WO2022089458A1 - Expression regulatory element and use thereof - Google Patents

Abstract

The present invention provides an expression regulatory element and a use thereof, and relates in particular to an expression regulatory element and a use thereof in regulating the expression of one or more genes in a cell. Further disclosed is a use of an expression regulatory element in plant breeding. Further disclosed is a method for improving the expression of a target gene by using an expression regulatory element of the invention. The method comprises: operably linking an expression regulatory element of the present invention to a target gene, so as to improve the expression of the target gene.

Description

An expression control element and its application technical field

The invention belongs to the field of biotechnology, and in particular relates to an expression control element and its application in regulating gene expression.

Background technique

In the process of crop genetic improvement, how to improve the expression level of some genes is one of the important issues that needs to be solved. A cis-acting element refers to a specific DNA sequence in series with a structural gene, which is the binding site of a transcription factor and regulates gene transcription by binding to a transcription factor. A cis-element that enhances transcription upon binding to a transcription factor is called an enhancer. By adding an enhancer to the regulatory region of the target gene, it may be possible to effectively increase the expression of the target gene without significantly changing the gene expression pattern.

The OCS gene encodes octopine synthase in Agrobacterium, and its promoter part has TATA box and CAAT box similar to eukaryotic promoters, which can be ubiquitously expressed in plant cells and has the characteristics of plant promoters. There is an important 16bp palindromic sequence (ACGTAAGCGCTTACGT) in the OCS promoter part, which has significant enhancer activity. After analyzing other strong promoter sequences widely used in plants, it was found that there are other expression control element sequences with certain homology to OCS. According to the characteristics of these sequences, the OCS elements were modified, in order to screen for genes that can improve the level of gene expression. Novel expression regulatory elements.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide an expression control element and its application. By changing the wild-type enhancer sequence, the present invention designs 64 expression control elements to be screened, connects to the expression vector for screening, and obtains 19 kinds of expression control elements with enhanced functions. These 19 kinds of expression control elements can improve the level of gene expression. have a significant effect.

expression regulatory element

In one aspect, the present invention provides an expression control element comprising the nucleotide sequence shown in any of SEQ ID NO.: 1-64 or a nucleotide sequence complementary thereto.

In one embodiment, the expression control element comprises 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 Polynucleotide sequence; preferably, the polynucleotide shown in any of SEQ ID NO.:28, SEQ ID NO.:33, SEQ ID NO.:44, SEQ ID NO.:45 or SEQ ID NO.:48 sequence.

In one embodiment, the expression control element is 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 Polynucleotide sequence; preferably, the polynucleotide shown in any of SEQ ID NO.:28, SEQ ID NO.:33, SEQ ID NO.:44, SEQ ID NO.:45 or SEQ ID NO.:48 sequence.

In one embodiment, the expression regulatory element is associated with 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 The homology of ≥80% (preferably ≥90%, more preferably ≥95%, ≥96%, ≥97%, ≥98%, optimally ≥99%).

In one embodiment, the expression control element is the same as SEQ ID NO.:2, SEQ ID NO.:5, SEQ ID NO.:7, SEQ ID NO.:8, SEQ ID NO.:10, SEQ ID NO.:10, SEQ ID NO.:10, SEQ ID NO. 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 sequence-complementary polynucleotides.

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

nucleic acid construct

In another aspect, the present invention provides a nucleic acid construct comprising the aforementioned expression regulatory element and a regulatory element or target gene operably linked thereto.

In one embodiment, the regulatory element is selected from one or more of the group consisting of promoter, enhancer, intron, 5'-untranslated region (5'-UTR, also known as leader sequence), 3'-UTR, transposon, terminator, polyadenylation sequence, marker gene, or a combination thereof.

In one embodiment, the gene of interest comprises herbicide tolerance, insect resistance, disease resistance, abiotic stress tolerance, yield stability, increased yield, carbohydrate metabolism, fatty acid metabolism, amino acid metabolism , Plant development, plant growth regulation, drought resistance, cold resistance, heat resistance, nutrient use efficiency, nitrogen use efficiency and salt resistance genes, microRNAs (small RNAs) and small RNA precursors.

carrier

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

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

In one embodiment, the gene of interest comprises herbicide tolerance, insect resistance, disease resistance, abiotic stress tolerance, yield stability, increased yield, carbohydrate metabolism, fatty acid metabolism, amino acid metabolism , Plant development, plant growth regulation, drought resistance, cold resistance, heat resistance, nutrient use efficiency, nitrogen use efficiency and salt resistance genes, microRNA (small RNA) and small RNA precursors.

In one embodiment, the vectors include cloning vectors, expression vectors, shuttle vectors, and integration vectors.

In one embodiment, the expression vector further contains at least one origin of replication to achieve 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 into which it is integrated.

Vectors can be of the type 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 present invention provides a host cell containing one or more of the aforementioned expression control elements, nucleic acid constructs and vectors.

In one embodiment, the aforementioned expression control element is integrated into the genome of the host cell.

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

In one embodiment, the host cells are eukaryotic cells, including animal cells, plant cells, fungi, etc.; preferably, the eukaryotic cells are plant cells, and preferably, the plants include angiosperms and gymnosperms .

In one embodiment, the plants include monocotyledonous and dicotyledonous plants.

In one embodiment, the plants include herbaceous and woody plants.

In one embodiment, the plant comprises Arabidopsis, tobacco, rice, corn, sorghum, barley, wheat, millet, soybean, tomato, potato, quinoa, lettuce, canola, cabbage, strawberry.

plant

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

The present invention also provides a plant with increased expression of a target gene, wherein the regulatory region of the target gene comprises the expression control element of the present invention.

The expression control element can be obtained by changing one or more nucleotides in the regulatory region of the target gene, or can be introduced by gene editing, or inserted into the regulatory region of the target gene. Expression regulatory elements are obtained.

method

The present invention also provides a method for regulating the expression of a target gene, the method comprising the step of regulating the expression of the target gene using the expression control element of the present invention.

In a preferred embodiment, the regulation of target gene expression is to increase target gene expression.

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

In one embodiment, the gene of interest comprises herbicide tolerance, insect resistance, disease resistance, abiotic stress tolerance, yield stability, increased yield, carbohydrate metabolism, fatty acid metabolism, amino acid metabolism , Plant development, plant growth regulation, drought resistance, cold resistance, heat resistance, nutrient use efficiency, nitrogen use efficiency and salt resistance genes, microRNA (small RNA) and small RNA precursors.

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

The term "regulatory region" generally refers to regulatory elements, such as promoters, enhancers, introns, 5'-untranslated regions (5'- UTR, also known as leader sequence) or 3'-UTR, transposon, region where terminator is located, or other non-coding region.

In a preferred embodiment, the regulatory region is the region where the promoter is located 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 following manner: by editing one or more nucleic acids in the regulatory region of the target gene, the regulatory region includes the expression regulatory element of the present invention Alternatively, the expression regulatory element of the present invention is inserted into the regulatory region of the target gene so that the regulatory region includes the expression regulatory element of the present invention. The editing includes changes, deletions, insertions and the like to nucleic acids.

In one embodiment, the expression regulatory element of the present invention is introduced into the regulatory region of the target gene, which can be achieved by homologous recombination, gene editing (eg, CRISPR technology, TALEN technology, ZFN technology).

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

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

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

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

In one embodiment, the expression control element can also be set in the enhancer, intron, 5'-untranslated region (5'-UTR, also called leader sequence) or 3'-UTR, Transposons, terminator regions or within genes.

In the present invention, the same expression control element or a combination of different types of expression control elements can be used to control the expression level of the target gene; the same expression control element can be one copy of the expression control element, or multiple copies of the expression control element. Expression regulatory elements.

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

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, there may be spacer sequences between multiple copies of the same or different expression control elements, and the spacer sequences may be 1-200 nucleotides, preferably 1-50 nucleotides.

In one embodiment, multiple copies of the same or different expression regulatory elements can 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 the expression of a target gene in plant cells, the method comprising the step of regulating the expression of the target gene using the expression control element of the present invention.

In a preferred embodiment, the modulation is to increase the expression of the target gene.

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

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

In another aspect, the present invention also provides a method for preparing a plant cell, plant tissue, plant part or plant with increased expression of a target gene, the method comprising utilizing the present invention in the plant cell, plant tissue, plant part or plant The expression regulatory element steps that increase the expression of the target gene.

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

In another aspect, the present invention also provides plant cells, plant tissues, plant parts or plants prepared by the aforementioned methods.

use

In another aspect, the present invention provides the use of the aforementioned expression regulatory elements, nucleic acid constructs or vectors in regulating gene expression. Preferably, the regulation of gene expression is to increase 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 cells with increased target gene expression; 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 elements, nucleic acid constructs or vectors in the preparation of plant cells, plant tissues, plant parts or plants with increased target 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 enhancing 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 plant cells, plant tissues or plants.

General Definition

Unless otherwise defined, 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 sequence," "nucleic acid molecule," and "nucleic acid" are used interchangeably and include DNA, RNA, or hybrids thereof, which may be double-stranded or single-stranded.

The terms "homology" or "identity" are used to refer to the matching of sequences between two polypeptides or between two nucleic acids. Accordingly, the compositions and methods of the present invention also comprise the nucleotide sequences and polypeptide sequences of the present invention (eg, 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 Homologue of SEQ ID NO.:55). "Homology" can be calculated by known methods including, but not limited to, Computational Molecular Biology (Lesk, A.M. ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects [Biocomputing: Informatics and Genome Projects] (Smith, D.W. ed.) Academic Press [Academic Press], New York (1993); Computer Analysis of Sequence Data, Part I [Computer Analysis of Sequence Data, Part I Section] (edited by Griffin, A.M. and Griffin, H.G.) Humana Press [Humana Press], New Jersey (1994); Sequence Analysis in Molecular Biology] (edited by von Heinje, G.) Academic Press [Academic Press] (1987); and Sequence Analysis Primer (edited by Gribskov, M. and Devereux, J.) Stockton Press [Stockton Press], New York (1991).

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

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

The term "regulatory element" may function in "cis" or "trans", and is usually "cis", ie it activates the expression of a gene located on the same nucleic acid molecule (eg, chromosome) as the regulatory element. Regulatory elements include promoters, enhancers, introns, 5'-untranslated regions (5'-UTRs, also known as leader sequences) or 3'-UTRs, transposons or terminators.

The term "regulatory region" generally includes regulatory elements such as promoters, enhancers, introns, 5'-untranslated regions (5'-UTRs) involved in regulating the transcription of nucleic acid molecules (eg, genes or target genes). , also known as the leader sequence) or 3'-UTR, transposons, regions where terminators are located, or other noncoding regions.

As used herein, the term "promoter" has the meaning well known to those skilled in the art and refers to a non-coding nucleotide sequence located upstream of a gene that initiates expression of a downstream gene. A constitutive promoter is a nucleotide sequence which, when operably linked to a polynucleotide encoding or defining the gene product, results in the gene product in a cell under most or all physiological conditions of the cell production. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in substantially only when an inducer corresponding to the promoter is present in a cell. The gene product is produced intracellularly. A tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in substantially only when the cell is of the tissue type to which the promoter corresponds. The gene product is produced in the cell.

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 (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" includes elements that allow 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 elements that ensure 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 depends on methods well known to those skilled in the art for transforming host cells. For example, plasmid vectors can be used.

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

The term "plant" is to be understood as any differentiated multicellular organism capable of photosynthesis, including crop plants at any stage of maturity or development, in particular monocotyledonous or dicotyledonous plants, vegetable crops including artichokes, cabbage, Arugula, leeks, asparagus, lettuce (eg, head lettuce, leaf lettuce, romaine), bok choy, yellow taro, melons (eg, melon, watermelon, crenshaw ), white melon, romaine melon), canola crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, kale, Chinese cabbage, pak choi), spinach, carrots, napa, Okra, onions, celery, parsley, chickpeas, parsnips, chicory, peppers, potatoes, gourds (e.g., zucchini, cucumbers, zucchini, zucchini, squash), radishes, dried bulb onions, rutabagas, Purple eggplant (also called eggplant), salsify, endive, shallots, endive, garlic, spinach, green onions, zucchini, greens, beets (sugar beets and fodder beets), sweet potatoes, chard, Wasabi, tomatoes, turnips, and spices; fruits and/or trailing crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quinces, almonds, chestnuts, hazelnuts, pecans, Pistachios, walnuts, citrus, blueberries, boysenberry, cranberries, currant, loganberry, raspberry, strawberry, blackberry, grape, avocado, banana, kiwi, persimmon, pomegranate, pineapple , tropical fruits, pomes, melons, mangoes, papayas, and lychees; field crops such as clover, alfalfa, evening primrose, jasmine, corn/maize (fodder corn, sweet corn, popcorn), hops, lotus Barley, peanuts, rice, safflower, small grain crops (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas, soybeans), oily plants (rapese, mustard, poppy, olive, sunflower, 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 flower bed plants, such as flowering plants, cacti, succulents and/or ornamentals, and trees such as forests (broadleaf and evergreen, such as conifers), fruit trees, ornamental Trees, and nut-bearing trees, as well as shrubs and other seedlings.

The term "plant tissue" or "plant part" includes plant cells, protoplasts, plant tissue cultures, plant callus, plant pieces as well as plant embryos, pollen, ovules, seeds, leaves, stems, flowers, shoots, seedlings, fruits , nucleus, ear, root, root tip, anther, etc.

The term "plant cell" is to be understood as 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, components of plants, 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 regularly interspaced short palindromic repeats), which are derived from the immune system of microorganisms. Among them, gene editing tools include guideRNA, Cas proteins (such as Cas9, Cpf1, Cas12b, etc.). Gene editing tools referred to in TALEN technology are restriction enzymes that can cut specific DNA sequences and include a TAL effector DNA binding domain and a DNA cleavage domain. Gene editing tools referred to in ZFN technology are also restriction enzymes that can cut specific DNA sequences, including a zinc finger DNA binding domain and a DNA cleavage domain. It is well known to those skilled in the art that by constructing nucleotides encoding gene editing tools and other regulatory elements in a suitable vector, and then transforming cells, the editing of the intracellular genome can be achieved, and the types of editing include gene knockout, insertion , base editing.

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, WI, USA) pQE70 , pQE60, pQE-9 (Qiagen), pD10, psiX174, pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene), ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pKK232-8, pCM7, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, pBPV, pMSG, and pSVL (Pharmacia), among others.

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

In the production method of the present invention, the cells are cultured on a nutrient medium suitable for the 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 medium, it can be recovered from cell lysates.

Table 1. Sequences of different expression regulatory elements

seq ID	name		sequence
1	A1	ACGTAAGCGATGACGT
2	A2	ACGTAAGCAATGACGT
3	A3	ACGTAAGCACTGACGT
4	A4	ACGTAAGCGCTGACGT
5	A5	ACGTAAGCGATTACGT
6	A6	ACGTAAGCAATTACGT
7	A7	ACGTAAGCACTTACGT
8	A8	ACGTAAGCGCTTACGT
9	B1	ACGTAAGGGATGACGT
10	B2	ACGTAAGGAATGACGT
11	B3	ACGTAAGGACTGACGT

12	B4	ACGTAAGGGCTGACGT
13	B5	ACGTAAGGGATTACGT
14	B6	ACGTAAGGAATTACGT
15	B7	ACGTAAGGACTTACGT
16	B8	ACGTAAGGGCTTACGT
17	C1	ACGCAAGCGATGACGT
18	C2	ACGCAAGCAATGACGT
19	C3	ACGCAAGCACTGACGT
20	C4	ACGCAAGCGCTGACGT
twenty one	C5	ACGCAAGCGATTACGT
twenty two	C6	ACGCAAGCAATTACGT
twenty three	C7	ACGCAAGCACTTACGT
twenty four	C8	ACGCAAGCGCTTACGT
25	D1	ACGCAAGGGATGACGT
26	D2	ACGCAAGGAATGACGT
27	D3	ACGCAAGGACTGACGT
28	D4	ACGCAAGGGCTGACGT
29	D5	ACGCAAGGGATTACGT
30	D6	ACGCAAGGAATTACGT
31	D7	ACGCAAGGACTTACGT
32	D8	ACGCAAGGGCTTACGT
33	E1	ACGTAAGCGATGACGC
34	E2	ACGTAAGCAATGACGC
35	E3	ACGTAAGCACTGACGC
36	E4	ACGTAAGCGCTGACGC
37	E5	ACGTAAGCGATTACGC
38	E6	ACGTAAGCAATTACGC
39	E7	ACGTAAGCACTTACGC
40	E8	ACGTAAGCGCTTACGC
41	F1	ACGTAAGGGATGACGC
42	F2	ACGTAAGGAATGACGC
43	F3	ACGTAAGGACTGACGC
44	F4	ACGTAAGGGCTGACGC

45	F5	ACGTAAGGGATTACGC
46	F6	ACGTAAGGAATTACGC
47	F7	ACGTAAGGACTTACGC
48	F8	ACGTAAGGGCTTACGC
49	G1	ACGCAAGCGATGACGC
50	G2	ACGCAAGCAATGACGC
51	G3	ACGCAAGCACTGACGC
52	G4	ACGCAAGCGCTGACGC
53	G5	ACGCAAGCGATTACGC
54	G6	ACGCAAGCAATTACGC
55	G7	ACGCAAGCACTTACGC
56	G8	ACGCAAGCGCTTACGC
57	H1	ACGCAAGGGATGACGC
58	H2	ACGCAAGGAATGACGC
59	H3	ACGCAAGGACTGACGC
60	H4	ACGCAAGGGCTGACGC
61	H5	ACGCAAGGGATTACGC
62	H6	ACGCAAGGAATTACGC
63	H7	ACGCAAGGACTTACGC
64	H8	ACGCAAGGGCTTACGC

The main advantages of the present invention:

1. The present invention screened out a group of expression control elements.

2. Connecting the expression control element of the present invention to the target gene can significantly increase the expression level of the target gene.

Description of drawings

Figure 1. The composition of the screening vector, the enhancer is the 64 expression control element sequences to be screened, Mini35S is the modified promoter with weak transcriptional activation ability, and GUS is the β-glucuronidase gene (β-glucuronidase) .

In Figures 2A-2F, the positive control is the Gus plasmid started with mini35S, and the negative control is the Gus plasmid started with the modified mini35S.

Figure 2A is an assay for the activity of A1-A8 expression regulatory elements.

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

Figure 2C is the detection of the activity of C1-C8, D1-D8 expression regulatory elements.

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

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

Figure 2F is the detection of G1-G8, H1-H8 expression regulatory element activity.

Figure 3. The composition of the relative fluorescence measurement vector, the enhancer is the sequence of the five expression control elements to be determined, Mini35S is a modified promoter with weak transcriptional activation ability, mCherry is a red fluorescent group, and 2×35s is normal The promoter of transcriptional activation ability, GPF is green fluorescent protein, Nos and E9 are common terminators.

Figure 4. Gus staining of transformed seedlings stably containing plasmids with different expression regulatory elements. WT did not do any treatment of Arabidopsis thaliana seedlings, mini35S is a transformed seedling with the transformed mini35S promoter as the promoter, and it is a negative control; 35S is a transformed seedling with the 35S promoter as a promoter, which is a positive control (Fig. "1301"); the other 3 transformed seedlings were respectively added with F4 expression control elements, F5 expression control elements and F8 expression control elements in the modified mini35 regulatory region to verify the F4 expression control elements, F5 expression control elements and F8 expression control elements function of the element.

Implementation

The present invention will be further described below in conjunction with the embodiments. The following descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention in other forms. Changes to equivalent embodiments with equivalent changes. Any simple modifications or equivalent changes made to the following embodiments according to the technical essence of the present invention without departing from the content of the solution of the present invention fall within the protection scope of the present invention.

Example 1. Screening of 64 sequences

The pCAMBIA1301-mini35S(82bp)-GUS screening plasmid was constructed, in which the mini35S(82bp) promoter has weak transcriptional activation ability. After the regulatory region of mini35s (82bp) is connected to the screened expression regulatory element, if the expression level of GUS is significantly increased, it indicates that the expression regulatory element has the ability to improve gene expression.

The wild-type enhancer sequence was expanded to 64 according to the sequence of its non-conserved site (the group number is AH, a total of 8 groups, each group is numbered 1-8 in sequence, the names and sequences are shown in Table 1). The pCAMBIA1301-mini35S(82bp)-GUS screening plasmids connected to 64 expression control element sequences constitute 64 different plasmids to be screened. The sequence after connecting with mini35s (82bp) is

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

Protoplast transformation was performed on the plasmid containing the sequence to be screened, followed by GUS staining:

(1) For every 10ug of the corresponding plasmid, add 200ul of protoplasts (about ⁴ *105 cells), then add 220ul of the newly prepared PEG solution, mix well, and place at room temperature in the dark for 10-20 minutes to induce transformation;

(2) Slowly add 880ul of W5 solution after induced transformation, gently invert and mix, centrifuge horizontally at 250g for 3 minutes, and discard the supernatant;

(3) Add 1ml of WI solution to resuspend, transfer to a six-well plate (1ml of WI solution has been added in advance) and cultivate in the dark at room temperature (or 28°C) for 6-16 hours. After removal, the protoplasts were stained with GUS, and elements with transcriptional enhancing activity were selected according to the staining results.

In Figures 2A-2F, the positive control is the Gus plasmid started with mini35S, and the negative control is the Gus plasmid started with the modified mini35S (82bp). The mini35S promoter can normally start the expression of the Gus gene, but it is basically lost after the transformation. promoter activity. If a more obvious blue color is observed after adding the expression control element before the modified mini35S (82bp), it indicates that the expression control element does have enhanced activity.

As shown in Figure 2A, A2, A5, A7, and A8 could significantly increase the expression of GUS.

As shown in Figure 2B, B2 and B4 could significantly increase the expression of GUS.

As shown in Figure 2C, C1, C4, C7, C8, D4 could significantly increase the expression of GUS.

As shown in Figure 2D, E1 could significantly increase the expression of GUS.

As shown in Figure 2E, F3, F4, F5, and F8 could significantly increase the expression of GUS.

As shown in Figure 2F, G1, G3, and G7 could significantly increase the expression of GUS.

In summary, through the above experiments, 19 expression regulatory elements that can enhance the expression of GUS were screened, A2, A5, A7, A8, B2, B4, C1, C4, C7, C8, D4, E1, F3, F4, F5, F8, G1, G3, G7, 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 verification of the 19 sequences screened out

The expression control elements D4, E1, F4, F5 and F8 in the above-mentioned embodiment are selected and connected with the mCherry fluorophore started with mini35 (82bp) respectively, and the GFP gene started with the 35S promoter is also included on the carrier, forming a relative Fluorescence measurement carrier. The expression control element to be verified is connected to the vector in the manner shown in Figure 3, and the connection between the enhancer and mini35s (82bp) is the same as that in Example 1, and five constructed relative fluorescence measurement vectors and a negative control ( Protoplast transformation was performed 24 hours after transformation, and the relative fluorescence intensity was measured with the normalized GFP fluorescence intensity as a reference. The results are shown in Table 2: The sequences of the five expression control elements determined, Mini35S is the modified promoter Mini35S (82bp) with weak transcriptional activation ability, mCherry is the red fluorescent group, 2X 35s is the promoter with normal transcriptional activation ability, GPF is the green fluorescent protein , Nos and E9 are common terminators.

Relative fluorescence intensity = FmCherry/FGFP

FmCherry is the average of all red fluorescent protein (mCherry) luminescence intensities in three different fields of view; FGFP is the average of all green fluorescent protein (GFP) luminescence intensities in three different fields of view.

Table 2. Relative fluorescence intensities of D4, E1, F4, F5 and F8

Numbering	Expression regulatory element name	Relative fluorescence intensity (1×)
1	D4	1.84
2	E1	2.02
3	F4	1.96
4	F5	1.98
5	F8	2.00
6	none	0

As shown in Table 2, the vectors containing D4, E1, F4, F5 and F8 expression control elements have significantly higher mCherry fluorescence and higher relative fluorescence intensity than those without expression control elements, which proves that 5 Each expression regulatory element does have a strong effect on improving the expression of the target gene.

Example 3. Verification of the effect of multi-copy expression regulatory elements

The vector double fluorescence detection vector containing 3 copies of F4 and 3 copies of F5 expression control elements was constructed respectively. Each expression regulatory element was placed end to end and inserted 75 bp upstream of the mini35S (82 bp) promoter. After inserting 3 copies of F4, the sequences of the mini35S (82bp) promoter and enhancer are:

(The underlined is 3 copies of F4, the bold italic is marked with mini35s (82bp), the TATA box and the most downstream F4 are separated by 75bp), the 3 copies of F5 expression regulatory elements are added in the same way as F4.

Transform protoplasts. 24 hours later, measured under fluorescence, with the normalized GFP fluorescence intensity as a reference, to determine the relative fluorescence intensity, after increasing the number of expression regulatory elements, the relative fluorescence intensity was significantly improved, and the effect was better than that of one copy of the expression regulatory element. strong, and the results are shown in Table 3.

Table 3. Relative fluorescence intensity of F4 and F5

Example 4. Verification of the expression control element of the present invention enhancing the effect of other promoters

The PUN1 core promoter in 319bp pepper was amplified, and F4, F5 and F8 were respectively fused to the TATA box upstream of the promoter of the capsaicin synthase gene PUN1 by BstEII digestion. The connected promoter and enhancer sequences were

(The underlined is 3 copies of F4, and the bold italics is the PUN1 core promoter); other expression control elements are added in the same manner as above. Then the modified promoter was fused with mCherry to form pCAMBIA1301-pPUN1(enhancer)::mCherry-Tnos-35S::GFP-TE9 dual fluorescent vector. The vector was transformed into capsicum placenta protoplasts by PEG-mediated method, and the relative fluorescence intensity was measured and calculated.

The experimental results showed that the expression of mCherry was increased after the expression regulatory elements were connected, compared with the control without the expression regulatory elements.

Example 5. Verification of the expression regulation of the expression regulatory element of the present invention in stably transformed plants Effect

The pCAMBIA1301-mini35S(enhancer)::GUS vector carrying the expression regulatory elements of F4, F5 and F8, as well as the pCAMBIA1301-mini35S::GUS negative control and pCAMBIA1301-35S::GUS positive control, were transformed into Pseudomonas by inflorescence infection method. mustard. GUS staining was performed on T1 transgenic Arabidopsis, and it was further confirmed that F4, F5 and F8 elements have good expression regulation element activity.

The results are shown in Figure 4. WT did not do any treatment, no Gus color development, the positive control used the common 35S promoter, Gus color development was obvious, the modified mini35S was a negative control, no Gus color development, adjusted in the modified mini35 If the F4 expression control element, the F5 expression control element and the F8 expression control element are added to the region, a certain degree of Gus coloration can be observed. All have the function of enhancing promoter activity.

Example 6. Insertion of the expression control element of the present invention before the target gene by gene editing

The expression control element of the present invention is inserted into the regulatory region of the target gene by means of gene editing, and the expression level of the gene is detected by RT-PCR and western methods, and an increase in the expression level can be obviously detected.

Claims (10)

1. An expression control element, characterized in that the expression control element comprises 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, Any of 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 A polynucleotide sequence shown or a polynucleotide sequence complementary thereto.
2. A nucleic acid construct, characterized in that the nucleic acid construct contains the expression regulatory element of claim 1 and a regulatory element or target gene operably linked to it.
3. A vector, characterized in that the vector comprises the expression control element of claim 1 or the nucleic acid construct of claim 2.
4. A host cell, characterized in that the host cell contains 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 the expression of a target gene, characterized in that the method comprises the step of regulating the expression of the target gene by using the expression control element of claim 1 .
6. A method for regulating the expression of a target gene in a plant cell, the method comprising the step of regulating the expression of the target gene using the expression control element described in claim 1.
7. A method for preparing a plant cell, plant tissue, plant part or plant with increased expression of a target gene, the method comprising utilizing the expression control element according to claim 1 to improve the target gene in the plant cell, plant tissue, plant part or plant steps of gene expression.
8. The method according to any one of claims 5-7, wherein the method comprises the step of introducing the expression control element according to claim 1 in the regulatory region of the target gene.
9. The method of claim 8, wherein the expression control element of claim 1 is introduced into the regulatory region of the target gene, which is achieved by any of the following methods:

   (1) by editing one or more nucleic acids in the regulatory region of the target gene so that the regulatory region contains the expression control element of claim 1;

   (2) inserting the expression control element of claim 1 into the regulatory region of the target gene so that the expression control element of claim 1 is included in the regulatory region.
10. The expression control element according to claim 1, the nucleic acid construct according to claim 2, the vector according to claim 3 or the host cell according to claim 4 are in any one or any of the following aspects a)-e). the use of:

    a) regulate gene expression,

    b) preparing cells with increased target gene expression,

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

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

    e) Preparation of reagents or kits for increasing the expression level of target genes in plant cells, plant tissues or plants.

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