CN113564163A

CN113564163A - Expression regulation element and application thereof

Info

Publication number: CN113564163A
Application number: CN202011172725.0A
Authority: CN
Inventors: 解光宁; 牛小牧; 梁亚峰
Original assignee: Shandong Shunfeng Biotechnology Co Ltd
Current assignee: Shandong Shunfeng Biotechnology Co Ltd
Priority date: 2020-10-28
Filing date: 2020-10-28
Publication date: 2021-10-29
Also published as: WO2022089458A1

Abstract

The invention provides an expression regulatory element and application thereof, and particularly relates to the expression regulatory element and application thereof in regulating expression of one or more genes in cells. The invention further discloses a method for improving the expression of a target gene by using the expression regulatory element, which comprises the step of operably connecting the expression regulatory element and the target gene to improve the expression of the target gene.

Description

Expression regulation element and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an expression regulation element and application thereof in regulating gene expression.

Background

In the process of crop genetic improvement, how to increase the expression level of partial gene expression is one of the important problems that need to be solved. Cis-acting elements refer to specific DNA sequences in tandem with structural genes, which are binding sites for transcription factors and regulate gene transcription by binding to transcription factors. Cis-elements that are capable of enhancing transcription upon binding of transcription factors are called enhancers. By adding an enhancer to the regulatory region of the target gene, it is possible to effectively improve the expression of the target gene without significantly changing the gene expression pattern.

The OCS gene codes octopine synthetase in agrobacterium, and the promoter part of the OCS gene has TATA box and CAAT box similar to eukaryotic promoters, can be universally expressed in plant cells, and has the characteristics of plant promoters. An important 16bp palindrome (ACGTAAGCGCTTACGT) is present in the OCS promoter portion, which has significant enhancer activity. After analyzing other strong promoter sequences widely used in plants, it is found that other expression regulatory element sequences having a certain homology with the OCS exist, and OCS elements are modified according to the characteristics of the sequences so as to screen new expression regulatory elements capable of improving the gene expression level.

Disclosure of Invention

The invention aims to provide an expression regulation element and application thereof. The invention designs 64 expression regulation and control elements to be screened by changing the wild enhancer sequence, and connects the expression regulation and control elements to an expression vector for screening to obtain 19 expression regulation and control elements with enhanced functions, and the 19 expression regulation and control elements have obvious effect on improving the gene expression level.

Expression regulatory elements

In one aspect, the present invention provides an expression control element comprising a nucleotide sequence set forth in any one of SEQ ID NOs 1-64 or a nucleotide sequence complementary thereto.

In one embodiment, the expression control element comprises a polynucleotide sequence as set forth in any one of SEQ ID NO 2, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 17, SEQ ID NO 20, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 28, SEQ ID NO 33, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 51 or SEQ ID NO 55; preferably, the polynucleotide sequence shown in any one of SEQ ID NO 28, SEQ ID NO 33, SEQ ID NO 44, SEQ ID NO 45 or SEQ ID NO 48.

In one embodiment, the expression control element is a polynucleotide sequence as set forth in any one of SEQ ID NO 2, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 17, SEQ ID NO 20, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 28, SEQ ID NO 33, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 51 or SEQ ID NO 55; preferably, the polynucleotide sequence shown in any one of SEQ ID NO 28, SEQ ID NO 33, SEQ ID NO 44, SEQ ID NO 45 or SEQ ID NO 48.

In one embodiment, the expression control element has 80% homology or more (preferably 90% or more, more preferably 95% or more, 96% or more, 97% or more, 98% or more), with any of SEQ ID NO 2, 5% or more, 7% or more, 8% or more, 10% or more, 12% or more, 17% or more, 20% or more, 23% or more, 24% or more, 28% or more, 33% or more, 43% or more, 44% or more, 45% or more, 48% or more, 49% or more, 51% or more, or 55% or more, with the sequence shown in SEQ ID NO.

In one embodiment, the expression control element is a polynucleotide complementary to any one of the sequences shown in SEQ ID NO 2, 5, 7, 8, 10, 12, 17, 20, 23, 24, 28, 33, 43, 44, 45, 48, 49, 51 or 55.

In one embodiment, the polynucleotide is preferably single-stranded or double-stranded.

Nucleic acid constructs

In another aspect, the invention provides a nucleic acid construct comprising the aforementioned expression control element operably linked to a regulatory element or gene of interest.

In one embodiment, the adjustment element is selected from one or more of the group consisting of: promoters, enhancers, introns, 5 ' -untranslated regions (5 ' -UTR, also known as leader sequences), 3 ' -UTR, transposons, terminators, polyadenylation sequences, marker genes, or combinations thereof.

In one embodiment, the target genes include genes for herbicide tolerance, insect resistance, disease resistance, abiotic stress tolerance, yield stability, yield increase, carbohydrate metabolism, fatty acid metabolism, amino acid metabolism, plant development, plant growth regulation, drought resistance, cold resistance, heat resistance, nutrient utilization efficiency, nitrogen utilization efficiency, and salt resistance, micrornas (small RNAs), and small RNA precursors.

Carrier

The present invention also provides a vector comprising the above expression control element and/or nucleic acid construct, and preferably, the vector further comprises a regulatory element or a target gene operably linked to the above expression control element and/or nucleic acid construct.

In one embodiment, the target gene is selected from plant genes or non-plant genes, including genes derived from eukaryotes or prokaryotes.

In one embodiment, the vector comprises a cloning vector, an expression vector, a shuttle vector, an integration vector.

In one embodiment, the expression vector further comprises at least one origin of replication for self-replication.

In one embodiment, the vector may be one that, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.

The vector may be of the type of plasmid, virus, cosmid, phage, etc., which are well known to those skilled in the art.

Preferably, the vector in the present invention is a plasmid.

Host cell

In another aspect, the invention provides a host cell comprising one or more of the foregoing expression control elements, nucleic acid constructs, and vectors.

In one embodiment, the host cell has the aforementioned expression control elements integrated into its genome.

In one embodiment, the host cell is a prokaryotic cell, such as E.coli, Agrobacterium.

In one embodiment, the host cell is a eukaryotic cell, including an animal cell, a plant cell, a fungus, and the like; preferably, the eukaryotic cell is a plant cell, and preferably, the plant includes angiosperms and gymnosperms.

In one embodiment, the plant includes a monocot and a dicot.

In one embodiment, the plant includes herbaceous plants and woody plants.

In one embodiment, the plant comprises arabidopsis, tobacco, rice, maize, sorghum, barley, wheat, millet, soybean, tomato, potato, quinoa, lettuce, rape, cabbage, strawberry.

Plant and method for producing the same

The invention also provides a plant stably transformed by the vector, wherein the vector comprises the expression regulation and control element; preferably, the vector comprises the expression control element of the present invention and a target gene.

The invention also provides a plant with improved target gene expression level, wherein a regulatory region of the target gene comprises the expression regulatory element.

The expression regulatory element may be obtained by changing one or more nucleotides in the regulatory region of the target gene, may be introduced by means of gene editing, or may be obtained by inserting the expression regulatory element of the present invention into the regulatory region of the target gene.

Method

The present invention also provides a method for regulating expression of a target gene, which comprises the step of regulating expression of the target gene using the expression regulatory element of the present invention.

In a preferred embodiment, the modulating target gene expression is increasing target gene expression.

In one embodiment, the target gene may be endogenous or exogenous to the host cell.

In one embodiment, the method of regulating a target gene of interest comprises the step of introducing an expression control element of the present invention into a regulatory region of the target gene.

The term "regulatory region" generally refers to regulatory elements (e.g., promoters, enhancers, introns, 5 ' -untranslated regions (5 ' -UTRs, also known as leader sequences) or 3 ' -UTRs, transposons, regions in which terminators or other noncoding regions are involved in regulating transcription of a nucleic acid molecule (e.g., a gene or target gene).

In a preferred embodiment, the regulatory region is the region of the promoter or upstream of the promoter.

The introduction of the expression regulatory element of the present invention into the regulatory region of the target gene can be achieved by: the expression regulatory element of the present invention is included in the regulatory region of the target gene by editing 1 or more nucleic acids in the regulatory region, or the expression regulatory element of the present invention is inserted into the regulatory region of the target gene so that the expression regulatory element of the present invention is included in the regulatory region. The editing includes changing, deleting, inserting and the like of nucleic acid.

In one embodiment, the introduction of the expression regulatory element of the present invention into the regulatory region of the target gene can be achieved by homologous recombination, gene editing (e.g., CRISPR technique, TALEN technique, ZFN technique).

In one embodiment, the method comprises inserting at least 1 copy of an expression control element of the invention into a regulatory region of a gene of interest.

In one embodiment, the expression control element is located within 0-5000bp, preferably within 0-2000bp, preferably within 0-550bp, preferably within 0-200bp upstream or downstream of the transcription start site of the target gene. Preferably, the expression control element is upstream or downstream of the transcription initiation site.

In one embodiment, the expression control element is provided in a promoter region; preferably, it is arranged 1bp to 200bp, more preferably 20bp to 100bp, more preferably 50bp to 90bp, more preferably 60bp to 80bp, e.g. 70bp, 71bp, 72bp, 73bp, 74bp or 75bp upstream of the promoter region TATA box.

In one embodiment, the expression control element is placed upstream of a promoter; preferably, it is placed 1bp to 200bp, more preferably, 20bp to 100bp, more preferably, 50bp to 90bp, more preferably, 60bp to 80bp, for example, 70bp, 71bp, 72bp, 73bp, 74bp or 75bp upstream of the promoter. .

In one embodiment, the expression regulatory element may also be located within an enhancer, intron, 5 ' -untranslated region (5 ' -UTR, also referred to as leader sequence) or 3 ' -UTR, transposon, terminator region or gene of the target gene.

In the present invention, the same expression control element or different types of expression control elements may be used in combination to control the expression level of a target gene; the same expression control element may be 1 copy of the expression control element or may be multiple copies of the expression control element.

In one embodiment, the expression regulatory element comprises at least one expression regulatory element of the invention, e.g., 1-19, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19.

In other embodiments, each of the expression regulatory elements comprises at least 1 copy of the expression regulatory element, e.g., 1-20 copies, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20.

In one embodiment, multiple copies of the same or different expression control elements may have a spacer sequence between them, which may be 1-200 nucleotides, preferably 1-50 nucleotides.

In one embodiment, multiple copies of the same or different expression control elements may be linked in one or more of head-to-head, head-to-tail, tail-to-head, and tail-to-tail.

In another aspect, the present invention also provides a method for regulating expression of a target gene in a plant cell, the method comprising the step of regulating expression of the target gene using the expression regulatory element of the present invention.

In a preferred embodiment, the modulation is an increase in the expression of a gene of interest.

In one embodiment, the method comprises the step of introducing an expression control element of the invention into a regulatory region of a gene of interest.

In another aspect, the present invention also provides a method for producing a plant cell, plant tissue, plant part or plant with increased expression of a target gene, the method comprising the step of increasing expression of the target gene in the plant cell, plant tissue, plant part or plant using the expression regulatory element of the present invention.

In one embodiment, the target gene may be an endogenous gene or an exogenous gene with respect to the plant. In one embodiment, the method comprises the step of introducing an expression control element of the invention into a regulatory region of a gene of interest.

In another aspect, the present invention also provides a plant cell, plant tissue, plant part or plant produced by the aforementioned method.

Use of

In another aspect, the invention provides the use of an expression control element, nucleic acid construct or vector as described above for regulating gene expression. Preferably, the regulated gene expression is increased gene expression.

In another aspect, the present invention provides the use of the aforementioned expression control element, nucleic acid construct or vector in the preparation of a cell having an increased expression level of a gene of interest; the cells include prokaryotic cells and eukaryotic cells, preferably, plant cells.

In another aspect, the present invention provides the use of the aforementioned expression control element, nucleic acid construct or vector in the preparation of a plant cell, plant tissue, plant part or plant with increased expression of a gene of interest.

In another aspect, the invention provides the use of an expression control element, nucleic acid construct or vector as described above in the preparation of a reagent or kit for increasing gene expression.

In another aspect, the present invention provides the use of the aforementioned expression control element, nucleic acid construct or vector in the preparation of a reagent or kit for increasing the expression level of a target gene in a plant cell, plant tissue or plant.

General definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

The terms "polynucleotide", "nucleotide sequence", "nucleic acid molecule" and "nucleic acid" are used interchangeably and include DNA, RNA or hybrids thereof, whether double-stranded or single-stranded.

The term "homology" or "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids. Thus, the compositions and methods of the invention also comprise homologs of the nucleotide and polypeptide sequences of the invention (e.g., in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:28, SEQ ID NO:33, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:51, or SEQ ID NO: 55). "homology" can be calculated by known methods including, but not limited to, the following: computational Molecular Biology [ Computational Molecular Biology ] (Lesk, A.M. ed.) Oxford University Press [ Oxford University Press ], New York (1988); biocontrol information and Genome Projects [ biologicals: informatics and genome project ] (Smith, d.w. eds.) Academic Press [ Academic Press ], new york (1993); computer Analysis of Sequence Data, Part I [ Computer Analysis of Sequence Data, Part I ] (Griffin, A.M. and Griffin, edited by H.G.) Humana Press [ Humasa Press ], New Jersey (1994); sequence Analysis in Molecular Biology [ Sequence Analysis in Molecular Biology ] (von Heinje, g. editor) Academic Press [ Academic Press ] (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J. eds.) Stockton Press (Stockton Press, New York (1991).

The term "encode" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, as a template for the synthesis of other polymers and macromolecules in biological processes having defined nucleotide sequences (i.e., rRNA, tRNA and mRNA) or defined amino acid sequences and the biological properties that result. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system.

The term "expression control element" refers to a nucleotide sequence that up-regulates or down-regulates the expression of one or more genes.

The term "regulatory element" may act in "cis" or "trans" and typically acts in "cis", i.e., it activates expression of a gene located on the same nucleic acid molecule (e.g., chromosome) on which the regulatory element is located. Regulatory elements include promoters, enhancers, introns, 5 ' -untranslated regions (5 ' -UTR, also known as leader sequences) or 3 ' -UTR, transposons or terminators.

The term "regulatory region" generally includes regulatory elements (e.g., promoters, enhancers, introns, 5 ' -untranslated regions (5 ' -UTRs, also known as leader sequences) or 3 ' -UTRs, transposons, regions in which terminators or other noncoding regions are involved in regulating transcription of a nucleic acid molecule (e.g., a gene or target gene).

As used herein, the term "promoter" has a meaning well known to those skilled in the art and refers to a non-coding nucleotide sequence located upstream of a gene that promotes expression of a downstream gene. Constitutive (constitutive) promoters are nucleotide sequences that: when operably linked to a polynucleotide that encodes or defines a gene product, it results in the production of the gene product in the cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or defines a gene product, causes the gene product to be produced intracellularly substantially only when an inducer corresponding to the promoter is present in the cell. A tissue-specific promoter is a nucleotide sequence that: when operably linked to a polynucleotide that encodes or defines a gene product, it results in the production of the gene product in the cell substantially only when the cell is of the tissue type to which the promoter corresponds.

As used herein, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).

The term "vector" is a vector that comprises elements that permit the vector to integrate into the host cell genome or to replicate autonomously within the cell, independent of the genome. The vector may contain any element which ensures self-replication. It usually carries genes that are not part of the central metabolism of the cell and is usually in the form of double-stranded DNA. The choice of vector will generally depend on the compatibility of the vector with the host cell into which the vector is to be introduced. If a vector is used, the choice of vector will depend on methods well known to those skilled in the art for transforming a host cell. For example, plasmid vectors can be used.

The term "host cell" is to be understood as any unicellular or multicellular organism, including, for example, bacteria such as E.coli, fungi such as yeasts (e.g., Saccharomyces cerevisiae), molds (e.g., Aspergillus), plant cells and plants, and the like.

The term "plant" is to be understood as including any differentiated multicellular organism capable of photosynthesis, in including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants, vegetable crops, including artichokes, corm cabbages, sesames, leeks, asparagus, lettuce (e.g. head lettuce, leaf lettuce), bok choy, yellow croaker, melons (e.g. melons, watermelons, crow's melon, honeydew melon, cantaloupe), rape crops (e.g. brussels sprouts, cabbage, cauliflower, broccoli, collards, headless cabbages, chinese cabbages, cephalanoplos, carrots, cabbage (napa), okra, onions, celery, chickpea, parsnip, endive, potato, cucurbits (e.g. zucchini, cucurbits, etc, Squash, pumpkin), radish, dried onion, turnip cabbage, purple eggplant (also called eggplant), salsify, endive, shallot, endive, garlic, spinach, green onion, squash, leafy vegetables (greens), beets (sugar and feed beets), sweet potato, lettuce, horseradish, tomato, turnip, and spices; fruit and/or vintage crops such as apple, apricot, cherry, nectarine, peach, pear, plum, prune, cherry, quince, almond, chestnut, hazelnut, pecan, pistachio, walnut, citrus, blueberry, boysenberry (boysenberry), raspberry, currant, loganberry, raspberry, strawberry, blackberry, grape, avocado, banana, kiwi, persimmon, pomegranate, pineapple, tropical fruit, pome, melon, mango, papaya, and lychee; field crops, such as clover, alfalfa, evening primrose, meadowfoam, corn/maize (fodder corn, sweet corn, popcorn), hops, jojoba, peanuts, rice, safflower, small grain crops (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas, soybeans), oleaginous plants (oilseed rape, mustard, poppy, olives, sunflowers, coconut, castor oil plants, cocoa beans, groundnuts), arabidopsis, fibrous plants (cotton, flax, hemp, jute), lauraceae (cinnamon, camphor), or a plant such as coffee, sugar cane, tea, and natural rubber plants; and/or bedding plants, such as flowering plants, cactus, fleshy plants and/or ornamental plants, and trees, such as forests (broad leaf and evergreen trees, such as conifers), fruit trees, ornamental trees, and nut-bearing trees, as well as shrubs and other plantlets.

The term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant calli, plant pieces, and plant embryos, pollen, ovules, seeds, leaves, stems, flowers, branches, seedlings, fruits, kernels, ears, roots, root tips, anthers, and the like.

The term "plant cell" is understood to mean any cell from or found in a plant, which is capable of forming, for example: undifferentiated tissue such as callus, differentiated tissue such as embryos, plant parts, plants or seeds.

The term "gene editing" technology includes CRISPR technology, TALEN technology, ZFN technology. CRISPR technology refers to Clustered, regularly interspaced short palindromic repeats (Clustered regular intercarried palindromic repeats) derived from the immune system of a microorganism. Wherein the gene editing tools comprise guideRNA, Cas protein (such as Cas9, Cpf1, Cas12b and the like). The gene editing tool referred to in TALEN technology is a restriction enzyme that can cleave a specific DNA sequence, which includes one TAL effector DNA binding domain and one DNA cleavage domain. The gene editing tool referred to in ZFN technology is also a restriction enzyme that can cut a specific DNA sequence, and includes a zinc finger DNA binding domain and a DNA cleavage domain. It is well known to those skilled in the art that editing of intracellular genomes can be achieved by constructing the nucleotides encoding gene editing tools and other regulatory elements into suitable vectors and transforming the cells, the types of editing including gene knock-outs, insertions, base edits.

Vectors suitable for use in the present invention include commercially available plasmids such as, but not limited to: pBR322(ATCC37017), pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden), GEM1(Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia), pKK232-8, pCM7, pSV2CAT, pOG44, pOG 1, pSG (VK 3), (pBPV, pMSG, and Strvl Pharmacia) and the like.

The nucleic acid sequences, nucleic acid constructs or expression vectors of the invention can be introduced into a host cell by a variety of techniques, including transformation, transfection, transduction, viral infection, gene gun or Ti-plasmid mediated gene delivery, as well as calcium phosphate transfection, DEAE-dextran mediated transfection, lipofection, electroporation, and the like.

In the production methods of the invention, the cells are cultured on a nutrient medium suitable for production of the polypeptide using methods well known in the art. If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted into the culture medium, it can be recovered from the cell lysate.

TABLE 1 sequences of different expression regulatory elements

The invention has the main advantages that:

1. the invention screens out a group of expression regulation and control elements.

2. The expression regulation and control element of the invention is connected with the target gene, so that the expression quantity of the target gene can be obviously improved.

Drawings

FIG. 1 shows the composition of the screening vector, wherein an enhancer is 64 expression regulatory element sequences to be screened, Mini35S is a promoter with weak transcriptional activation capability after being modified, and GUS is a beta-glucuronidase gene (beta-glucuronidase).

In FIGS. 2A-2F, the positive control is a mini 35S-initiated Gus plasmid, and the negative control is an engineered mini 35S-initiated Gus plasmid.

FIG. 2A is an assay for the activity of the A1-A8 expression regulatory element.

FIG. 2B is an assay for the activity of B1-B8 expression regulatory elements.

FIG. 2C is a test for the activity of expression regulatory elements against C1-C8, D1-D8.

FIG. 2D is an assay for the activity of the expression regulatory elements for E1-E8.

FIG. 2E is an assay for the activity of F1-F8 expression regulatory elements.

FIG. 2F is a test of the activity of expression regulatory elements against G1-G8, H1-H8.

FIG. 3 is a diagram showing the relative fluorescence measurement vector composition, wherein an enhancer is 5 expression regulatory element sequences to be determined, Mini35S is a promoter with weak transcriptional activation capability after modification, mCherry is a red fluorescent group, 2 × 35s is a promoter with normal transcriptional activation capability, GPF is green fluorescent protein, and Nos and E9 are common terminators.

FIG. 4 GUS staining of transformed shoots stably containing different expression control element plasmids. The WT is not processed with any Arabidopsis seedlings, and the mini35S is a transformed seedling taking the modified mini35S promoter as a promoter and is used as a negative control; 35S is a transformed seedling taking a 35S promoter as a promoter, and is a positive control; f4 expression regulatory elements, F5 expression regulatory elements and F8 expression regulatory elements are added to the modified mini35 regulatory regions of the other 3 transformed seedlings respectively and are used for verifying the functions of the F4 expression regulatory elements, the F5 expression regulatory elements and the F8 expression regulatory elements.

Detailed description of the preferred embodiments

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

Example 1 screening of 64 sequences

The pCAMBIA1301-mini35S (82bp) -GUS screening plasmid is constructed, wherein the transcriptional activation capability of a mini35S (82bp) promoter is weak. After the regulatory region of the mini35s (82bp) is connected with the screened expression regulatory element, if the expression level of GUS is obviously improved, the expression regulatory element has the capability of improving gene expression.

The wild-type enhancer sequence was extended to 64 pieces according to the sequence of its non-conserved site (group numbering)A-H, total 8 groups, each group numbered 1-8 in sequence, the names and sequences are shown in Table 1). The 64 expression regulatory element sequences are connected to pCAMBIA1301-mini35S (82bp) -GUS screening plasmids to form 64 different plasmids to be screened, the connection mode of main elements is shown in figure 1, wherein the sequences after the connection of the expression regulatory element A1 to be screened and mini35s (82bp) are

(underlined is A1, bold italics is mini35s (82bp), TATA box and A1 are 75bp apart), other expression control elements are added in the same way as A1.

The plasmids containing the sequences to be screened were protoplast transformed, followed by GUS staining:

(1) 200ul protoplasts (approximately 4 x 10) per 10ug of plasmid were added⁵Cells), adding 220ul of newly-prepared PEG solution, uniformly mixing, and standing at room temperature in a dark place for 10-20 minutes to induce transformation;

(2) slowly adding 880ul of W5 solution after the induction transformation is finished, slightly reversing and uniformly mixing, horizontally centrifuging for 3 minutes at 250g, and removing supernatant;

(3) adding 1ml of WI solution for resuspension, transferring to a six-well plate (1 ml of WI solution is added in advance), and culturing for 6-16 hours at room temperature (or 28 ℃) in the dark. And taking out the protoplast, carrying out GUS staining on the protoplast, and selecting an element with transcription enhancing activity according to a staining result.

In fig. 2A-2F, the positive control is a Gus plasmid started by mini35S, the negative control is a Gus plasmid started by modified mini35S (82bp), the mini35S promoter originally can normally start expression of Gus gene, but the activity of the promoter is basically lost after modification. If more obvious blue color is observed after the expression regulatory element is added before the mini35S (82bp) after modification, the expression regulatory element is proved to have the enhancement activity.

As shown in FIG. 2A, A2, A5, A7 and A8 can significantly improve GUS expression.

As shown in FIG. 2B, B2 and B4 can significantly improve GUS expression.

As shown in fig. 2C, C1, C4, C7, C8, and D4 could significantly increase GUS expression.

As shown in fig. 2D, E1 can significantly increase GUS expression.

As shown in fig. 2E, F3, F4, F5, and F8 significantly increased GUS expression.

As shown in fig. 2F, G1, G3, G7 could significantly increase GUS expression.

In conclusion, through the above experiments, 19 expression regulatory elements capable of enhancing GUS expression were screened, including A2, A5, A7, A8, B2, B4, C1, C4, C7, C8, D4, E1, F3, F4, F5, F8, G1, G3 and G7, and the sequences are shown in sequence as SEQ ID NO 2, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 17, SEQ ID NO 20, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 28, SEQ ID NO 33, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 51 or SEQ ID NO 55.

Example 2 further validation of 19 sequences selected

The expression regulatory elements D4, E1, F4, F5 and F8 in the above examples were selected and linked to the mCherry fluorescence group started by mini35(82bp), and the vector also included the GFP gene started by 35S promoter, to constitute a relative fluorescence measurement vector. The expression regulatory element to be verified was linked to the vector in the manner shown in fig. 3, and the enhancer was linked to mini35s (82bp) in the same manner as in example 1, and 5 constructed vectors for relative fluorescence measurement and a negative control (not including the expression regulatory element) were transformed into protoplasts, and after 24 hours of transformation, the vector was measured under fluorescence, and the relative fluorescence intensity was measured using the normalized GFP fluorescence intensity as a reference, and the results are shown in table 2: the illustrated enhancer is 5 expression regulatory element sequences to be determined, the Mini35S is a promoter Mini35S (82bp) with weak transcriptional activation capability after being modified, the mCherry is a red fluorescent group, the 2X 35s is a promoter with normal transcriptional activation capability, the GPF is green fluorescent protein, and the Nos and E9 are common terminators.

Relative fluorescence intensity of FmCherry/FGFP

FmCherry is the average of the luminescence intensity of all red fluorescent proteins (mCherry) in three different fields of view; FGFP is the average of the luminescence intensity of all Green Fluorescent Protein (GFP) in three different fields.

TABLE 2 relative fluorescence intensities of D4, E1, F4, F5 and F8

Numbering	Expression regulatory element names	Relative fluorescence intensity (1X)
			1	D4	1.84
2	E1	2.02
			3	F4	1.96
4	F5	1.98
			5	F8	2.00
6	Is free of	0

As shown in Table 2, the vectors containing the expression regulatory elements D4, E1, F4, F5 and F8 all measured significantly high mCherry fluorescence and higher relative fluorescence intensity compared with the vectors not containing the expression regulatory elements, which proves that the 5 expression regulatory elements have stronger effect of improving the expression of the target gene.

Example 3 validation of the Effect of multiple copies of expression regulatory elements

And respectively constructing vector dual-fluorescence detection vectors containing 3 copies of F4 and 3 copies of F5 expression regulatory elements. Each expression control element is connected end to end and inserted into 75bp upstream of a mini35S (82bp) promoter. After inserting 3 copies of F4, the sequences of the mini35S (82bp) promoter and enhancer are:

(underlined are 3 copies of F4, bold italics are mini35s (82bp), the TATA box and the downstream-most F4 are 75bp apart), 3 copies of F5 expression control elements are added in the same way as F4.

And (4) transforming the protoplast. After 24 hours, the relative fluorescence intensity was measured under fluorescence with the normalized GFP fluorescence intensity as a reference, and the relative fluorescence intensity was significantly improved after increasing the number of expression regulatory elements and was more potent than that of 1 copy of the expression regulatory element, with the results shown in Table 3.

Table 3 relative fluorescence intensities of F4 and F5

Example 4 verification of the Effect of the expression regulatory element of the present invention on enhancing the Effect of other promoters

Amplifying a PUN1 core promoter in 319bp pepper, fusing F4, F5 and F8 at the upstream 71bp of a TATA box of a promoter of a capsaicin synthase gene PUN1 by BstEII enzyme digestion, wherein the connected promoter and enhancer sequence is

(underlined 3 copies of F4, bold italics, the PUN1 core promoter); other expression regulatory elements were added in the same manner as described above. Then, the modified promoter is fused with mCBRy to form pCAMBIA1301-pPUN1(enhancer), mCBRy-Tnos-35S and GFP-TE9 double-fluorescence vector. The vector was transferred to pepper placenta protoplasts by a PEG-mediated method, and the relative fluorescence intensity was measured and calculated.

The experimental result shows that after the mCherry is connected with the expression regulation element, the expression level of the mCherry is improved compared with a control without the expression regulation element.

Example 5 verification of the Effect of Gene expression control of the expression control element of the present invention in stably transformed plants

Arabidopsis thaliana is respectively transformed by an inflorescence infection method through pCAMBIA1301-mini35S (enhancer) carrying F4, F5 and F8 expression control elements, GUS vectors, pCAMBIA1301-mini35S, GUS negative control, pCAMBIA1301-35S and GUS positive control. GUS staining is carried out on transgenic arabidopsis thaliana of T1 generation, and the F4, F5 and F8 elements are further determined to have better activity of expression regulatory elements.

The result is shown in fig. 4, WT is not treated, Gus is not developed, a common 35S promoter is used as a positive control, Gus is developed obviously, modified mini35S is a negative control, Gus is not developed, a certain degree of Gus development can be observed by adding an F4 expression regulatory element, an F5 expression regulatory element and an F8 expression regulatory element in a modified mini35 regulatory region, and the connection mode is consistent as in example 1, which indicates that the F4 expression regulatory element, the F5 expression regulatory element and the F8 expression regulatory element all have the function of enhancing the promoter activity.

Example 6 insertion of an expression control element of the present invention in front of a target Gene by means of Gene editing

The expression regulation and control element is inserted into the regulation region of the target gene in a gene editing mode, and the expression quantity of the gene is detected in an RT-PCR and western mode, so that the increase of the expression quantity can be obviously detected.

Sequence listing

<110> Shunheng Biotech Co., Ltd

<120> expression regulatory element and application thereof

<130> JH-CNP201702DJ

<160> 64

<170> PatentIn version 3.5

<210> 1

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> A1

<400> 1

acgtaagcga tgacgt 16

<210> 2

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> A2

<400> 2

acgtaagcaa tgacgt 16

<210> 3

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> A3

<400> 3

acgtaagcac tgacgt 16

<210> 4

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> A4

<400> 4

acgtaagcgc tgacgt 16

<210> 5

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> A5

<400> 5

acgtaagcga ttacgt 16

<210> 6

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> A6

<400> 6

acgtaagcaa ttacgt 16

<210> 7

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> A7

<400> 7

acgtaagcac ttacgt 16

<210> 8

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> A8

<400> 8

acgtaagcgc ttacgt 16

<210> 9

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> B1

<400> 9

acgtaaggga tgacgt 16

<210> 10

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> B2

<400> 10

acgtaaggaa tgacgt 16

<210> 11

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> B3

<400> 11

acgtaaggac tgacgt 16

<210> 12

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> B4

<400> 12

acgtaagggc tgacgt 16

<210> 13

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> B5

<400> 13

acgtaaggga ttacgt 16

<210> 14

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> B6

<400> 14

acgtaaggaa ttacgt 16

<210> 15

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> B7

<400> 15

acgtaaggac ttacgt 16

<210> 16

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> B8

<400> 16

acgtaagggc ttacgt 16

<210> 17

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> C1

<400> 17

acgcaagcga tgacgt 16

<210> 18

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> C2

<400> 18

acgcaagcaa tgacgt 16

<210> 19

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> C3

<400> 19

acgcaagcac tgacgt 16

<210> 20

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> C4

<400> 20

acgcaagcgc tgacgt 16

<210> 21

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> C5

<400> 21

acgcaagcga ttacgt 16

<210> 22

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> C6

<400> 22

acgcaagcaa ttacgt 16

<210> 23

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> C7

<400> 23

acgcaagcac ttacgt 16

<210> 24

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> C8

<400> 24

acgcaagcgc ttacgt 16

<210> 25

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> D1

<400> 25

acgcaaggga tgacgt 16

<210> 26

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> D2

<400> 26

acgcaaggaa tgacgt 16

<210> 27

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> D3

<400> 27

acgcaaggac tgacgt 16

<210> 28

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> D4

<400> 28

acgcaagggc tgacgt 16

<210> 29

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> D5

<400> 29

acgcaaggga ttacgt 16

<210> 30

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> D6

<400> 30

acgcaaggaa ttacgt 16

<210> 31

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> D7

<400> 31

acgcaaggac ttacgt 16

<210> 32

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> D8

<400> 32

acgcaagggc ttacgt 16

<210> 33

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> E1

<400> 33

acgtaagcga tgacgc 16

<210> 34

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> E2

<400> 34

acgtaagcaa tgacgc 16

<210> 35

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> E3

<400> 35

acgtaagcac tgacgc 16

<210> 36

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> E4

<400> 36

acgtaagcgc tgacgc 16

<210> 37

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> E5

<400> 37

acgtaagcga ttacgc 16

<210> 38

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> E6

<400> 38

acgtaagcaa ttacgc 16

<210> 39

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> E7

<400> 39

acgtaagcac ttacgc 16

<210> 40

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> E8

<400> 40

acgtaagcgc ttacgc 16

<210> 41

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> F1

<400> 41

acgtaaggga tgacgc 16

<210> 42

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> F2

<400> 42

acgtaaggaa tgacgc 16

<210> 43

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> F3

<400> 43

acgtaaggac tgacgc 16

<210> 44

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> F4

<400> 44

acgtaagggc tgacgc 16

<210> 45

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> F5

<400> 45

acgtaaggga ttacgc 16

<210> 46

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> F6

<400> 46

acgtaaggaa ttacgc 16

<210> 47

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> F7

<400> 47

acgtaaggac ttacgc 16

<210> 48

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> F8

<400> 48

acgtaagggc ttacgc 16

<210> 49

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> G1

<400> 49

acgcaagcga tgacgc 16

<210> 50

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> G2

<400> 50

acgcaagcaa tgacgc 16

<210> 51

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> G3

<400> 51

acgcaagcac tgacgc 16

<210> 52

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> G4

<400> 52

acgcaagcgc tgacgc 16

<210> 53

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> G5

<400> 53

acgcaagcga ttacgc 16

<210> 54

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> G6

<400> 54

acgcaagcaa ttacgc 16

<210> 55

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> G7

<400> 55

acgcaagcac ttacgc 16

<210> 56

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> G8

<400> 56

acgcaagcgc ttacgc 16

<210> 57

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> H1

<400> 57

acgcaaggga tgacgc 16

<210> 58

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> H2

<400> 58

acgcaaggaa tgacgc 16

<210> 59

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> H3

<400> 59

acgcaaggac tgacgc 16

<210> 60

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> H4

<400> 60

acgcaagggc tgacgc 16

<210> 61

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> H5

<400> 61

acgcaaggga ttacgc 16

<210> 62

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> H6

<400> 62

acgcaaggaa ttacgc 16

<210> 63

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> H7

<400> 63

acgcaaggac ttacgc 16

<210> 64

<211> 16

<212> DNA

<213> Artificial Sequence

<220>

<223> H8

<400> 64

acgcaagggc ttacgc 16

Claims

1. An expression regulatory element comprising a polynucleotide sequence as set forth in any of SEQ ID NO 2, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 17, SEQ ID NO 20, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 28, SEQ ID NO 33, SEQ ID NO 43, SEQ ID NO 44, SEQ ID NO 45, SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 51 or SEQ ID NO 55, or comprising a polynucleotide sequence as set forth in any of SEQ ID NO 2, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 17, SEQ ID NO 20, SEQ ID NO 55, 23, 24, 28, 33, 43, 44, 45, 48, 49, 51 or 55.

2. A nucleic acid construct comprising the expression control element of claim 1 operably linked to a regulatory element or gene of interest.

3. A vector comprising the expression control element of claim 1 or the nucleic acid construct of claim 2.

4. A host cell comprising the expression control element of claim 1, the nucleic acid construct of claim 2, or the vector of claim 3.

5. A method for regulating expression of a target gene, comprising the step of regulating expression of the target gene using the expression control element according to claim 1.

6. A method of regulating expression of a target gene in a plant cell, the method comprising the step of regulating expression of the target gene using the expression regulatory element of claim 1.

7. A method of producing a plant cell, plant tissue, plant part, or plant with increased expression of a gene of interest, the method comprising the step of increasing expression of the gene of interest in the plant cell, plant tissue, plant part, or plant using the expression control element of claim 1.

8. The method according to any one of claims 5 to 7, comprising the step of introducing the expression control element according to claim 1 into a regulatory region of a target gene.

9. The method of claim 8, wherein the introduction of the expression control element of claim 1 into the regulatory region of the target gene is achieved by any one of:

(1) comprising the expression control element of claim 1 in a regulatory region of a target gene by editing 1 or more nucleic acids in the regulatory region;

(2) inserting the expression regulatory element of claim 1 into a regulatory region of a target gene such that the expression regulatory element of claim 1 is contained in the regulatory region.

10. Use of the expression control element of claim 1, the nucleic acid construct of claim 2, the vector of claim 3, or the host cell of claim 4 in any one or any of a) to e) below:

a) the expression of the gene is regulated and controlled,

b) preparing a cell with an increased expression level of a target gene,

c) preparing plant cells, plant tissues, plant parts or plants with increased expression of the target gene,

d) preparing a reagent or a kit for improving gene expression,

e) preparing a reagent or a kit for increasing the expression level of a target gene in a plant cell, a plant tissue or a plant.