Method for targeted inhibition of gene expression and application

Abstract

The present invention provides a method for conditionally regulating gene expression, comprising: introducing a miRNA target sequence into a non-coding region of a target gene in a plant cell, the non-coding region comprising a 5 'UTR, 3' UTR or intron region.

Description

A method for targeted inhibition of gene expression and its application Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular, to a method and application for targeted inhibition of gene expression, and in particular, to a method and application for conditionally reducing target gene expression by introducing a specific miRNA target sequence into a target gene region through genome editing technology. Background Art

[0002] Gene expression regulation is a crucial mechanism for maintaining homeostasis in cells and living systems, and is also closely related to biological development and the occurrence of disease. Artificially regulating gene expression is a crucial tool for studying gene function and a fundamental approach for developing new drugs and enhancing plant and animal germplasm. Traditional gene silencing methods typically utilize RNAi (RNA interference) technology.

[0003] RNA interference (RNAi) is a phenomenon in which double-stranded RNA (dsRNA) induces the efficient and specific degradation of homologous mRNA. It is a gene silencing defense mechanism used by organisms to resist nucleic acid invasions such as viruses, transposons, and repetitive genomes. In plants, RNAi primarily involves two types of small RNA molecules: siRNA (small interference RNA) and miRNA (microRNA). RNAi technology involves the introduction of synthetic or vector-expressed dsRNA into eukaryotic cells, which promotes the degradation of endogenous mRNA of specific genes, effectively and specifically blocking the expression of specific genes in the body, inducing cells to exhibit a specific gene deletion phenotype and obtaining mutants with loss or reduced function. RNA interference technology (RNAi) is now very mature, but it also has certain problems and limitations: (1) RNA interference technology may have off-target effects; (2) The knockdown of its target gene cannot be stably inherited: Since RNA interference requires the additional expression or introduction of small molecule RNA in the cell, it must rely on transient transfection or transgenic technology; even if transgenic technology is used to stably express small molecule RNA, its knockdown effect is not stable because its expression is affected by many factors (temporal and spatial diversity of promoters, effects of exogenous fragments in the genome, different epigenetic modifications and gene silencing between generations, etc.). (3) The knockdown effect of different target genes / sites varies greatly, making it difficult to predict the knockdown effect.

[0004] Therefore, there is an urgent need in this field to develop a method for conditional knockdown of endogenous gene expression by precisely knocking in specific miRNA target sequences in the non-coding region of the target gene using precise gene editing technology tools.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a method for conditional knockdown of endogenous gene expression by precisely knocking in specific miRNA target sequences in the non-coding region of the target gene using precise gene editing technology tools.

[0007] The first aspect of the present invention provides a method for conditionally regulating gene expression, comprising:

[0008] The miRNA target sequence is introduced into the non-coding region of the target gene in the plant cell, wherein the non-coding region includes the 5'UTR, 3'UTR or intron region.

[0009] In another preferred embodiment, the non-coding region is 3'UTR.

[0010] In another preferred embodiment, the miRNA target sequence is introduced into the position before the middle position of the 3'UTR of the target gene in the plant cell, preferably, within 150 bp from the first base near the stop codon (preferably, 5 bp to 150 bp, more preferably, 5-100 bp, further preferably, 5-20 bp).

[0011] In another preferred embodiment, the miRNA target sequence is introduced into the non-coding region of the target gene in the plant cell within the first base to 150 bp near the stop codon.

[0012] In another preferred embodiment, the regulating gene expression includes reducing or inhibiting the expression of the target gene.

[0013] In another preferred embodiment, the target gene is an endogenous plant gene.

[0014] In another preferred embodiment, the endogenous plant gene is a gene involved in plant growth and development, biotic or abiotic stress.

[0015] In another preferred embodiment, the length of the introduced miRNA target sequence is between 5 and 100 bases, preferably, between 6 and 50 bases, and more preferably, between 7 and 24 bases.

[0016] In another preferred embodiment, the sequence of the introduced miRNA target sequence is shown as SEQ ID NO. 1-4.

[0017] In another preferred embodiment, the introduced miRNA target sequence is complementary to the endogenous miRNA sequence, preferably, completely complementary.

[0018] In another preferred embodiment, the plants include monocotyledonous plants and dicotyledonous plants.

[0019] In another preferred embodiment, the plant includes a grass plant.

[0020] In another preferred embodiment, the plants include rice, corn, wheat, sorghum, barley, soybean, peanut, rapeseed, cotton, Arabidopsis, tomato, tobacco, potato, and cassava.

[0021] In another preferred embodiment, the plant includes rice.

[0022] In another preferred embodiment, the introduced miRNA target sequence exhibits spatiotemporal specific expression in the organism, preferably, exhibits tissue specific expression.

[0023] In another preferred embodiment, the introduced miRNA target sequences include target sequences that specifically express miRNA in tissues and organs such as roots, stems, leaves, flowers, seeds, endosperm, embryos, leaf sheaths, and axillary buds (i.e., specific miRNA target sequences).

[0024] In another preferred embodiment, the root-specific miRNA target sequence is osa-miR156k, osa-miR171h, osa-miR160e, osa-miR535, osa-miR162a, osa-miR171g, osa-miR1432, osa-miR1318, osa-miR156l, osa-miR164e, osa-miR166g, osa-miR535 and other miRNA target sequences.

[0025] In another preferred example, the leaf-specific miRNA target sequence is Osa-miR156d / f / j / m, osa-miR172a / b / d, osa-miR164a / b / f, osa-miR396d / e / f and other miRNA target sequences.

[0026] In another preferred embodiment, the endosperm-specific miRNA target sequence is miRNA target sequences such as osa-miR827b and osa-miR1866-5p.

[0027] In another preferred embodiment, the embryo-specific miRNA target sequence is osa-miR528, osa-miR397b*, osa-miR390*, osa-miR397b, osa-miR408 and other miRNA target sequences.

[0028] In another preferred embodiment, the ear-specific miRNA target sequence is Osa-miR166g / h, Osa-miR319a / b, Osa-miR529a, Osa-miR166k / l and other miRNA target sequences.

[0029] In another preferred embodiment, the constitutively expressed miRNA target sequence is Osa-miR156a / b / c / e / g / i, Osa-miR166a / b / c / d / f, Osa-miR168 and other miRNA target sequences.

[0030] In another preferred embodiment, the introduction is to introduce the miRNA target sequence through DNA modification such as sequence insertion, replacement or deletion.

[0031] In another preferred embodiment, the introduction is based on CRISPR / Cas, Talen or ZFN gene editing methods.

[0032] In another preferred embodiment, the introduction is based on single-base editing and guided editing methods.

[0033] In another preferred embodiment, one or more miRNA target sequences are introduced into the non-coding region of the target gene.

[0034] In another preferred embodiment, after the miRNA target sequence is introduced, a new transcript is formed, and the protein encoded by the transcript is completely identical to the protein encoded by the target gene.

[0035] The second aspect of the present invention provides an engineered cell, which is obtained by treating the cell using the method described in the first aspect of the present invention.

[0036] In another preferred embodiment, the cells include eukaryotic cells or prokaryotic cells.

[0037] In another preferred embodiment, the cells include plant or animal cells.

[0038] In another preferred embodiment, the cells include monocotyledonous plant cells and dicotyledonous plant cells.

[0039] In another preferred embodiment, the cells are cells of rice, corn, wheat, sorghum, barley, soybean, peanut, rapeseed, cotton, Arabidopsis, tomato, tobacco, potato, or cassava.

[0040] A third aspect of the present invention provides a method for improving agronomic traits of plants, comprising:

[0041] Plant cells are treated with the method described in the first aspect of the present invention to reduce or inhibit the expression of target genes in the plant cells, thereby improving the agronomic traits of the plants.

[0042] In another preferred embodiment, the plant agronomic traits include: leaf green, grain yield, grain quality, growth rate, total biomass or accumulation rate, fresh weight at maturity, dry weight at maturity, fruit yield, seed yield, plant nitrogen content, plant free amino acid content, plant protein content, drought resistance, heat resistance, nitrogen absorption, root lodging, harvest index, stem lodging, plant height, ear height, ear length, disease resistance, cold resistance, salt resistance and tiller number.

[0043] In another preferred embodiment, the plant is non-transgenic, for example, a non-transgenic genetically modified plant can be obtained by transient transformation using a genome editing system; or, after obtaining a genetically modified plant integrated with an exogenous transgene, a non-transgenic genetically modified plant can be obtained by genetic segregation of the offspring.

[0044] In another preferred embodiment, the method further comprises the step of regenerating the plant cells into plants.

[0045] The fourth aspect of the present invention provides a plant or its progeny, which is obtained by treating plant cells with the method described in the first aspect of the present invention.

[0046] In another preferred embodiment, the method further comprises the step of regenerating the plant cells into plants.

[0047] A fifth aspect of the present invention provides a method for producing plant seeds, comprising hybridizing a plant cell obtained by the method of the first aspect of the present invention with itself or with another plant of the same crop.

[0048] The sixth aspect of the present invention provides a method for regulating the level of targeted transcripts in cells, comprising introducing a gene editing system targeting the non-coding region of a target gene into the cell, whereby the new transcripts produced contain endogenous miRNA target sequences, causing the new transcripts to be recognized by the corresponding endogenous miRNA and degraded or inhibit protein translation, thereby conditionally reducing the expression of the target gene.

[0049] In another preferred embodiment, the gene editing system includes a gene modification tool, which modifies the non-coding region of the target gene. Preferably, the modification is gene editing. More preferably, the modification is based on CRISPR / Cas, Talen and ZFN gene editing methods.

[0050] In another preferred embodiment, the modification is performed based on single-base editing and guided editing methods.

[0051] In another preferred embodiment, the cells include eukaryotic cells or prokaryotic cells.

[0052] In another preferred embodiment, the cells include plant and animal cells.

[0053] In another preferred embodiment, the cells include monocotyledonous plant cells and dicotyledonous plant cells.

[0054] In another preferred embodiment, the cells are cells of rice, corn, wheat, sorghum, barley, soybean, peanut, rapeseed, cotton, Arabidopsis, tomato, tobacco, potato, or cassava.

[0055] The seventh aspect of the present invention provides a method for producing a genetically modified plant, comprising the step of regenerating a whole plant from the cell of the second aspect of the present invention, wherein the expression of a target gene in the genetically modified plant is altered relative to that in a non-genetically modified plant.

[0056] In another preferred embodiment, the change includes a decrease in the expression level of the target gene.

[0057] An eighth aspect of the present invention provides a method for improving plants, the method comprising the steps of:

[0058] (a) providing a plant cell, plant tissue, or plant part, and introducing a miRNA target sequence into a non-coding region of a target gene in the plant cell, plant tissue, or plant part, wherein the non-coding region includes a 5'UTR, a 3'UTR, or an intron region;

[0059] (b) regenerating the plant cells, plant tissues, or plant parts of step (a) into plants.

[0060] In another preferred embodiment, in step (a), the plant cells, plant tissues, and plant parts are modified using gene editing technology, so that the expression or activity of the target gene in the plant cells, plant tissues, and plant parts is reduced or inhibited.

[0061] In another preferred embodiment, the gene editing technology is selected from the following group: CRISPR gene editing system, error-prone PCR, gene recombination, TALEN and ZFN.

[0062] In another preferred embodiment, the method is used to improve agronomic traits of plants.

[0063] A ninth aspect of the present invention provides a method for preparing genetically engineered plant tissues or plant cells, comprising the steps of:

[0064] The miRNA target sequence is introduced into the non-coding region of the target gene in the plant tissue or plant cell, wherein the non-coding region includes 5'UTR, 3'UTR or intron region, thereby obtaining a genetically engineered plant tissue or plant cell.

[0065] In another preferred embodiment, the method is used to conditionally reduce or inhibit the expression of a target gene.

[0066] A tenth aspect of the present invention provides a method for preparing a genetically engineered plant, comprising the steps of:

[0067] The genetically engineered plant tissue or plant cell prepared by the method described in the ninth aspect of the present invention is regenerated into a plant body, thereby obtaining a genetically engineered plant.

[0068] The eleventh aspect of the present invention provides a genetically engineered plant, which is prepared using the method described in the seventh aspect or the tenth aspect of the present invention.

[0069] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1. Schematic diagram of the method for targeted inhibition of gene expression, where promoter refers to the promoter, TGA refers to the stop codon of the target gene, target gene refers to the target gene, and 3'UTR is the region where the specific miRNA target sequence is inserted through gene editing.

[0071] Figure 2. Effects of introducing miRNA target sequences at different positions in the gene 3'UTR on gene expression. Figure 2A is a schematic diagram of the dual-luciferase assay vector used to evaluate the effects of introducing miRNA target sequences at different positions in the 3'UTR on upstream FLuc expression. Figure 2B shows the Osa-MIR156a target sequence introduced immediately downstream of the GID1 3'UTR at positions +5bp, +20bp, and +400bp after the stop codon of the FLuc gene, and analyzed in rice embryonic calli using the dual-luciferase system. WT represents an empty vector without the introduced miRNA target sequence. Average FLuc / NLuc activity conferred by WT was normalized to that of the GID1 3'UTR. miRNA156a Figure 2C. The Osa-MIR156a target sequence was introduced immediately downstream of the IPK1 3'UTR, at positions +5, +20, and +200 bp after the stop codon of the FLuc gene. The results were analyzed in rice embryonic calli using the dual-luciferase system. WT represents an empty vector without the introduced miRNA target sequence. The average FLuc / NLuc activity conferred by WT was normalized to that of the IPK1 3'UTR. miRNA156a - Average FLuc / NLuc activity of LUC.

[0072] Figure 3. In situ introduction of specific miRNA target sequences into plant genes conditionally reduces target gene expression. Figure 3A shows the introduction of the rice constitutively expressed Osa-MIR156a target sequence into the 3'UTR of the target gene GID1. WT represents the expression result of GID1 without genetic modification. gid1_miRNA156a is the gene editing at the +15bp position downstream of the stop codon of the GID1 gene. After the formation of a new transcript, the fluorescence quantitative results of the gene expression of the GID1 gene in different tissues of wild-type rice plants and edited plants are shown. Figure 3B shows the introduction of the rice root-specific Osa-MIR171h target sequence into the 3'UTR of the target gene GID1. WT represents the expression result of GID1 without genetic modification. gid1_miRNA171h is the gene editing at the +15bp position downstream of the stop codon of the GID1 gene. After the formation of a new transcript, the fluorescence quantitative results of the gene expression of the GID1 gene in different tissues of wild-type rice plants and edited plants are shown. Figure 3C shows the introduction of the rice leaf-specific Osa-MIR164a target sequence into the 3'UTR of the target gene GID1. WT represents the expression result of GID1 without genetic modification. gid1_miRNA164a represents the gene expression fluorescence quantification results of the GID1 gene in different tissues of wild-type rice plants and edited plants after gene editing at the +15bp position downstream of the stop codon of the GID1 gene to form a new transcript. Figure 3D shows the introduction of the rice panicle-specific Osa-MIR166g target sequence into the 3'UTR of the target gene GID1. WT represents the expression result of GID1 without genetic modification. gid1_miRNA166g represents the gene expression fluorescence quantification results of the GID1 gene in different tissues of wild-type rice plants and edited plants after gene editing at the +15bp position downstream of the stop codon of the GID1 gene to form a new transcript. DETAILED DESCRIPTION

[0073] The present inventor has been through extensive and in-depth research, and for the first time unexpectedly discovered that by in the target gene non-coding region, targeting knocks in miRNA target sequence, and then reduces the method and application of target gene expression, relatively traditional gene expression regulation method, the present invention does not need to carry out transgenic operation, gene regulation characteristics can stably inherit, gene expression regulation effect is more efficient, reliable and accurate, is more widely used, is expected to replace traditional gene silencing technology in some fields, for applications such as gene therapy, animal and plant breeding and disease model preparation provide a kind of effective, universal method. The present invention can be used in the field of genetic engineering, for the large-scale creation of plant research and germplasm resources, especially with the creation of the germplasm resource material of crops and forestry crops of economic value. On this basis, the present inventor has completed the present invention.

[0074] Terminology

[0075] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0076] As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0077] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0078] As used herein, the term "target gene" or "sequence of interest" refers to any gene that, when transferred to a plant, confers a desired characteristic on the plant (e.g., antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other harmful organisms, herbicide tolerance, improved nutritional value, improved performance of an industrial process, or altered reproductive capacity). A "target gene" may also be a gene transferred to a plant for the production of a commercially valuable enzyme or metabolite in the plant.

[0079] As used herein, "exogenous" refers to a nucleic acid molecule or nucleotide sequence that is not naturally associated with the host cell into which it is introduced, and the sequence is derived from another species or from the same species or organism, but has been modified from its original form or the form primarily expressed in the cell, including multiple copies of non-naturally occurring nucleic acid sequences. Thus, a nucleotide sequence derived from an organism or species different from the organism or species to which the cell into which it is introduced belongs is heterologous relative to that cell or the progeny of the cell. In addition, a heterologous nucleotide sequence includes a nucleotide sequence that is derived from and inserted into the same natural original cell type, but exists in a non-natural state, for example, exists in a different copy number, and / or is under the control of regulatory sequences different from those found in the natural state of the nucleic acid molecule. A nucleic acid sequence can also be heterologous to other nucleic acid sequences associated therewith, for example, in a nucleic acid construct, such as an expression vector. As a non-limiting example, a promoter can be present in a nucleic acid construct in combination with one or more regulatory elements and / or coding sequences that do not naturally exist in relation to that particular promoter, i.e., they are heterologous to the promoter.

[0080] As used herein, the term "cell" refers to any living cell. The cell can be a prokaryotic cell or a eukaryotic cell. The cell can be isolated. The cell may or may not be able to regenerate into an organism. The cell can be in the context of a tissue, callus, culture, organ, or part. In certain embodiments, the cell can be a plant cell. The plant cell of the present invention can be in an isolated single-cell form, or can be a cultured cell, or can be a part of a higher-level organizational unit (e.g., plant tissue or plant organ). The plant cell can be derived from angiosperms or gymnosperms or a part thereof. In other embodiments, the plant cell can be a monocotyledonous plant cell or a dicotyledonous plant cell. The monocotyledonous plant cell can be, for example, maize, rice, sorghum, sugarcane, barley, wheat, oats, turfgrass, or an ornamental grass cell. The dicotyledonous plant cell can be, for example, a rice, tobacco, pepper, eggplant, sunflower, cruciferous plant, flax, potato, cotton, soybean, sugar beet, or rapeseed cell.

[0081] As used herein, the term "plant part" includes, but is not limited to, embryos, pollen, ovules, seeds, leaves, stems, buds, flowers, branches, fruits, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, plant cells (including intact plant cells in plants and / or parts of plants), plant protoplasts, plant tissues, plant cell tissue cultures, plant callus, plant masses, and the like. As used herein, "bud" refers to the above-ground parts including leaves and stems. Additionally, as used herein, "plant cell" refers to the structural and physiological units of a plant, including cell walls and may also refer to a protoplast.

[0082] In the context of a cell, prokaryotic cell, bacterial cell, eukaryotic cell, vegetable cell, plant and / or plant part, the term "introducing" (introducing or introducing) means contacting a nucleic acid molecule with the cell, eukaryotic cell, plant, plant part and / or plant cell in such a way that the nucleic acid molecule is able to enter the inside of the cell, eukaryotic cell, vegetable cell and / or plant and / or plant part. In the case of introducing more than one nucleic acid molecule, these nucleic acid molecules can be assembled into a part of a single polynucleotide or nucleic acid construct, or assembled into separate polynucleotides or nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Therefore, these polynucleotides can be introduced into the plant cell in a single transformation event, in a separate transformation event, or, for example, as part of a breeding scheme by conventional hybridization.

[0083] "MicroRNAs" (abbreviated as miRNAs) are small, non-coding RNA molecules (containing between approximately 20 and approximately 24 nucleotides, typically approximately 22 nucleotides) found in plants, animals, and some viruses. Their functions are RNA silencing and post-transcriptional regulation of gene expression. MiRNA genes are typically transcribed by RNA polymerase II (Pol II). The polymerase often binds to a promoter found near a DNA sequence, encoding a hairpin loop that will become the pre-miRNA. The resulting transcript is capped at the 5' end with specially modified nucleotides, polyadenylated with multiple adenosines (poly-A tail), and spliced.

[0084] Conditionally reduce target gene expression

[0085] The present invention precisely introduces the preferred miRNA target sequence of the present invention into the non-coding region of the target gene to generate new transcripts containing the endogenous miRNA target sequence. By utilizing the spatiotemporal expression specificity of the endogenous miRNA, the new transcripts are recognized by the corresponding endogenous miRNA and degraded or protein translation is inhibited, thereby conditionally reducing the expression of the target gene.

[0086] Specifically, the present invention provides a method for conditionally regulating gene expression, comprising:

[0087] The miRNA target sequence is introduced into the non-coding region of the target gene in the plant cell, wherein the non-coding region includes the 5'UTR, 3'UTR or intron region.

[0088] The main advantages of the present invention include:

[0089] (1) The present invention is the first to discover a method and application for reducing target gene expression by targeted knock-in of miRNA target sequences in the non-coding region of a target gene. Compared with traditional gene expression regulation methods, the present invention does not require transgenic manipulation, and the gene regulation characteristics can be stably inherited. The gene expression regulation effect is more efficient, reliable and accurate, and has a wider application. It is expected to replace traditional gene silencing technology in some fields and provide an effective and universal method for applications such as gene therapy, plant and animal breeding, and disease model preparation. The present invention can be used in the field of genetic engineering, for plant research and large-scale creation of germplasm resources, especially for the creation of germplasm resources of economically valuable crops and forestry crops.

[0090] (2) The present invention proposes and develops for the first time the use of precise gene editing technology tools to precisely knock in specific miRNA target sequences in the non-coding region of the target gene to achieve conditional knockdown of endogenous gene expression.

[0091] The present invention will be further described below in conjunction with specific implementations. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0092] Unless otherwise specified, the reagents and materials used in the examples of the present invention are commercially available products.

[0093] Materials and methods

[0094] Plasmid construction

[0095] To test the inhibitory effect of introduced miRNA target sequences on target gene expression, we developed a dual-luciferase reporter system (Figure 2). The backbone vector PCBSG032 (described in Tian Y, Zhong D, Li X, Shen R, Han H, Dai Y, Yao Q, Zhang X, Deng Q, Cao X, Zhu JK, Lu Y. High-throughput genome editing in rice with a virus-based surrogate system. J Integr Plant Biol. 2022 Oct 11. doi:10.1111 / jipb.13381. Epub ahead of print. PMID: 36218268) was first digested with HindIII and BamHI to remove the sgRNA and Cas9 expression elements. The 35S mini-promoter and FLuc were then constructed. The sequenced plasmid was then digested again with XmaI to construct NLuc behind the 35S promoter (Figure 2A). For the construction of vectors for inserting miRNA target sequences at different positions in the 3'UTR, the 3'UTR sequences of the target genes GID1 and IPK1 (as shown in SEQ ID NO.5-6) ​​were first constructed at the end of the luciferase FLUC as a control group (WT). The Osa-MIR156a target sequence was then used as a primer to insert into different positions of the GID1 3'UTR sequence by PCR amplification of the plasmid to evaluate the inhibitory effect of endogenous Osa-MIR156a on the expression of genes containing target sequences in these 3'UTRs. As shown in Figure 2B, the Osa-MIR156a target sequence was introduced at +5bp, +20bp, and +400bp of the GID1 3'UTR (this position is in the middle of the entire GID1 3'UTR sequence). The test results showed that the introduction of the Osa-MIR156a target sequence near the stop codon had a stronger inhibitory effect on FLuc expression than the introduction at the middle position +400 of the GID1 3'UTR. Similarly, as shown in Figure 2C, the Osa-MIR156a target sequence was introduced at +5bp, +20bp, and +200bp of the IPK1 3'UTR (this position is in the middle and later part of the entire IPK1 3'UTR sequence). The test results showed that the introduction of the Osa-MIR156a target sequence near the stop codon had a stronger inhibitory effect on FLuc expression than the introduction at the middle position +200bp of the IPK1 3'UTR.

[0096] SEQ ID NO.5 GID1 3'UTR sequence:

[0097] SEQ ID NO.6 IPK1 3'UTR sequence:

[0098] In order to generate a knock-in plant of the miRNA target sequence of rice GID1, the present invention uses Cas9 / sgRNA combined with donor DNA to make a knock-in plant in the embryonic callus of rice of Zhonghua 11. For the construction of CRISPR-Cas9 plasmid, preparation was performed as described previously (Lu, et al. Targeted, efficient sequence insertion and replacement in rice. Nat. Biotechnol. 38.1402-1407 (2020)). The vector PCBSG032 used in the present invention was digested with BsaI endonuclease to obtain a linear vector, and then sgRNA (chemically synthesized by GenScript Biotechnology Co., Ltd.) (as shown in Table 1) was recombined into the vector to form a binary expression vector. Osa-miRNA target sequences SEQ ID NOs. 1-4 (as shown in Table 2) were designed as donor DNA sequences, and the ends were phosphorothioated. The two phosphodiester bonds between the three bases at the 5' and 3' ends of the primers were thiolated, and the C5 positions of the first and last bases were phosphorylated. The modified donor DNA sequences were synthesized at Sangon Biotech (Shanghai) Co., Ltd.

[0099] Table 1 Target sites of GID1 gene in rice

[0100] Table 2 Donor DNA sequences for introducing different miRNA target sequences into rice

[0101] Gene gun-mediated knock-in of miRNA target sequences

[0102] The CRISPR / Cas9 plasmid and annealed donor DNA were used to bombard embryogenic calli of rice Zhonghua 11. Biolistic transformation was performed as previously described (Lu, et al. Targeted, efficient sequence insertion and replacement in rice. Nat. Biotechnol. 38:1402-1407 (2020)). After bombardment, tissue culture was used to regenerate plants without the use of resistance selection agents.

[0103] Agrobacterium transformation of rice callus cells

[0104] The dual-luciferase reporter vector was transformed into Agrobacterium tumefaciens EHA105 by electroporation. Agrobacterium-mediated transformation, tissue culture, and regeneration of rice Zhonghua 11 were performed according to the methods described in the literature (Nishimura, A et al. A protocol for Agrobacterium-mediated transformation in rice. Nat. Protoc. 1, 2796–2802 (2006)). Hygromycin selection (50 mg / L) was used during subsequent tissue culture.

[0105] Assay of luciferase reporter gene activity

[0106] The calli transformed with Agrobacterium were harvested, frozen in liquid nitrogen, and ground thoroughly. The FLuc / NLuc activity was measured using the Dual-Reporter Assay System (N1610, Promega, Madison, USA).

[0107] Detection and verification of the editing effect of the target gene GID1

[0108] Progeny plants obtained through gene gun-mediated genetic transformation were verified by extracting genomic DNA using conventional methods and sequencing the PCR products amplified with targeted site-specific primers (Table 3). The knock-in sites were then further confirmed by Hi-Tom sequencing. The resulting stable homozygous positive lines were subsequently analyzed for GID1 expression.

[0109] Table 3. Primers used

[0110] RNA preparation and qRT-PCR

[0111] RNA was extracted from plant samples using the Novozyme Plant RNA Kit (RC411). Reverse transcription of cDNA was performed using the Novozyme RNA Reverse Transcription Kit R333. qRT-PCR was performed using the Novozyme Fluorescent Quantitative PCR Kit Q712. The primers used are listed in Table 3.

[0112] The present invention is by in target gene non-coding region, targeting knocks in specific miRNA target sequence, and then reduces the method for target gene expression.As shown in Figure 1, target gene non-coding region 3'UTR introduces specific miRNA, forms a new transcript containing miRNA target sequence compared to wild type (WT), is identified by endogenous miRNAs and then reduces gene expression.The present embodiment compares the impact (Fig. 2A) of gene expression knocking low level by introducing miRNA target sequence at different positions in 3'UTR region.Specific experimental result is as shown in Figure 2B, compares 3'UTR different positions and introduces Osa-MIR156a target sequence, finds that compared to wild type (WT), on GID1 3'UTR, near the stop codon position (i.e., 5bp, 20bp region starting next to the first base of the stop codon), introducing miRNA target sequence effect is better than introducing (GID1 3'UTR middle position back region, i.e., 400bp region starting next to the first base of the stop codon) at 400bp. As shown in Figure 2C, compared to the wild type (WT), the introduction of the miRNA target sequence near the stop codon on the IPK1 3'UTR (i.e., the 5bp and 20bp regions immediately following the first base of the stop codon) was significantly more effective than the introduction at 200bp (i.e., the 200bp region immediately following the first base of the stop codon) (the region closer to the middle of the IPK1 3'UTR). These results indicate that the best knockdown effect of the target gene expression is achieved when the miRNA target sequence is introduced near the stop codon, but the optimal knock-in position is within 150bp, which has a better effect.

[0113] This example demonstrates that the introduction of different specific miRNA target sequences into the 3'UTR region can conditionally knock down gene expression. Specific experimental results are shown in Figure 3. The present invention selects GID1 as the target editing gene. This gene encodes a soluble gibberellin receptor that mediates GA signal transduction in rice and affects rice plant height. Four tissue-specific miRNA target sequences were knocked into the GID1 3'UTR 15bp downstream of the stop codon by gene gun genetic transformation to obtain positive transgenic plants. The GID1 gene expression level was then detected by fluorescence quantitative experiments. Compared with the wild type (WT), after the constitutively expressed Osa-MIR156a target sequence was introduced, the expression level of GID1 in the edited rice plants was significantly decreased in different tissues (Figure 3A); after the root-specific expression Osa-MIR171h was introduced, the expression level of GID1 in the edited rice plants was significantly decreased only in the roots (Figure 3B); after the leaf-specific expression Osa-MIR164ah was introduced, the expression level of GID1 in the edited rice plants was significantly decreased only in the leaves (Figure 3C); after the panicle-specific expression Osa-MIR166g was introduced, the expression level of GID1 in the edited rice plants was significantly decreased only in the panicle (Figure 3D).

[0114] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for conditionally regulating gene expression, characterized in that: include: The miRNA target sequence is introduced into the non-coding region of the target gene in the plant cell, and the non-coding region includes the 5'UTR, 3'UTR or intron region.

2. The method according to claim 1, characterized in that The miRNA target sequence is introduced into the non-coding region of the target gene in the plant cell within the first base to 150 bp near the stop codon (preferably, 5 bp to 150 bp, more preferably, 5-100 bp, and further preferably, 5-20 bp).

3. An engineered cell, characterized in that The cells are obtained by processing according to the method of claim 1 or 2.

4. A method for improving agronomic traits of plants, characterized in that: include: Plant cells are treated with the method according to claim 1 or 2, thereby reducing or inhibiting the expression of target genes in plant cells, thereby improving plant agronomic traits.

5. A method for producing plant seeds, characterized in that: A plant comprising crossing a plant cell obtained by the method of claim 1 or 2 with itself or with another plant of the same crop.

6. A method for regulating the level of a targeted transcript in a cell, characterized in that The method comprises introducing a gene editing system targeting the non-coding region of the target gene into the cell, whereby the new transcripts produced contain endogenous miRNA target sequences, causing the new transcripts to be recognized by the corresponding endogenous miRNA and degraded or inhibit protein translation, thereby conditionally reducing the expression of the target gene.

7. A method for producing a genetically modified plant, characterized in that The method comprises the step of regenerating a whole plant from the cell of claim 3, wherein the expression of a target gene in the genetically modified plant is altered relative to a plant that has not been genetically modified.

8. A method for improving plants, characterized in that: The method comprises the steps of: (a) providing a plant cell, plant tissue, or plant part, and introducing a miRNA target sequence into a non-coding region of a target gene in the plant cell, plant tissue, or plant part, wherein the non-coding region includes a 5'UTR, a 3'UTR, or an intron region; (b) regenerating the plant cells, plant tissues, or plant parts in step (a) into plants.

9. A method for preparing genetically engineered plant tissues or plant cells, characterized in that: Includes steps: The miRNA target sequence is introduced into the non-coding region of the target gene in the plant tissue or plant cell, wherein the non-coding region includes 5'UTR, 3'UTR or intron region, thereby obtaining a genetically engineered plant tissue or plant cell.

10. A method for preparing a genetically engineered plant, characterized in that: Includes steps: The genetically engineered plant tissue or plant cell prepared by the method of claim 9 is regenerated into a plant body, thereby obtaining a genetically engineered plant.

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