.5-0.8, infection medium (MS salt 2.15g/L, B5 vitamin, sucrose 20g/L, glucose 10g/L, acetosyringone (AS) 40mg/L, 2-morpholinoethanesulfonic acid (MES) 4g/L, zeatin (ZT) 2mg/L, ph 5.3)) to initiate inoculation. The cotyledonary node tissue was co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-culturing step). Preferably, the cotyledonary node tissue is cultivated after the infection step on a solid medium (MS salt 4.3g/L, B vitamin, sucrose 20g/L, glucose 10g/L, MES g/L, ZT2mg/L, agar 8g/L, pH 5.6). After this co-cultivation stage, there may be an optional "recovery" step. In the "recovery" step, at least one antibiotic (cephalosporin 150-250 mg/L) known to inhibit the growth of Agrobacterium is present in the recovery medium (B5 salt 3.1g/L, B vitamin, MES1g/L, sucrose 30g/L, ZT mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L, pH 5.6) without the addition of a selection agent for plant transformants (step 3: recovery step). Preferably, the tissue mass regenerated from cotyledonary nodes is cultured on a solid medium with antibiotics but no selection agent to eliminate agrobacterium and provide a recovery period for the infected cells. Next, the cotyledonary node regenerated tissue pieces are cultured on a medium containing a selection agent (glyphosate) and the grown transformed calli are selected (step 4: selection step). Preferably, the cotyledonary node regenerated tissue pieces are cultured on a selective solid medium (B5 salt 3.1g/L, B vitamin, MES1g/L, sucrose 30g/L, 6-benzyladenine (6-BAP) 1mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 100mg/L, aspartic acid 100mg/L, N- (phosphonomethyl) glycine 0.25mol/L, pH 5.6) with a selective agent, resulting in selective growth of the transformed cells. then, the transformed cells are regenerated into plants (step 5: regeneration step), and preferably, the cotyledonary node regenerated tissue pieces grown on the medium containing the selection agent are cultured on solid media (B5 differentiation medium and B5 rooting medium) to regenerate the plants.

The selected resistant tissue blocks were transferred to the B5 differentiation medium (B5 salt 3.1g/L, B vitamin, MES1g/L, sucrose 30g/L, ZT mg/L, agar 8g/L, cephalosporin 150mg/L, glutamic acid 50mg/L, aspartic acid 50mg/L, gibberellin 1mg/L, auxin 1mg/L, N- (phosphonomethyl) glycine 0.25mol/L, pH 5.6) and cultured at 25 ℃. The differentiated plantlets were transferred to the B5 rooting medium (B5 salt 3.1g/L, B vitamin, MES1g/L, sucrose 30g/L, agar 8g/L, cephalosporin 150mg/L, indole-3-butyric acid (IBA) 1 mg/L), cultured at 25℃to a height of about 10cm, and transferred to a greenhouse for cultivation until set. In the greenhouse, the cells were cultured at 26℃for 16 hours per day and at 20℃for 8 hours.

3. Verification of transgenic soybean plants Using TaqMan

Soybean plants into which prBnH4-01 promoter sequence was transferred, soybean plants into which prGm17gTsf1 control promoter sequence was transferred, soybean plants into which pr35S control promoter sequence was transferred, and soybean plants into which prAtH a748 were transferred were respectively taken: about 100mg leaf of soybean plant with 1TEV chimeric control promoter sequence was used as a sample, its genomic DNA was extracted with the DNEASY PLANT Maxi Kit of Qiagen, and EPSPS gene copy numbers were detected by Taqman probe fluorescent quantitative PCR method to determine prBnH-01, prGml7gTsf, pr35S and prAtH A748: copy number of 1TEV gene. Meanwhile, wild type soybean plants were used as a control, and detection and analysis were performed as follows. Experiments were repeated 3 times and averaged.

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

Step 6, respectively taking soybean plants with prBnH4-01 promoter sequences, soybean plants with prGm17gTsf1 control promoter sequences, soybean plants with pr35S control promoter sequences and soybean plants with prAtH A748:1TEV chimeric control promoter sequence soybean plants and wild soybean plants leaf 100mg each, respectively in a mortar with liquid nitrogen to homogenate, each sample taken 3 replicates;

step 7, extracting genomic DNA of the sample by using DNEASY PLANT MINI KIT of Qiagen, wherein the specific method refers to the product instruction;

Step 8, determining the concentration of the genomic DNA of the sample by using NanoDrop 2000 (Thermo Scientiiic);

Step 9, adjusting the concentration of the genome DNA of the sample to the same concentration value, wherein the concentration value ranges from 80 ng/mu L to 100 ng/mu L;

Step 10, identifying the copy number of the sample by adopting a Taqman probe fluorescent quantitative PCR method, taking the sample with the identified known copy number as a standard substance, taking the sample of a wild type soybean plant as a control, repeating each sample for 3 times, and taking an average value; the fluorescent quantitative PCR primer and the probe sequences are respectively as follows:

The following primers and probes were used to detect EPSPS gene sequences:

Primer 1: GGTGTGCAGGTGAAGTCTGAAG is shown as SEQ ID NO: shown at 24;

Primer 2: GTCTTTGGTCCACGCAAGGT is shown as SEQ ID NO: shown at 25;

Probe 1: CGGTGATCGTCTTCCAGT is shown as SEQ ID NO: 26;

the PCR reaction system is as follows:

the 50 Xprimer/probe mixture contained 45. Mu.L of each primer at a concentration of 1mM, 50. Mu.L of probe at a concentration of 100. Mu.M, and 860. Mu.L of 1 XTE buffer, and was stored in amber tubes at 4 ℃.

The PCR reaction conditions were:

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

By analyzing the experimental results of the copy number of the EPSPS gene, prBnH4-01 promoter sequence, prGm17gTsf1 control promoter sequence, pr35S control promoter sequence, prAtH A748 were confirmed: the 1TEV chimeric control promoter sequences were all integrated into the chromosome set of the soybean plants tested, and soybean plants with the prBnH4-01 promoter sequence, soybean plants with the prGm17gTsf1 control promoter sequence, soybean plants with the pr35S control promoter sequence, prAtH a748: soybean plants with 1TEV chimeric control promoter sequence all obtained single copy transgenic soybean plants.

4. Effect detection of constitutive promoter of the present invention for driving LUC reporter gene expression in soybean tissues

Soybean plants with the prBnH4-01 promoter sequence, soybean plants with the prGm17gTsf1 control promoter sequence, soybean plants with the pr35S control promoter sequence and soybean plants with the prAtH a748 were obtained in part 3 of this example: soybean plants with 1TEV chimeric control promoter sequence were sampled as test samples at different locations at different times of the transgenic soybean plants, respectively:

Samples were taken from 3 sites during the vegetative growth period (V3 phase) as test samples: roots, stems and leaves;

6 sites were sampled during reproductive growth period as test samples: roots, stems, leaves, flowers, pod skin, fruits;

wild-type soybean plants (CK 3) at the same site in the same growth period were sampled as negative control samples.

Test samples of different parts in different periods and negative control samples of the same part in the same period of a wild type soybean plant (CK 3) (three strains are sampled in each period, 3 repeats of the same mass are sampled in each part of each strain) are subjected to liquid nitrogen freeze-grinding, then 1 XPLB buffer solution is respectively added, and centrifugation is carried out at 12000rpm for 10min at 4 ℃ to respectively suck supernatant for later use.

The test samples and the negative control samples were subjected to double luciferase assay according to the methods of step 5 and step 6 in the second example 4, and the relative activity intensity of the promoter was reflected by the level of the LUC/REN ratio using the REN gene as an internal reference (the definition of the LUC/REN ratio was the same as that of the LUC/REN ratio in Table 2 of the third example described above). The LUC/REN ratios at different sites of the stably transformed soybeans at different periods are shown in Table 3.

TABLE 3 LUC/REN ratio at different sites of stably transformed soybeans at different periods

The results in Table 3 show that: (1) The promoter prBnH-01 of the invention is expressed in roots, stems, leaves, flowers, pod skin and fruits of soybean plants, which indicates that the promoter prBnH-01 can drive the target heterologous gene to be expressed in the plants in a constitutive mode; (2) In the stems and roots of soybean during vegetative growth, and prAtH a748: prBnH4-01 has higher activity of driving the expression of the LUC gene compared with lTEV control promoter; (3) In stems, roots, flowers and fruits during the reproductive growth period of soybean, and prAtH a748: prBnH4-01 has higher activity in driving LUC gene expression than the 1TEV control promoter.

Fifth example herbicide resistance Effect detection of transgenic Arabidopsis plants

1. And constructing prBnH-01 recombinant expression vector for driving herbicide-resistant gene HTG.

PrBnH4-01 promoter sequence, prGm17gTsf1 control promoter sequence, pr35S control promoter sequence and prAtH a748: the 5 'and 3' ends of lTEV chimeric control promoter sequences were ligated to universal adapter primer 2:

5' Universal adaptor primer 2:5'-cacgtgaccctagtcacttaaagcttggcgcgcc-3' as set forth in SEQ ID NO: shown at 27;

3' -terminal universal adaptor primer 2:5'-cagtagctggtgttggaggcat-3' as set forth in SEQ ID NO: 28.

The construction of vectors using conventional methods of cleavage is well known to those skilled in the art. The structural diagram of the vector DBNBC-HTG (vector backbone: pCAMBIA2301 with a modified resistance tag (available from CAMBIA Co.)) containing herbicide-resistant gene HTG is shown in FIG. 3 (Spec: spectinomycin resistance gene; RB: right border; ascI: restriction enzyme AscI recognition site; HTG: hydroxyphenylpyruvate dioxygenase gene (SEQ ID NO: 29), t35s: cauliflower virus 35s terminator (SEQ ID NO: 17), prAtUbi10: promoter of Arabidopsis Ubiquitin (Ubiquintin) 10 gene (SEQ ID NO: 20), spAtCTP2: arabidopsis chloroplast transit peptide (SEQ ID NO: 21), cEPSPS: 5-enolpyruvylshikimate-3-phosphate synthase gene (SEQ ID NO: 22), tNos: terminator of nopaline synthase gene (SEQ ID NO: 23), LB: left border).

The vector DBNBC-HTG is subjected to enzyme digestion reaction by utilizing restriction enzyme AscI, so that the vector DBNBC-HTG is linearized, the enzyme digestion product is purified to obtain a linearized DBNBC-HTG expression vector, the prBnH4-01 promoter sequence connected with the universal joint primer 2 and the linearized DBNBC-HTG expression vector are subjected to recombination reaction, and the operation steps are carried out according to the instruction of Takara In-Fusion Snap Assembly Master Mix kit (Clontech, CA, JPN, CAT: 638949) to construct a recombinant expression vector DBN18-D, and the structural schematic diagram of the recombinant expression vector is shown In FIG. 4.

The recombinant expression vector DBN18-D is used for transforming escherichia coli DH5 alpha competent cells by a heat shock method, and the heat shock conditions are as follows: 100 mu L of escherichia coli DH5 alpha competent cells, 20 mu L of recombinant plasmid DNA (recombinant expression vector DBN 18-D), carrying out light flick, uniformly mixing, carrying out water bath heat shock at 42 ℃ for 30s, and immediately placing on ice for 2min; mu.L of antibiotic-free LB liquid medium (tryptone 10g/L, yeast extract 5g/L, naCl g/L, pH was adjusted to 7.5 with NaOH) was added and incubated with shaking (200 rpm) at 37℃for 1 h. Then, the positive clone colonies were picked up by culturing in an inverted manner on the LB solid plate containing 50mg/L of spectinomycin (Spectinomycin) at 37℃for 12 hours, and cultured overnight in LB liquid medium containing 50mg/L of spectinomycin at 37℃with shaking (200 rpm). Extracting the plasmid by an alkaline lysis method: centrifuging the bacterial solution at 12000rpm for 1min, removing supernatant, and suspending the precipitated bacterial cells with 100 μl of ice-precooled solution I (25 mM Tris-HCl, 10mM EDTA (ethylenediamine tetraacetic acid), 50mM glucose, pH=8.0); 200. Mu.L of freshly prepared solution II (0.2M NaOH, 1% SDS (sodium dodecyl sulfate)) was added, the tube was inverted 4 times, mixed, and placed on ice for 3-5min; adding 150 μl ice-cold solution III (3M potassium acetate, 5M acetic acid), immediately mixing, and standing on ice for 5-10min; centrifuging at 4deg.C and 12000rpm for 5min, transferring supernatant to new 2mL centrifuge tube, adding 2 times volume of absolute ethanol, mixing, and standing at room temperature for 5min; centrifuging at 4deg.C and 12000rpm for 5min, removing supernatant, washing precipitate with 70% ethanol (V/V), and air drying; mu.L of TE (10 mM Tris-HCl, 1mM EDTA, pH=8.0) containing RNase (20. Mu.g/mL) was added to dissolve the precipitate; digesting RNA in a water bath at 37 ℃ for 30 min; preserving at-20deg.C. Sequencing and identifying the extracted plasmid, and the result shows that the recombinant expression vector DBN18-D contains SEQ ID NO:1, i.e., the prBnH-01 promoter sequence.

Following the procedure described above for constructing recombinant expression vector DBN18-D comprising prBnH4-01 promoter sequence, said prGm17gTsf1 control promoter sequence linked to said universal adaptor primer 2, said pr35S control promoter sequence linked to said universal adaptor primer 2, and said prAtH A748 linked to said universal adaptor primer 2: and (3) respectively carrying out recombination reaction on the 1TEV chimeric control promoter sequences and the linearized DBNBC-HTG expression vector to sequentially obtain recombinant expression vectors DBN19-D to DBN21-D, and sequencing to verify that the nucleotide sequences in the recombinant expression vectors DBN19-D to DBN21-D are correctly inserted.

2. Recombinant expression vector transformation of agrobacterium

According to the method of transforming Agrobacterium with the recombinant expression vector in part 1 of the third example described above, the recombinant expression vectors DBN18-D to DBN21-D which have been constructed correctly were transformed into Agrobacterium GV3101 by liquid nitrogen method, respectively, and sequencing verification results showed that the recombinant expression vectors DBN18-D to DBN21-D were completely correct in structure.

3. The promoter drives herbicide resistance effect detection of herbicide resistance gene HTG in transgenic arabidopsis plants

Immersing an arabidopsis inflorescence in the agrobacterium solution of part 2 of the present embodiment according to the method of part 2 of the third embodiment described above to transfer the T-DNA in the recombinant expression vectors DBN18-D to DBN21-D constructed in part 2 of the present embodiment into the arabidopsis chromosome to obtain corresponding transgenic arabidopsis plants, namely an arabidopsis T ₁ plant transferred with the prBnH-01 promoter sequence, an arabidopsis T ₁ plant transferred with the prGm gTsf1 control promoter sequence, an arabidopsis T ₁ plant transferred with the pr35S control promoter sequence, and an arabidopsis T3826 a748 transferred with the prAtH a748:1TEV chimeric Arabidopsis T ₁ plant of control promoter sequence.

The T ₁ transformants were selected from the untransformed seed background using the glyphosate selection protocol. An arabidopsis T ₁ plant into which a prBnH4-01 promoter sequence was transferred, an arabidopsis T ₁ plant into which a prGm17gTsf control promoter sequence was transferred, an arabidopsis T ₁ plant into which a pr35S control promoter sequence was transferred, and an arabidopsis T prAtH a748: arabidopsis T ₁ plants and wild type Arabidopsis plants (CK 4) with 1TEV chimeric control promoter sequences (18 days after sowing) were sprayed with 2-fold field concentration (50 gai/ha) of topramezone to examine herbicide tolerance of Arabidopsis. After 7 days of spraying, the degree of damage of each plant by the herbicide was counted according to the leaf whitening area ratio (leaf whitening area ratio=leaf whitening area/total leaf area×100%). The substantially non-whitening phenotype was rated 0, the proportion of leaf whitening area was rated 1, the proportion of leaf whitening area was rated more than 50% was rated 2, and the proportion of leaf whitening area was rated 100% was rated 3.

The transformation event resistance performance of each recombinant expression vector was scored (X-phytotoxicity score, N-peer number of victims, S-phytotoxicity grade number, T-total plant number, M-highest phytotoxicity grade) according to the formula x= [ Σ (n×s)/(t×m) ]×100, and the resistance was evaluated according to the score: high resistant plants (0-15 min), medium resistant plants (16-33 min), low resistant plants (34-67 min), and non-resistant plants (68-100 min). The experimental results are shown in table 4.

TABLE 4 results of experiments on the tolerance of transgenic Arabidopsis thaliana T ₁ plants to topramezone

The results in table 4 show that: (1) Compared with CK4, the arabidopsis thaliana plants transformed into prBnH-01 promoter sequences have tolerance to topramezone for topramezone with 2 times of field concentration. Thus, the constitutive promoter prBnH-01 of the present invention can drive the expression of a desired heterologous gene in plants. (2) and prAtH a748: compared with lTEV control promoter, prBnH4-01 drives herbicide resistance effect of herbicide resistance gene HTG in transgenic Arabidopsis plants.

In summary, the invention discloses a constitutive promoter from rape Histone gene for the first time, which shows activity in most tissues and many types of cells of plants, especially in roots, stems, leaves, flowers, pod skin and fruits of plants, and has wide application prospect on plants.

Finally, it should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.

Claims (18)

1. A constitutive promoter, characterized in that its nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:3, said constitutive promoter is derived from SEQ ID NO:1.

2. The constitutive promoter according to claim 1, characterized in that its nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:3 and is selected from SEQ ID NO: 1.

3. The constitutive promoter according to claim 1 or 2, characterized in that its nucleotide sequence is set forth in SEQ ID NO: 1. SEQ ID NO:2 or SEQ ID NO: 3.

4. A recombinant DNA construct comprising the constitutive promoter of any one of claims 1-3 operably linked to a heterologous nucleotide sequence of interest.

5. The recombinant DNA construct according to claim 4, wherein said heterologous nucleotide sequence of interest encodes a protein of interest.

6. An expression cassette comprising the recombinant DNA construct of claim 4 or 5.

7. A recombinant vector comprising the expression cassette of claim 6.

8. A method of expressing a heterologous nucleotide sequence of interest in a plant comprising: stably integrating a heterologous nucleotide sequence of interest operably linked to a constitutive promoter of any one of claims 1-3 into a plant cell.

9. The method of expressing a heterologous nucleotide sequence of interest in a plant according to claim 8, wherein the plant is arabidopsis thaliana, brassica napus, tobacco, soybean, cotton, capsicum, beet, pumpkin, eggplant, chinese cabbage, carrot, tomato, pea, spinach, potato, or peanut.

10. The method of expressing a heterologous nucleotide sequence of interest in a plant according to claim 8, wherein the heterologous nucleotide sequence of interest is constitutively expressed in the plant tissue.

11. The method of expressing a heterologous nucleotide sequence of interest in a plant according to claim 8, wherein the heterologous nucleotide sequence of interest encodes a protein of interest.

12. The method of expressing a heterologous nucleotide sequence of interest in a plant according to claim 11, wherein the heterologous nucleotide sequence of interest encodes a herbicide tolerance protein.

13. The method of expressing a heterologous nucleotide sequence of interest in a plant according to claim 11, wherein the heterologous nucleotide sequence of interest encodes an insect-resistant protein.

14. A plant or part comprising the constitutive promoter of any one of claims 1 to 3.

15. A method of obtaining a processed agricultural product comprising treating the harvest of the plant or part of claim 14 to obtain a processed agricultural product.

16. Use of a constitutive promoter according to any one of claims 1-3 for constitutive expression of a heterologous nucleotide sequence of interest in plant tissue.

17. Use of a constitutive promoter according to claim 16 for constitutive expression of a heterologous nucleotide sequence of interest in plant tissue, wherein said plant is arabidopsis thaliana, brassica napus, tobacco, soybean, cotton, capsicum, beet, pumpkin, eggplant, chinese cabbage, carrot, tomato, pea, spinach, potato or peanut.

18. Use of a constitutive promoter according to claim 16 for constitutive expression of a heterologous nucleotide sequence of interest in plant tissue, wherein said heterologous nucleotide sequence of interest encodes a protein of interest.