Constitutive promoters and uses thereofTechnical Field
The invention relates to a constitutive promoter and application thereof, in particular to a constitutive promoter from Artemisia annua and application thereof.
Background
One of the goals of plant genetic engineering is to achieve modifications to plants that can be made as desired to produce plants with a desired characteristic or trait. Traits that are often desired include improved nutritional quality, increased yield, conferring pest resistance, improved drought and stress tolerance, improved horticultural quality, and conferring herbicide resistance, among others. Researchers have now been able to acquire exogenous genes (e.g., genes of heterologous or natural origin) and integrate the genes into the plant genome for expression to exhibit the corresponding traits.
When expressing a gene of interest in plants, it is important to select appropriate regulatory signals. Typical regulatory signals include promoter regions, 5 'untranslated leader sequences, and 3' transcription terminator/polyadenylation sequences, among others. Wherein the promoter is a DNA sequence located upstream of the 5' end of the structural gene that is recognized, bound by RNA polymerase and initiates transcription of the gene. In plant genetic engineering, promoters are often classified into three types, constitutive promoters, specific promoters and inducible promoters, according to their mode of action and function. Constitutive promoters are promoters that direct RNA synthesis at a level of expression to a degree throughout most or all tissues and/or growth stages of a plant, and can be classified as strong, medium, and weak promoters according to their effect on directing RNA synthesis. Constitutive promoters are particularly advantageous in these cases, since in many cases it is necessary to express the gene of interest (or chimeric gene) 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 (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 CaMV35S promoter with repeat enhancer 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, the discovery and isolation of more sources and safe, efficient, while enabling control of expression of the gene of interest (or chimeric gene) at different levels or application to the superposition of multiple genes in the same transgenic plant remains a hotspot of continued interest in the art.
Disclosure of Invention
The invention aims to provide a novel constitutive promoter and application thereof, wherein the promoter is derived from Artemisia princeps Pampanini (Aegilops tauschii.) and can regulate and control the high-efficiency expression of target nucleic acid in plant tissues such as roots, stems, leaves, filaments, pollen, seeds, spike tops and the like, thereby providing a novel tool and selection for plant genetic engineering and having very wide application prospect.
In a first aspect, the invention provides a constitutive promoter comprising the nucleotide sequence of SEQ ID NO 39.
Preferably, the constitutive promoter has a nucleotide sequence comprising SEQ ID NO. 39 and the constitutive promoter is derived from SEQ ID NO. 1.
More preferably, the constitutive promoter comprises the nucleotide sequence of SEQ ID NO. 39 and is a part or a fragment of SEQ ID NO. 1.
More preferably, the constitutive promoter has a nucleotide sequence comprising SEQ ID NO. 39 and is a truncated sequence of SEQ ID NO. 1.
Further preferably, the nucleotide sequence of the constitutive promoter comprises SEQ ID NO. 39 and is a truncated sequence at the 5' end (N-terminal) of SEQ ID NO. 1.
Further preferably, the 5' end (N-terminal) is truncated by no more than (less than or equal to) 1720bp.
Further preferably, the nucleotide sequence of the constitutive promoter is shown as SEQ ID NO. 1, SEQ ID NO. 37, SEQ ID NO. 38 and SEQ ID NO. 39.
In a second aspect, the invention also provides a chimeric gene comprising the above constitutive promoter operably linked to a nucleic acid of interest.
Preferably, the nucleic acid of interest encodes a protein of interest.
In a third aspect, the invention also provides an expression cassette comprising a constitutive promoter or a chimeric gene of the invention.
In a fourth aspect, the invention also provides a recombinant vector comprising a constitutive promoter or a chimeric gene or an expression cassette of the invention. In a fifth aspect, the invention also provides a host cell comprising a constitutive promoter, a chimeric gene, an expression cassette or a recombinant vector of the invention.
In a sixth aspect, the invention also provides a method of expressing a nucleic acid of interest in a plant comprising stably integrating the nucleic acid of interest operably linked to a constitutive promoter as described above into a plant cell.
Preferably, the nucleic acid of interest is constitutively expressed in plant tissue.
Preferably, the nucleic acid of interest encodes a protein of interest.
More preferably, the nucleic acid of interest encodes a herbicide tolerance protein.
More preferably, the nucleic acid of interest encodes an insect-resistant protein.
Preferably, the plant is maize, arabidopsis thaliana, canola, tobacco, soybean, cotton, capsicum, beet, pumpkin, eggplant, chinese cabbage, carrot, tomato, pea, spinach, potato, or peanut.
In a seventh aspect, the invention also provides a plant or plant part comprising the constitutive promoter, chimeric gene, expression cassette or recombinant vector described above.
In an eighth aspect, the present invention also provides a method of obtaining a processed agricultural product comprising treating a harvest of the plant or plant part described above to obtain a processed agricultural product.
In a ninth aspect, the present invention also provides a use of the constitutive promoter described above for constitutive expression of a nucleic acid of interest in plant tissue.
Preferably, the nucleic acid of interest encodes a protein of interest.
More preferably, the nucleic acid of interest encodes a herbicide tolerance protein.
More preferably, the nucleic acid of interest encodes an insect-resistant protein.
Preferably, the plant is maize, arabidopsis thaliana, canola, tobacco, soybean, cotton, capsicum, beet, pumpkin, eggplant, chinese cabbage, carrot, tomato, pea, spinach, potato, or peanut.
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" or "nucleic acid" 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) within a cell under the control of a suitable regulatory region (e.g., a plant expressible promoter region). The gene or nucleic acid may contain several operably linked DNA fragments, such as a promoter, a 5 'untranslated leader sequence, a coding region, and a 3' untranslated region containing a polyadenylation site. Endogenous plant genes are genes found naturally in plant species. A chimeric gene is any gene that is not normally found in a plant species, or is 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 "gene of interest" or "nucleic acid of interest" in the present invention may be an endogenous gene or nucleic acid, or may be an exogenous or heterologous gene or nucleic acid. Endogenous gene or nucleic acid refers to a gene that is naturally found or present in a plant species or plant host, and exogenous or heterologous gene or nucleic acid refers to any gene that is not or is not present in a plant species or plant host. Alternatively, the gene of interest or nucleic acid of interest may be a natural sequence or a selectively synthetic sequence.
The gene or nucleic acid of interest of the application may be homologous with respect to the promoter of the application, e.g. the gene or nucleic acid of interest and the promoter are derived from the same plant species or are naturally linked together. The gene or nucleic acid of interest of the application may also be exogenous or heterologous with respect to the promoter of the application, i.e. the gene or nucleic acid of interest and the promoter are derived from different plant species or are non-naturally linked together or present together. For example, a chimeric gene comprises a promoter sequence of the application operably linked to a coding sequence that differs from the promoter sequence of the application. Although the gene or nucleic acid sequence of interest is heterologous to the promoter sequence, it may be homologous or heterologous (exogenous) to the plant host.
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/nucleic acid expression. Constitutive promoters are used to operably link a gene of interest, a nucleic acid of interest, or a gene editing system guide RNA (gRNA) for expression 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 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 terms "constitutive expression" and "constitutively expressed" in the present invention mean that the nucleic acid of interest or the gene of interest is expressed at a substantially uniform level in different tissues and/or different stages of growth and development of the plant. The terms "constitutive expression" and "constitutive expression" in the context of the present invention mean that the gene of interest or nucleic acid of interest exhibits a relatively stable and sustained expression in most or all tissues and/or growth and development stages of the plant, e.g., the CaMV35S promoter of cauliflower mosaic virus, which is capable of promoting high-strength expression of foreign genes in plants in most organs and at different developmental stages, and that the constitutive promoter also ensures broad expression of gRNA in host cells, improving the efficiency and accuracy of gene editing. The promoters of the invention may be homologous or exogenous or heterologous to the plant species or plant host. Homologous refers to a promoter derived from a plant species or plant host into which the transcription initiation region has been introduced, exogenous or heterologous refers to the absence of the promoter or transcription initiation region in the native plant or plant host into which the transcription initiation region has been introduced.
The invention provides a constitutive promoter, the nucleotide sequence of which comprises SEQ ID NO. 39. Further, the nucleotide sequence of the constitutive promoter includes SEQ ID NO. 37, SEQ ID NO. 38 or SEQ ID NO. 1. In a preferred embodiment of the invention, the nucleotide sequence of the constitutive promoter comprises SEQ ID NO 39 and is derived from SEQ ID NO 1. Further, the promoter of the present invention comprises the nucleotide sequence of SEQ ID NO. 39 and is at least a portion of SEQ ID NO. 1 or is at least a fragment of SEQ ID NO. 1. In another preferred embodiment, the nucleotide sequence of the constitutive promoter comprises SEQ ID NO. 39 and is a truncated sequence of SEQ ID NO. 1, wherein the truncated sequence is a truncated sequence of the 5' end (N-terminal) of SEQ ID NO. 1 and the truncated length is not more than (less than or equal to) 1720bp.
Specifically, SEQ ID NO. 39 is a fragment of 1649bp in length located at the 3 '-end of SEQ ID NO. 1, and is the remaining fragment (truncated fragment) after truncating 1720bp at the 5' -end of SEQ ID NO. 1. The constitutive promoter of the present application may extend to its 5 'end or 3' end on the basis of SEQ ID NO. 39. In a preferred embodiment, the constitutive promoter of the present application can be referred to as SEQ ID NO. 1 and extends to its 5' end on the basis of SEQ ID NO. 39, but its nucleotide sequence extending to its 5' end cannot be longer than the 5' end of SEQ ID NO. 1 itself. In other words, the constitutive promoter of the present application is a truncated nucleotide fragment obtained by truncating or removing nucleotides having a length of 1720bp or less from the 5' end of SEQ ID NO. 1, and the truncated fragment comprises SEQ ID NO. 39. The constitutive promoters obtained above are all within the scope of the present application.
For the above-mentioned promoters, the other nucleotides included in the nucleotide sequence except SEQ ID NO:39 do not affect the activity of the promoter itself. The fifth example of the present application also demonstrates that prAetUbi-04 promoter (SEQ ID NO: 39) has activity, while promoters prAetUbi5-03 (SEQ ID NO: 38), prAetUbi5-02 (SEQ ID NO: 37) and prAetUbi-01 (SEQ ID NO: 1) containing SEQ ID NO:39 have good activity. As one of ordinary skill in the art can reasonably expect from the disclosure herein, SEQ ID NO. 39 is the shortest fragment (smallest unit) that functions as a promoter, and when SEQ ID NO. 39 is included in the nucleotide sequence of the promoter, it has both the function and the activity of the promoter. Similarly, when the nucleotide sequence of the promoter comprises SEQ ID NO. 39 and is selected from at least a portion of SEQ ID NO. 1 or is a truncated sequence of SEQ ID NO. 1, those skilled in the art can reasonably expect that these promoters all have the same or similar function and activity as the promoters shown in SEQ ID NO. 39.
Promoter sequences having promoter activity and being at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the promoter sequences of the present invention are all included in the present invention, i.e., the range of sequence identity is distributed over at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.
Isolated sequences that have promoter activity and hybridize under stringent conditions to the promoter sequences of the present invention or fragments thereof are also encompassed by the present invention. These sequences are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to the sequences of the present invention, i.e., the sequence identity ranges are distributed over at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.
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 nucleic acid 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 nucleotide sequence of interest constituting the chimeric gene in an expression cassette for expression in the plant of interest. Such an expression cassette provides a large number of restriction sites for insertion of a 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 nucleotide sequence of interest and transcription and translation termination regions which function in plants.
The termination region may be derived from the promoter sequences of the present invention, may be derived from operably linked 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 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, 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), potexviral leader sequences such as Tobacco Etch Virus (TEV) leader sequences, maize Dwarf Mosaic Virus (MDMV) leader sequences, and human immunoglobulin heavy chain binding protein (BiP), non-translated leader sequences from alfalfa mosaic virus coat protein mRNA (AMV RNA 4), tobacco Mosaic Virus (TMV) leader sequences, and maize chlorosis spot virus (MCMV) leader sequences. 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 nucleic acid 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 nucleic acid or nucleic acid sequence operably linked to the promoter of the present invention may encode a protein of interest. Examples of such nucleic acids or nucleic acid 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 preventing 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-resistant proteins of interest are well known in the art and include, but are not limited to, bacillus toxic proteins, lectins, wherein the lectins include vanishing lotus 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, etc.
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, nucleotide sequences that have anti-pathogenic activity or minimize the effects of viral pathogens can be selected for constitutive expression in plant tissue.
The promoter sequences and methods of the present invention can be used for expression regulation of any nucleic acid 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.
Host cells of the invention include, but are not limited to, plant cells or bacteria. Bacteria include, but are not limited to, agrobacterium, bacillus, escherichia, salmonella, pseudomonas or Rhizobium cells. Plant cells are non-regenerable cells or regenerable cells. 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 living cell with an incomplete structure, such as a plant cell without a nucleus. Specifically, the plant cells are cell-free screen-like cells (mature screen cells). 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. In another aspect, the plant cell is a somatic cell.
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 another aspect, the plant part is a plant cell as described above.
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, which have the following beneficial effects:
The invention discloses a constitutive promoter from Artemisia annua for the first time, the constitutive promoter shows good activity in most tissues and cells of plants, especially in the tissues such as roots, stems, leaves, flowers, pollen, seeds, spike tops and the like of plants, can drive the efficient expression of exogenous genes or nucleic acids, provides new tools and choices for plant genetic engineering, and has very excellent application potential.
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 of the structure of recombinant dual luciferase expression vector DBN13844 containing promoter prZmUbi 1;
FIG. 3 is a schematic diagram of the structure of recombinant dual luciferase expression vector DBN14192 containing promoter prAetUbi-01;
FIG. 4 is a schematic diagram of the structure of recombinant vector DBN15003 driving the expression of EPSPS gene from constitutive promoter prAetUbi-01;
FIG. 5 is a graph showing the effect of transgenic maize transformed with a constitutive promoter prAetUbi-01 driven EPSPS gene on tolerance to glyphosate herbicide.
Detailed Description
The constitutive promoter of the present invention and its technical scheme of use are further described below by specific examples.
Example one, constitutive promoter prAetUbi-01 was obtained
The nucleotide sequence of the maize ubiquitin protein is used as a reference sequence, and homologous genes are searched and aligned in EnsemblPlants (https:// plants. Ensembl. Org/index. Html) to obtain candidate festival ubiquitin protein genes (AET 7Gv 21096800). A sequence about 3kb upstream of the node wheat ubiquitin gene is selected as a target sequence, the node wheat genome DNA is used as a PCR amplification template, and a primer 1 (SEQ ID NO: 2) and a primer 2 (SEQ ID NO: 3) are designed for PCR amplification.
The PCR used a 50. Mu.L reaction system in which 0.5. Mu.L of Q5 polymerase, 10. Mu.L of reaction buffer, 1. Mu.L of 10mM dNTPs, 2.5. Mu.L each of 10. Mu.M primer 1 and 10. Mu.M primer 2, and <1000ng of genomic DNA, was supplemented with nuclease-free water to 50. Mu.L.
The PCR reaction conditions were:
Wherein the Q5 polymerase is NEW ENGLANDThe high-fidelity Q5DNA polymerase of the company, the reaction buffer solution isThe specific operation steps of the reaction buffer solution in the High-FIDELITY DNA Polymerase kit of the company are as followsCompany PCR UsingHigh-FIDELITY DNA Polymerase (M0491) kit instructions.
The PCR product was ligated into a Blunt-ended Blunt vector (Beijing full-size gold company), the procedure was carried out according to the full-size gold company Blunt vector product instruction, and then the ligation product was sequenced (Sanger method), and it was confirmed that the constructed Blunt vector contained a promoter fragment of the Artemisia interna ubiquitin gene having a length of 3369bp, the sequence of which was shown in SEQ ID NO:1 and designated prAetUbi-01.
Example two, effect of constitutive promoter prAetUbi-01 in maize callus verification
1. Construction of a double luciferase expression vector containing the constitutive promoter prAetUbi-01
A double luciferase report system (Dual-Luciferase Reporter) containing firefly luciferase (Firefly luciferase, LUC) and Ren (Rellina luciferase, REN) is introduced, the expression efficiency of a target gene driven by a promoter is judged by detecting the activity of the LUC, and REN is used as an internal reference, so that the inter-group error of plant tissues caused by different factors such as agrobacterium infection conversion efficiency can be eliminated.
The structure of vector DBNBC-Dual_LUC (vector backbone: modified pCAMBIA2301, supplied by CAMBIA Co.) containing LUC and REN reporter genes is shown in FIG. 1. Wherein cSpec is a spectinomycin gene, RB is a right border, cLUC is a firefly luciferase gene (SEQ ID NO: 4), tNos is an Agrobacterium tumefaciens nopaline synthase gene terminator (SEQ ID NO: 5), prOsUBQ2 is a rice UBQ2 gene promoter (SEQ ID NO: 6), cREN is a Renilla luciferase gene (SEQ ID NO: 7), tPinII is a potato proteinase inhibitor II gene terminator (SEQ ID NO: 8), prOsActl is a rice actin gene promoter (SEQ ID NO: 9), cPAT is a phosphinothricin acetyl transferase gene (SEQ ID NO: 10), t35S is a cauliflower mosaic virus gene terminator (SEQ ID NO: 11), and LB is a left border.
A vector was constructed which driven expression of the LUC gene by the prAetUbi-01 promoter and transformed into maize, with the constitutive promoter prZmUbi (SEQ ID NO: 12) known in the art as a control. The expression pattern and expression intensity of the promoter prAetUbi-01 were compared and analyzed by performing luciferase activity assay on each tissue cell during each growth period of maize, and the construction of vectors using conventional cleavage methods is well known to those skilled in the art. Specifically, DBNBC-Dual_LUC vector was linearized with restriction enzyme SalI and amplified using the primer pair shown in SEQ ID NO. 13 and SEQ ID NO. 14, respectively, and the primer pair shown in SEQ ID NO. 15 and SEQ ID NO. 16 to give prZmUbi element with a linker sequence (SEQ ID NO. 12) and prAetUbi5-01 element with a linker sequence (SEQ ID NO. 1). The linearized DBNBC-Dual_LUC vector was subjected to mixed recombination reactions with the prZmUbi element with the linker sequence and the prAetUbi5-01 element with the linker sequence, respectively, and the procedure was performed according to the instructions of Takara In-Fusion Snap Assembly Master Mix kit (Clontech, calif., JPN, CAT: 638949) to construct recombinant Dual luciferase expression vectors DBN13844 and DBN14192, the structures of which are shown In FIGS. 2 and 3.
2. Recombinant double luciferase expression vector for transforming agrobacterium
The correctly constructed recombinant double luciferase expression vectors DBN13844 and DBN14192 were transformed into Agrobacterium LBA4404 (INVITRGEN, chicago, USA; cat. No. 18313-015), respectively, by liquid nitrogen. The transformation condition is that 100 mu L of agrobacterium LBA4404 and 3 mu L of recombinant expression vector are placed in liquid nitrogen for 10min, then warm water bath is carried out at 37 ℃ for 10min, the transformed agrobacterium LBA4404 is inoculated in an LB test tube and cultured for 2h under the conditions of 28 ℃ and 200rpm, the transformed agrobacterium LBA4404 is coated on an LB solid plate containing 50mg/L of Rifampicin (RIFAMPICIN) and 50mg/L of spectinomycin (Spectinomycin) until positive monoclonal is grown, the monoclonal culture is selected and plasmids thereof are extracted, sequencing identification is carried out on the extracted plasmids, and the result shows that the structures of the recombinant double luciferase expression vectors DBN13844 and DBN14192 are completely correct.
3. Transient transformation of maize callus
The corn callus is a biological cell reactor for efficiently expressing proteins, and exogenous genes are introduced into the corn callus by using an agrobacterium infection method, so that the functions of the exogenous genes are primarily studied.
For Agrobacterium-mediated maize transformation, briefly, immature chick embryos are isolated from maize, the chick embryos are contacted with an Agrobacterium suspension, wherein the Agrobacterium is capable of transferring T-DNA in the DBN13844 and DBN14192 vectors to at least one cell of one of the chick embryos (step 1: an infection step), in which the chick embryo is preferably immersed in an Agrobacterium suspension (OD660 = 0.4-0.6, infection medium (MS salts 4.3g/L, MS vitamin, casein 300mg/L, sucrose 68.5g/L, glucose 36g/L, acetosyringone (AS) 40mg/L, 2, 4-dichlorophenoxyacetic acid (2, 4-D) 1mg/L, pH 5.3)) to initiate inoculation. The young embryo is co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-culturing step). Preferably, the young embryos are cultured after the infection step on solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 20g/L, glucose 10g/L, AS100mg/L, 2,4-D1mg/L, agar 8g/L, pH 5.8). 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 (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, 2,4-D1mg/L, cephalosporin 250mg/L, plant gel 3g/L, pH 5.8) without addition of a selection agent for plant transformants (step 3: recovery step). Young embryos are cultured on solid medium with antibiotics but no selection agent to eliminate agrobacterium and provide a recovery period for the infected cells while callus is formed.
4. Detection of LUC/REN double luciferase activity in maize callus
Maize calli transformed with recombinant double luciferase expression vectors DBN13844 and DBN14192 are taken respectively and reference is made to Promega companyThe detection is carried out by using a report ASSAY SYSTEM (E1960) kit, and the specific method is as follows:
The preparation method comprises the steps of (1) diluting 5× Passivelysisbuffer (PLB) with 4 times of ultrapure water to obtain 1×PLB, (LAR II) dissolving a freeze-dried powder luciferase assay Substrate by using a luciferase assay buffer II (10 mL), storing the freeze-dried powder luciferase assay Substrate at-80 ℃ in a dark place, and (II) dissolving 200 mu L of Stop & Glo Substrate (50×) in 10mL of Stop & Globuffer to obtain a 1×stop & Glo Reagent solution, and storing the solution at-80 ℃ in a dark place;
Step 2, taking the callus after finishing infection and culturing for 5 days, pressing and absorbing water on absorbent paper, dividing the callus transferred into each carrier into 3 parts, placing the 3 parts into a 2.0mL centrifuge tube added with 1 large steel ball and 1 small steel ball, freezing in liquid nitrogen, and grinding for 1min under the condition of 1200rpm by using a vibration grinder;
Step 3, adding 150uL of 1 XPLB into a centrifuge tube after grinding, uniformly mixing, swirling for 20s, standing for 10min for full cracking, centrifuging at 12000rpm for 10min at 4 ℃, taking 100 uL of supernatant extract, adding into an ELISA plate, and setting for 3 repetitions;
Step 4, adding 100 mu L of 1 Xfirefly luciferase reaction solution, uniformly mixing by shaking plates, detecting the activity of firefly luciferase by an enzyme-labeling instrument, and finishing the detection within 30min, wherein the unit of the fluorescence intensity of the detected firefly luciferase is RLU (relative fluorescence unit);
And 5, adding 100 mu L of 1 XRenilla luciferase reaction solution, uniformly mixing by shaking plates, detecting the activity of Renilla luciferase by using an enzyme-labeling instrument, and finishing the detection within 30min, wherein the unit of the fluorescence intensity of the detected Renilla luciferase is RLU (relative fluorescence unit).
Wherein, the enzyme-labeled instrument (BioTek-H1 MF) detection program is a full spectrum light intensity reading.
The results of the firefly Luciferase (LUC) and Renilla luciferase (REN) activity assays in transiently transformed maize calli are shown in Table 1. Wherein the REN gene is an internal reference, and the ratio of LUC/REN reflects the relative activity intensity of the promoter (LUC/REN ratio= (LUC value of maize callus transformed with recombinant expression vector-LUC value of maize callus wild type)/(REN value of maize callus transformed with recombinant expression vector-REN value of maize callus wild type)).
TABLE 1 luciferase Activity in transient transformed maize callus
The results in Table 1 show that in transiently transformed maize callus cells, promoters prAetUbi-01 and prZmUbi1 are both capable of normally driving expression of the LUC gene. Therefore, the promoter prAetUbi-01 can normally regulate and control gene expression, and has good application prospect.
Example III, effect of constitutive promoter prAetUbi-01 in transgenic maize plants verification
1. Corn stable transformation of recombinant double luciferase vectors DBN13844 and DBN14192
Transformation was performed using conventional agrobacterium infection, and the aseptically cultured maize young embryos were co-cultured with agrobacterium as described in example two to transfer the T-DNA in the constructed recombinant expression vector into the maize genome to generate transgenic maize events.
For Agrobacterium-mediated maize transformation, briefly, immature chick embryos are isolated from maize, the chick embryos are contacted with an Agrobacterium suspension, wherein the Agrobacterium is capable of transferring the nucleotide sequence of interest and the nucleotide sequence of the pat gene to at least one cell of one of the chick embryos (step 1: an infection step), in which the chick embryo is preferably immersed in an Agrobacterium suspension (OD660 = 0.4-0.6, infection medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 68.5g/L, glucose 36g/L, acetosyringone (AS) 40mg/L, 2, 4-di-chlorophenoxyacetic acid (2, 4-D) 1mg/L, pH 5.3)) to initiate inoculation. The young embryo is co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-culturing step). Preferably, the young embryos are cultured after the infection step on solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 20g/L, glucose 10g/L, AS100mg/L, 2,4-D1mg/L, agar 8g/L, pH 5.8). 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 (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, 2,4-D1mg/L, cephalosporin 250mg/L, plant gel 3g/L, pH 5.8) without addition of a selection agent for plant transformants (step 3: recovery step). Young embryos are cultured on solid medium with antibiotics but no selection agent to eliminate agrobacterium and provide a recovery period for the infected cells while callus is formed. Next, the callus is inoculated on a medium containing a selection agent (4- [ hydroxy (methyl) phosphono ] -DL-homoalanine) and the grown transformed callus is selected (step 4: selection step). Preferably, the callus is cultured on selective solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, cephalosporin 250mg/L, 4- [ hydroxy (methyl) phosphono ] -DL-homoalanine 10mg/L, 2,4-D1mg/L, plant gel 3g/L, pH 5.8) with a selective agent, resulting in selective growth of the transformed cells. Then, the callus is regenerated into a plant (step 5: regeneration step), preferably, the callus grown on the medium containing the selection agent is cultured on a solid medium (MS differentiation medium and MS rooting medium) to regenerate the plant.
The selected resistant callus is transferred to the MS differentiation medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, 6-benzyl adenine 2mg/L, cephalosporin 250mg/L, 4- [ hydroxy (methyl) phosphono ] -DL-homoalanine 5mg/L, plant gel 3g/L, pH 5.8) and cultured and differentiated at 25 ℃. The differentiated plantlets were transferred to the MS rooting medium (MS salt 2.15g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, cephalosporin 250mg/L, indole-3-acetic acid 1mg/L, plant gel 3g/L, pH 5.8), cultured to about 10cm high at 25℃and transferred to a greenhouse for cultivation until set. In the greenhouse, the cells were cultivated at 28℃for 16 hours per day and at 20℃for 8 hours.
2. Verification of maize plants transformed with recombinant double luciferase vectors DBN13844 and DBN14192
Extracting genome DNA by DNEASY PLANT MaxiKit (Qiagen), detecting copy numbers of the screening marker PAT genes in the vectors DBN13844 and DBN14192 by a Taqman probe fluorescent quantitative PCR method, and taking wild type corn plants as a control, setting 3 times of repetition in the experiment, and taking an average value.
The specific method for detecting the PAT gene copy number is as follows:
Step 1, respectively taking 100mg of leaves of corn plants transferred into recombinant double luciferase vectors DBN13844 and DBN14192, grinding into homogenate in a mortar by liquid nitrogen, and taking 3 samples for each sample;
Step 2, extracting genome DNA of the sample, wherein the specific method refers to the instruction;
Step 3, determining the concentration of the genomic DNA by using NanoDrop2000 (Thermo Scientific);
Step 4, adjusting the concentration of the genome DNA to the same concentration value, wherein the concentration value ranges from 80 ng/mu L to 100 ng/mu L;
And 5, 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 corn plant as a reference, repeating 3 times of each sample, taking the average value of the samples, and detecting the fluorescent quantitative PCR primer of the PAT gene as shown in SEQ ID NO. 17-18, wherein the probe is shown in SEQ ID NO. 19.
The PCR reaction was carried out using JumpStartTMTaq ReadyMixTM (Sigma) at 10. Mu.L, 50 Xprimer/probe mixture at 1. Mu.L, genomic DNA at 3. Mu.L, and water (ddH2 O) at 6. Mu.L. 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 an amber tube at 4 ℃.
The PCR reaction conditions were:
The data were analyzed using SDS2.3 software (Applied Biosystems). Experimental results show that PAT gene is integrated into the chromosome group of the tested corn in a single copy form, so that transgenic corn plants containing single copy promoter prAetUbi-01 and promoter prZmUbi1 are obtained.
3. Detection of LUC/REN double luciferase activity in each tissue of maize plants
Root and leaf were sampled at the seedling stage, stem and leaf were sampled at the trumpet-shaped stage, flower filaments and pollen were sampled at the flowering stage, seed was sampled at the filling stage, and 3 replicates were sampled per tissue for each stage of maize T0 plants transformed with promoters prAetUbi-01 and prZmUbi1, respectively. The samples were subjected to the detection of double luciferase activity according to the method of the above-described embodiment II of the present application, and the detection results are shown in Table 2.
TABLE 2 luciferase Activity at different times and different sites in stably transformed maize plants
The results in Table 2 show that promoter prAetUbi-01 is able to normally drive constitutive expression of the LUC gene in various tissues and higher expression in leaves and roots, filaments and pollen at seedling stage during various stages of maize growth and development. Specifically, expression of LUC driven by prAetUbi-01 was 0.5-fold and 0.9-fold, respectively, in leaves and roots in seedling stage compared to that driven by prZmUbi1, and expression of LUC driven by prAetUbi5-01 was 0.5-fold and 0.6-fold, respectively, in filaments and pollen in flowering stage compared to that driven by prZmUbi. Therefore, the promoter prAetUbi-01 has stronger transcriptional activity in corn, and the activity in partial tissues is equivalent to that of the promoter prZmUbi1 commonly used in the field, and the novel constitutive promoter prAetUbi5-01 provides a novel tool and a novel choice for the expression of exogenous genes in plant species, and has great application prospects.
Example IV, constitutive promoter prAetUbi-01 verification of the effect of driving expression of EPSPS Gene in maize
1. Construction of expression vector containing constitutive promoter prAetUbi-01 and EPSPS Gene
To further determine the utility of constitutive promoter prAetUbi-01, the promoter is operably linked to a herbicide tolerance gene and verified whether it can drive expression of the gene, thereby conferring herbicide tolerance traits to plants.
Specifically, a plant expression vector was constructed which drives the EPSPS gene from promoter prAetUbi-01. The backbone vector DBNBC-01 (resistance tag modified pCAMBIA2301, available from CAMBIA) was linearized using restriction enzyme SalI and amplified using primer pair SEQ ID NO:20 and SEQ ID NO:21 for prAetUbi-01 (SEQ ID NO: 1) with the adaptor sequence, primer pair SEQ ID NO:22 and SEQ ID NO:23, primer pair SEQ ID NO:24 and SEQ ID NO:25, primer pair SEQ ID NO:26 and SEQ ID NO:27, primer pair SEQ ID NO:28 and SEQ ID NO:29, primer pair SEQ ID NO:30 and SEQ ID NO:31, primer pair SEQ ID NO:32 and SEQ ID NO:33 for the amplified adaptor sequence spActCTP (SEQ ID NO: 34) element cEPSPS (SEQ ID NO: 35) element tNos (SEQ ID NO: 5) element, pr35s (SEQ ID NO: 36) element, cPAT) element and 5311 (SEQ ID NO: 11) respectively.
The linearized backbone vector DBNBC-01 fragment was subjected to mixed recombination reactions with the prAetUbi-01 element with the linker sequence, the spActCTP element with the linker sequence, the cEPSPS element with the linker sequence, the tNos element with the linker sequence, the pr35S element with the linker sequence, the cPAT element with the linker sequence, and the t35S element with the linker sequence, respectively, according to the instructions of Takara In-Fusion Snap Assembly Master Mix kit (Clontech, CA, JPN, CAT: 638949), and the construction of a recombinant vector DBN15003 containing the EPSPS gene was performed, the structure of which is schematically shown In FIG. 4.
2. Recombinant expression vector DBN15003 transformed agrobacterium
Referring to the second embodiment of the application, the recombinant expression vector DBN15003 is transformed into the agrobacterium LBA4404 by a liquid nitrogen method, and plasmids are extracted for sequencing identification, and the result shows that the structure of the recombinant expression vector DBN15003 is completely correct.
3. Recombinant expression vector DBN15003 stably transformed corn
Referring to the third embodiment of the application, agrobacterium transformed with recombinant expression vector DBN15003 is used to infect corn, and the T-DNA of the recombinant expression vector DBN15003 is inserted into corn chromosome to obtain the corresponding transgenic corn T0 plant.
About 100mg of leaves of the transgenic maize T0 plant are taken as a sample, the genome DNA of the transgenic maize T0 plant is extracted by using the DNEASY PLANT Maxi Kit of Qiagen, the copy number of the EPSPS gene of the vector DBN15003 is detected by a TaqMan probe fluorescent quantitative PCR method, and the wild maize plant is taken as a control. Experiments were repeated 3 times and averaged.
Experimental results show that the fragment of the EPSPS gene driven by prAetUbi-01 has been successfully integrated into the genome of the detected transgenic maize plant in single copy form, thereby obtaining a transgenic maize plant containing the single copy promoter prAetUbi 5-01.
4. Evaluation of herbicide resistance in transgenic maize
Transgenic maize T0 plants and wild type maize plants (18 days after sowing) transformed with prAetUbi-01 promoter and EPSPS gene were sprayed evenly with 3360g ae/ha (4 x field concentration) of glyphosate herbicide. After 7 days of herbicide application (7 DAT), the plants were evaluated for herbicide tolerance/resistance based on leaf and plant damage phenotypes, leaf bases exhibited slightly transparent or translucent films, leaves or leaf bases were slightly chlorosis, and could recover to grade 1 within 14 days, leaves or leaf bases were significantly chlorosis and yellow, recovery times were greater than 14 days, possibly accompanied by slight leaf curl, shrinkage to grade 2, significant leaf curl, shrinkage, or plant deformity, or reduced plant height, inhibited growth to grade 3, severe plant deformity, or severely inhibited growth, or leaf wilting, and withered death to grade 4.
The transformation event resistance performance was scored according to the formula x= [ Σ (n×s)/(t×m) ]×100 (X-phytotoxicity score, N-peer number of victims, S-phytotoxicity grade, T-total number of plants, M-highest phytotoxicity grade), and the resistance was evaluated according to the scores as high resistance (0-25 points), medium resistance (26-75 points), low resistance (76-100 points). The experimental results are shown in table 3 and fig. 5.
TABLE 3 results of tolerance experiments on glyphosate in transgenic maize T0 plants
From the results of Table 3, it was found that transgenic corn into which EPSPS gene driven by constitutive promoter prAetUbi-01 was transferred had a lower leaf damage degree and had a high effect of resistance to glyphosate, compared to wild type corn, which had a more serious leaf damage degree and did not have herbicide resistance, in the case of treatment with glyphosate at 4-fold field concentration. Meanwhile, as shown in fig. 5, the transgenic corn (right five plants) transformed with EPEPS gene driven by constitutive promoter prAetUbi-01 can grow normally, almost has no leaf damage, and shows that the transgenic corn has good tolerance to herbicide, while the wild corn (leftmost first plant) has dry and dead leaves, namely has no herbicide tolerance. That is, the constitutive prAetUbi-01 of the present application can normally drive the expression of a target gene in plant species and impart corresponding characteristics to plants.
Example five, construction of truncations and Activity detection of constitutive promoter prAetUbi-01
1. Construction of a double luciferase expression vector containing a promoter prAetUbi-01 truncate
The sequence of the constitutive promoter prAetUbi-01 is subjected to bioinformatics analysis by using TSSP and other online analysis websites, and the promoter sequence is determined to contain hormone response cis-acting elements, optical signal response elements, a plurality of cis-acting elements such as CAATBox related to transcriptional activity and TATA BOX related to transcription initiation.
In order to further identify transcriptional regulatory elements controlling the activity of constitutive promoter prAetUbi-01, i.e. the sequence with highest efficiency of the promoter to drive the expression of the target gene and the shortest sequence with transcriptional activity, full-length promoter prAetUbi-01 is subjected to deletion truncation of 5' -end unidirectional fragments to different extents according to element distribution conditions in the promoter sequence. The sizes of the 4 truncated promoter fragments are 2860bp, 2143bp, 1649bp and 874bp, which correspond to SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39 and SEQ ID NO. 40 respectively and are named prAetUbi-02, prAetUbi5-03, prAetUbi5-04 and prAetUbi-05.
With reference to the double luciferase expression vector containing the promoter prAetUbi-01 constructed in example II of the present application, the backbone vector DBNBC-Dual_LUC containing LUC and REN double luciferin was linearized with restriction enzyme SalI using the primer pair shown in SEQ ID NO. 41 and SEQ ID NO. 16, the primer pair shown in SEQ ID NO. 42 and SEQ ID NO. 16, the primer pair shown in SEQ ID NO. 43 and SEQ ID NO. 16, the primer pair shown in SEQ ID NO. 44 and SEQ ID NO. 16, and the truncated promoters of the full-length promoters prAetUbi5-01, respectively. The linearized backbone vector DBNBC-Dual_LUC fragment was subjected to mixed recombination reactions with truncated promoters with the linker sequence, respectively, and the procedure was followed according to the instructions of Takara In-Fusion Snap Assembly Master Mix kit (Clontech, calif., JPN, CAT: 638949) to obtain the Dual luciferase expression vectors DBN15061, DBN15062, DBN15063 and DBN15064 containing truncated promoters prAetUbi-02, prAetUbi5-03, prAetUbi5-04, prAetUbi-05.
2. Activity detection of truncations of promoter prAetUbi-01 in maize callus the vectors DBNl4192, DBN15061, DBN15062, DBN15063 and DBN15064 were subjected to transient transformation of maize callus using the method of example two of the application, and the corresponding supernatants were prepared for use and assayed for luciferase activity as shown in Table 4.
TABLE 4 transient transformation of luciferase Activity in maize callus with truncations of different promoters
As can be seen from the data in Table 4, the truncated promoters prAetUbi5-02, prAetUbi5-03 and prAetUbi-04 of the full-length promoter prAetUbi-01 all had good transcriptional activity in transiently transformed maize callus cells. Specifically, LUC expression driven by prAetUbi-02 was 0.7 times that driven by prAetUbi5-01, LUC expression driven by prAetUbi5-03 was 0.6 times that driven by prAetUbi5-01, and LUC expression driven by prAetUbi5-04 was 0.8 times that driven by prAetUbi 5-01. While, under the drive of prAetUbi-05, LUC was hardly expressed, indicating that the truncated promoter shown in SEQ ID NO. 40 had little transcriptional activity. Thus, it can be seen that SEQ ID NO. 39 is the shortest sequence with promoter activity, and in combination with analysis of the active elements and element distribution of the full-length prAetUbi-01 promoter, it can be reasonably expected by those skilled in the art that when SEQ ID NO. 39 is contained in the promoter sequence, it has transcriptional activity, and thus can normally function as a promoter, thereby driving expression of the target gene.
In summary, the invention discloses a constitutive promoter from Artemisia annua for the first time, the constitutive promoter of the invention shows activity in most tissues and cells of many types of plants, especially in roots, stems, leaves, filaments, pollen, seeds and spike tops of plants, can normally drive the expression of target genes, provides a new tool and selection for plant genetic engineering, and has good application prospect.
While only exemplary embodiments of the present invention have been described above, it will be understood by those skilled in the art that these are by way of example only and that the scope of the present invention is defined by the appended claims. Various changes or modifications to these embodiments may be made by those skilled in the art without departing from the principles and spirit of the invention, but such changes or modifications fall within the scope of the invention.