Use of ENE modulators in modulating plant transposons

Abstract

The present invention relates to the use of ENE modulators for modulating expression or activity of transposon in plants. It is found for the first time that ENE in a plant can form a triple helix structure with RNA, and a poly-A tail structure can be protected from deadenylation, so that the stability of transposon is enhanced. By deleting ENE, the transposon activity of the transposon can be inhibited. The new discovery of the invention provides a new way for inhibiting the transposon activity of the transposon, is beneficial to reducing the risk of unstable genome caused by transposon activation in the plant breeding process, and increases the controllability of genetic engineering breeding.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and more specifically, the present invention relates to the use of an ENE regulator in regulating plant transposons. Background Art

[0002] Transposons (TEs) are mobile DNAs that are widely present in eukaryotic genomes. The movement of transposons is also called transposon mobilization. In plants, due to the mobility characteristics of transposons (also called transposition activity or jumping activity), the presence of a large number of transposons can cause genome instability. For example, transposons can destroy gene structures and cause gene function loss, or they can change plant phenotypes by regulating gene expression levels and changing their expression patterns. Therefore, transposons are strongly activated during in vitro tissue culture processes commonly used for vegetative propagation and gene editing of crops, which can produce a series of genetic and epigenetic variations, including gene modification, gene deletion, and changes in gene expression patterns, and even the generation of new genes, thus bringing great potential risks to plant breeding.

[0003] In eukaryotes, transposons are usually strictly controlled by epigenetic factors. For example, in plants, transposons are mainly silenced by RNA-mediated DNA methylation (RdDM) pathway, and may also be controlled by siRNA-mediated post-transcriptional inhibition (e.g., when transposons escape RdDM silencing). However, not all transposons are strictly controlled by strict epigenetic factors. Some transposons can show strong transposition activity, such as Evade. The Copia-type long terminal repeat (LTR) retrotransposon Evade was originally discovered in met1-derived epigenetic recombination inbred lines, which showed that the insertion of Evade caused abnormal developmental defects. At the same time, its strong jumping activity was also observed in mutants of DDM1 and siRNA production factors. A recent study also showed that Evade had experienced abnormally active proliferation in history, which can be evidenced by the large number of Evade insertions in natural populations of Arabidopsis.

[0004] Therefore, the field needs to explore the mechanism of action of the transposition activity of transposons, so as to reduce the risk of genome instability caused by transposon activation during plant breeding. Summary of the invention

[0005] The object of the present invention is to provide the use of an ENE regulator for regulating the expression or activity of a transposon in a plant.

[0006] In a first aspect of the present invention, there is provided use of an ENE regulator for regulating the expression or activity of a transposon or a gene in a plant.

[0007] In one or more embodiments, the regulator includes an up-regulator and a down-regulator, wherein the up-regulator enhances the expression or activity of a transposon or a gene, and the down-regulator inhibits the expression or activity of a transposon or a gene.

[0008] In one or more embodiments, regulating the expression or activity of the transposon or gene includes: enhancing or inhibiting the expression or activity of the ENE gene; enhancing or inhibiting the formation of a triple helix structure between the ENE gene and poly-A RNA; enhancing or inhibiting the deadenylation of the poly-A tail of the transposon or gene.

[0009] In one or more embodiments, the modulator is an up-regulator or a down-regulator of ENE.

[0010] In one or more embodiments, the ENE downregulator includes: ENE knockout or silencing agents, ENE activity inhibitors.

[0011] In one or more embodiments, the ENE up-regulator includes: an ENE overexpression agent, an ENE activation or promotion agent.

[0012] In one or more embodiments, the ENE activating or promoting agent includes: DNA sequence, RNA sequence, and active fragments thereof of ENE.

[0013] In one or more embodiments, the expression or activity of a gene (including a reporter gene) can be enhanced by adding an ENE activating or promoting agent to the gene (including a reporter gene) (e.g., adding an ENE DNA sequence to a DNA without an ENE sequence, or adding an ENE RNA sequence to an RNA without an ENE RNA sequence).

[0014] In one or more embodiments, the down-regulator of ENE includes: a CRISPR gene editing reagent, a homologous recombination reagent or a site-directed mutagenesis reagent for ENE, which causes a loss-of-function mutation in ENE; and an interfering molecule that specifically interferes with the expression of the gene encoding ENE.

[0015] In one or more embodiments, the up-regulator of ENE includes: an expression vector or expression construct that can express (preferably overexpress) ENE after being transferred into cells, a regulatory factor (such as an enhancer) that specifically promotes the expression of ENE, and a CRISPR gene editing reagent or homologous recombination reagent for ENE.

[0016] In a second aspect of the present invention, a method for regulating the expression or activity of a transposon or gene in a plant is provided, the method comprising: regulating the expression or activity of an ENE sequence; regulating the ENE sequence to form a triple helix structure with RNA; and / or regulating the deadenylation of the transposon poly-A tail.

[0017] In one or more embodiments, the method for regulating the expression or activity of an ENE sequence comprises: overexpressing, knocking out or silencing the ENE, or enhancing or inhibiting the activity of the ENE.

[0018] In one or more embodiments, the method for promoting the expression or activity of the ENE sequence comprises: transferring an expression vector or expression construct capable of expressing (preferably overexpressing) ENE into a cell; introducing a regulatory factor (such as an enhancer) that specifically promotes the expression of ENE; and performing gene editing using a CRISPR system or homologous recombination reagent to introduce an ENE coding gene or an ENE RNA sequence.

[0019] In one or more embodiments, the method of inhibiting the expression or activity of the ENE sequence comprises: performing gene editing with a CRISPR system to knock out the ENE coding gene, knocking out the ENE coding gene by homologous recombination; silencing ENE with an interfering molecule that specifically interferes with the expression of the ENE coding gene; and performing a loss-of-function mutation on ENE in a plant containing ENE.

[0020] In one or more embodiments, the nucleotide sequence of the ENE comprises a nucleotide sequence selected from the group consisting of:

[0021] (a) the nucleotide sequence shown in SEQ ID NO:21;

[0022] (b) a nucleotide sequence derived from (a) having the function of (a) formed by replacing, deleting or adding one or more (e.g., 1-20; preferably 1-15; more preferably 1-10, such as 5, 3) nucleotides of the nucleotide sequence shown in SEQ ID NO: 21; or

[0023] (c) a nucleotide sequence derived from (a) that has more than 80% (preferably more than 85%; more preferably more than 90%; such as more than 95%, more than 96%, more than 97%, more than 98%, more than 99%) homology with the nucleotide sequence defined in (a) and has the function of (a); or

[0024] (d) A nucleotide sequence fragment defined in (a), or a nucleotide sequence fragment formed by adding or deleting one or several (usually within 20, preferably within 10, and more preferably within 5) nucleotides at its 3' end and / or 5' end (for example, adding a primer, a tag sequence, an active element).

[0025] In one or more embodiments, the (b) includes: a nucleotide sequence derived from (a) having the function of (a) and formed after a mutation occurs at one or more of positions 20, 21, 22, 36, 37, 38, 39, 50, 53, 54, 55, 58, 66, 69, 70, 71, and 74 of the amino acid sequence shown in SEQ ID NO: 21; preferably, in the (b), the mutated nucleotide is C; more preferably, the (b) includes: a nucleotide sequence as shown in SEQ ID NO: 22-28.

[0026] In one or more embodiments, the transposon is a LTR retrotransposon.

[0027] In one or more embodiments, the LTR retrotransposon includes (but is not limited to): Copia type, Gypsy type, BEL type, DIRS type, ERV type.

[0028] In one or more embodiments, the LTR retrotransposon includes (but is not limited to): Evade, ATGP7.

[0029] In one or more embodiments, the plant includes (but is not limited to): a plant expressing the Evade gene; a plant containing an ENE sequence; and / or a cruciferous plant.

[0030] In one or more embodiments, the plants expressing the Evade gene or containing the ENE sequence include (but are not limited to): Arabidopsis, turnip, strawberry, rice, highland barley, apple, alfalfa, Sichuan mulberry, Physcomitrella patens, sorghum, tomato, potato, truffle, grape, and corn; the cruciferous plants include (but are not limited to): Arabidopsis, turnip, watercress, rapeseed, broccoli, cabbage, white ribbon grass, wild wood ear vegetable, oil cabbage, English cabbage, curled flower, and tower vegetable.

[0031] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 , Evade's ENE sequence can form a triple helix structure.

[0033] a. Schematic diagram of the sequence structure of the Arabidopsis thaliana Evade retrotransposon. The light grey, dark grey and black rectangles represent the LTR, ORF (open reading frame) and ENE sequences, respectively. The U-rich part of the ENE sequence is marked in light grey.

[0034] b. Secondary structure of ENE RNA predicted by mfold. The U-rich part is marked in dark grey.

[0035] c. Schematic diagram of the principle of in vitro thermal denaturation experiment. The fluorescence intensity of the triple-helix structure of SYBR Gold changes with increasing temperature, and its melting curve shows a biphasic change. The derivative of the melting curve shows two corresponding melting peaks.

[0036] d. In vitro thermal denaturation experiment. The melting curves of polyadenylic acid A20, ENE, and their mixture are represented by black, dark gray, and light gray, respectively. The SYBR Gold fluorescence signal was detected every 0.5°C increase in temperature. The shaded area represents the 95% confidence interval.

[0037] Figure 2 , the specific characterization of the triple helix structure formed by the ENE sequence of Evade.

[0038] a. Secondary structure of ENE RNA predicted by mfold. Dark grey indicates the U-rich part of the ENE sequence, and light grey indicates the polyadenylation sequence. The dotted lines indicate the corresponding mutation sites.

[0039] bh. Melting curves of ENE binding to A20 after mutation of corresponding sites. The black curve represents the melting curve of A20 alone; the light gray represents the melting curve of the mixture of ENE and A20; the dark gray represents the melting curve of the mixture of ENE mutant (Mut1-7) and A20.

[0040] Figure 3 , ENE RNA triple helix structure stabilizes RNA.

[0041] a. Schematic diagram of the structure of the eGFP reporter system. The ENE sequence is represented by a black box and the arrows indicate the RT-qPCR primer sites.

[0042] b. RT-qPCR results of the transient expression experiment of eGFP in tobacco shown in Figure a. The ordinate represents the relative RNA level of eGFP, with basta as the internal reference. The bar graph shows the mean ± standard deviation of three biological replicates, and the circles represent the raw data. P values ​​were obtained by two-tailed Welch's t-test.

[0043] c. Schematic diagram of the luciferase reporter system used for transient expression experiments in tobacco. The Evade ORF sequence is located upstream of the luciferase gene, with or without the Evade ENE sequence inserted downstream. The ENE sequence is indicated by a black box.

[0044] d. Tobacco transient expression experiment of the luciferase reporter system shown in panel c, showing two representative biological replicates.

[0045] e. Quantitative measurement of firefly luciferase (Fluc) activity relative to Renilla luciferase (Rluc) activity. The data shown in the bar graph are the mean ± SD of three biological replicates, and the circles represent the raw data. P values ​​were obtained by two-tailed Welch's t-test.

[0046] Figure 4 , the triple helix structure of ENE RNA promotes the transposition of Evade.

[0047] a. Evade mRNA levels in ddm1, evade-1ddm1, and evade-2ddm1 mutants, with Actin2 as an internal control. Bar graph data are the mean ± sem of three biological replicates, and circles represent raw data. P values ​​were obtained using two-tailed Welch's t-test.

[0048] b. Evade mRNA stability in ddm1-L1 and ddm1-L4 mutants. Degradation curves of Evade RNA in ddm1 and evade-1 mutants: relative RNA levels before and after treatment with the transcription inhibitor (cordycepin), and the curves show the results of nonlin linear fitting. 1 / 2 Represents half-life.

[0049] c. Evade eclDNA levels in ddm1 and evade-1ddm1 mutants. ALE-qPCR was used to determine the eclDNA levels. PCR-amplified poprice DNA was used as a control. Values ​​are the mean ± SD of three biological replicates, and P values ​​were obtained by two-tailed Welch's t-test.

[0050] d. Evade copy number of ddm1 and evade-1ddm1 mutants in T4 (n=10,10,10,9,10,10) and T5 (n=9,10,10,5,9,9) generations. Copy number was determined by ddPCR using Actin2 as a single-copy reference gene. Each circle represents the copy number of an individual plant. Data are shown as mean ± SD. P values ​​were determined by two-tailed Welch's t-test. DETAILED DESCRIPTION

[0051] After in-depth research, the inventors took the plant (e.g. Arabidopsis) retrotransposon Evade as the research object and found that the RNA deadenylation pathway is a key process to limit the transposition activity of the transposon. The formation of the ENE RNA triple helix structure can protect the poly-A tail structure from deadenylation, thereby enhancing RNA stability, and adding ENE to the end of the GFP reporter gene can significantly enhance the reporter gene expression activity. The new discovery of the present invention provides a new way to inhibit the transposition activity of the transposon, which is conducive to reducing the risk of genome instability caused by transposon activation during plant breeding and increasing the controllability of genetic engineering breeding. At the same time, it also provides a new way to enhance gene expression, which is conducive to artificially enhancing the stability of gene expression and increasing the possibility of studying low-expression genes.

[0052] Transposon Evade

[0053] As used herein, the term "transposon" is also called "transposable element" or "jumping element", which is essentially a DNA fragment of a certain length, and is named because it can move in the host genome, for example, it can "jump" from one site of a chromosome to another site in the chromosome group of an organism, or transfer from one chromosome to another chromosome. According to different transposition mechanisms, transposons can be divided into retrotransposons (Class I) and DNA transposons (Class II). Retrotransposons can be further divided into long terminal repeat (LTR) retrotransposons and non-LTR retrotransposons.

[0054] "LTR retrotransposon" is the most common transposon family in many plants, and is divided into multiple subtypes, including Copia type, Gypsy type, BEL type, DIRS type, ERV type, etc. In a preferred embodiment of the present invention, the retrotransposon Evade belongs to a Copia type LTR retrotransposon.

[0055] As used herein, the term "Evade" refers to a gene having Evade transposon activity, which may be an Evade gene from Arabidopsis thaliana, or a gene homologous to the Evade gene from Arabidopsis thaliana, containing substantially the same domains, and having substantially the same functions. An exemplary Evade gene from Arabidopsis thaliana may refer to NC_003076.

[0056] In the present invention, the Evade gene also includes fragments, derivatives and analogs thereof. As used herein, the terms "fragment", "derivative" and "analog" refer to gene fragments that substantially maintain the same biological function or activity as the gene, which may be (i) a gene in which one or more nucleotides are replaced, and such replaced nucleotides may or may not be located in the ORF region, or (ii) a gene formed by the fusion of an additional gene sequence to this gene sequence, or (iii) a gene lacking (e.g., truncated) one or more nucleotides (e.g., a degenerate sequence thereof), etc. According to the definition herein, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.

[0057] It should be understood that although the Evade gene of the present invention is preferably obtained from a cruciferous plant, especially Arabidopsis thaliana, other genes highly homologous to the Arabidopsis thaliana Evade gene (such as having more than 80%, such as 85%, 90%, 95%, or even 98% sequence identity) obtained from other plants or genes with degeneracy with the gene are also within the scope of the present invention. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0058] In the present invention, the term "ENE" refers to a unique T-rich repeat sequence at the 3' end of the Evade gene. Herein, "ENE sequence" generally refers to the sequence shown in SEQ ID NO:21 having the function of ENE. The term also includes variant forms of the SEQ ID NO:21 sequence having the same function as the ENE sequence. These variant forms include (but are not limited to): deletion, insertion and / or substitution of several (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, and even more preferably 1-8, 1-5) nucleotides, and addition or deletion of one or several (usually within 20, preferably within 10, and more preferably within 5) nucleotides at the 3' end and / or 5' end.

[0059] In one or more embodiments, the variant sequence of ENE includes mutations at one or more positions of positions 20, 21, 22, 36, 37, 38, 39, 50, 53, 54, 55, 58, 66, 69, 70, 71, and 74 of the amino acid sequence shown in SEQ ID NO: 21, and preferably the nucleotide after the mutation is C. In some preferred embodiments, the variant sequence of ENE includes sequences shown in SEQ ID NO: 22-28.

[0060] It should be understood that although the ENE sequence of the present invention is preferably obtained from cruciferous plants, especially Arabidopsis thaliana, other sequences obtained from other plants that are highly homologous to the Arabidopsis thaliana ENE sequence (such as having more than 80%, such as 85%, 90%, 95%, or even 98% sequence identity) or their variant forms are also within the scope of the present invention. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST.

[0061] The vector containing the Evade gene and / or ENE sequence, and the host cell produced by genetic engineering using the vector or polypeptide Evade gene and / or ENE sequence are also included in the present invention. Methods well known to those skilled in the art can be used to construct suitable expression vectors.

[0062] Host cells are usually plant cells. Plants can generally be transformed using methods such as Agrobacterium transformation or gene gun transformation, such as the leaf disc method, rice embryo transformation method, etc.; Agrobacterium method is preferred. Transformed plant cells, tissues or organs can be regenerated into plants using conventional methods to obtain plants with altered traits relative to the wild type.

[0063] As used herein, the "plant" of the present invention refers to a plant that can have a transposon. In some preferred embodiments, the plant includes but is not limited to a plant selected from the group consisting of: a plant expressing an Evade gene; a plant containing an ENE sequence; and / or a cruciferous plant.

[0064] The plants expressing the Evade gene or containing the ENE sequence include (but are not limited to): Arabidopsis, turnip, strawberry, rice, highland barley, apple, alfalfa, Sichuan mulberry, Physcomitrella patens, sorghum, tomato, potato, truffle, grape, corn, etc. Those skilled in the art should understand that the plants applicable to the technical solution of the present invention are not limited to the above-mentioned plants, and the appropriate plants can be determined by identifying the presence of the Evade gene or the ENE sequence therein.

[0065] The cruciferous plants include (but are not limited to): Arabidopsis, turnip, watercress, rapeseed, broccoli, cabbage, leucophylla, wild auricularia, Chinese cabbage, English cabbage, curled lily, and tower vegetable.

[0066] With regard to "control plants", the selection of appropriate control plants is a routine part of experimental design and may include corresponding wild-type plants or corresponding transgenic plants without the gene of interest. Control plants are generally the same plant species or even varieties that are the same or belong to the same class as the plant to be evaluated. As used herein, control plants refer not only to complete plants, but also to plant parts, including leaves and leaf parts.

[0067] Methods for regulating transposon expression or activity

[0068] The inventors found during the research that there is an ENE sequence in the 3'UTR of the retrotransposon Evade, and the ENE sequence can form a triple helix structure with poly A RNA in vitro. And through further experiments and observations, it was proved that the sequence can form a triple helix structure with poly A RNA to improve the stability and transposition activity of its mRNA. Adding the sequence to the 3' end of the eGFP reporter gene can increase the mRNA level of eGFP. The inventors found that the RNA deadenylation pathway is a key process that limits the transposition activity of the transposon, and the ENE sequence can affect RNA deadenylation, thereby affecting the transposition activity of the transposon Evade. These findings of the present invention have not been studied before in the art.

[0069] Based on the new discovery of the present inventors, a method for regulating transposon expression or activity is provided, the method comprising: regulating the expression or activity of ENE sequence; regulating the ENE sequence to form a triple helix structure with RNA; and / or regulating the deadenylation of the transposon poly-A tail.

[0070] In one or more embodiments, the regulation includes up-regulation or down-regulation.

[0071] In one or more embodiments, the upregulation, enhancement, promotion indicates significant upregulation, enhancement, promotion, such as upregulation, enhancement, promotion by 5%, 10%, 20%, 40%, 60%, 80%, 90% or more.

[0072] In one or more embodiments, the downregulation, inhibition or reduction means a significant downregulation, inhibition or reduction, such as a downregulation, inhibition or reduction of 5%, 10%, 20%, 40%, 60%, 80%, 90% or more.

[0073] In one or more embodiments, the regulated expression includes: deletion of expression, increase of expression.

[0074] In one or more embodiments, the method for promoting the expression or activity of the ENE sequence includes: transferring an expression vector or expression construct that can express (preferably overexpress) ENE into a cell; introducing a regulatory factor (such as an enhancer) that specifically promotes the expression of ENE; using a CRISPR system or homologous recombination reagent to perform gene editing to introduce an ENE coding gene or ENE RNA sequence. In one or more embodiments, ENE is overexpressed (for example, using an overexpression system); an ENE sequence is added to a gene or transposon that does not originally contain an ENE sequence for fusion expression; and an ENE RNA sequence is added to RNA.

[0075] In one or more embodiments, the method for inhibiting the expression or activity of the ENE sequence includes: knocking out or silencing ENE, or inhibiting the activity of ENE; preferably includes: performing gene editing with a CRISPR system to knock out the coding gene of ENE, knocking out the coding gene of ENE by homologous recombination; silencing ENE with an interfering molecule that specifically interferes with the expression of the coding gene of ENE; and performing a loss-of-function mutation on ENE in a plant containing ENE.

[0076] In one or more embodiments, regulating the formation of a triple helix structure between the ENE sequence and RNA means significantly increasing / decreasing the formation of a triple helix structure between the ENE sequence and RNA, for example, the ratio of depolymerization of the triple helix structure between the ENE sequence and RNA is reduced / increased by 1 to 100%; more specifically, for example, reduced / increased by 2%, 4%, 6%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 80% or more.

[0077] In one or more embodiments, regulating transposon poly-A tail deadenylation means significantly enhancing / inhibiting transposon poly-A tail deadenylation, such as enhancing / inhibiting by 5%, 10%, 20%, 40%, 60%, 80%, 90% or more.

[0078] In the present invention, substances that regulate the expression or activity of ENE sequence, regulate the formation of triple helix structure between ENE sequence and RNA, or regulate the deadenylation of poly-A tail of transposon include activators, promoters, enhancers, inhibitors, antagonists, and blockers. The "up / down regulation", "enhancement / inhibition", and "increase / decrease" include "up / down regulation", "enhancement / inhibition" of transposon / gene activity or "up / down regulation", "enhancement / inhibition", and "increase / decrease" of transposon / gene expression. Any substance that can enhance / inhibit the expression or activity of ENE sequence, enhance / inhibit the formation of triple helix structure between ENE sequence and RNA, or enhance / inhibit the deadenylation of poly-A tail of transposon can be used in the present invention as a substance useful for inhibiting the expression or activity of transposon. They can be compounds, small chemical molecules, and biological molecules. The biological molecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.

[0079] It should be understood that based on the experimental data and regulatory mechanisms provided by the present invention, a variety of methods well known to those skilled in the art can be used to regulate (eg, inhibit) the expression or activity of the transposon, and these methods are all included in the present invention.

[0080] The main advantages of the present invention are:

[0081] The inventors have studied the mechanism of action of the transposition activity of the transposon Evade in the plant Arabidopsis thaliana, and found that the formation of the triple helix structure of ENE RNA in Evade can protect the poly-A tail structure from deadenylation, thereby enhancing RNA stability, and adding ENE to the end of the GFP reporter gene can significantly enhance the reporter gene expression activity. The new discovery of the present invention provides a new way to inhibit transposon activity in plants, which is conducive to reducing the risk of genome instability caused by transposon activation during plant breeding. It also provides a new way to enhance gene expression, which is conducive to artificially enhancing the stability of gene expression, increasing the possibility of studying low-expression genes, and increasing the controllability of genetic engineering breeding, and has important application value.

[0082] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0083] Experimental Materials and Methods

[0084] 1. Plant materials and growth conditions

[0085] The Arabidopsis materials of the present invention are all in the Columbia (Col-0) background. The ccr4a-1 (SAIL_802_A10), ccr4b-1 (SALK_151541C), caf1a-1 (SALK_070336) and caf1b-3 (SALK_044043) mutants were obtained from the Arabidopsis Biological Resource Center (https: / / abrc.osu.edu / ). The caf1a-1 / caf1b-3 double mutant was obtained by hybridization. The evade-1 and evade-2 mutants were generated by CRISPR-Cas9 targeting the Evade ENE sequence. The ddm1 mutant was generated by CRISPR-Cas9 in the evade-1 and evade-2 mutant backgrounds, respectively. The sgRNA and primer sequences for generating evade-1, evade-2, and ddm1 mutants by the CRISPR-Cas9 method are shown in Table 1.

[0086] Table 1

[0087]

[0088]

[0089] Seeds were sterilized with 75% ethanol, sown in Murashige and Skoog (MS) medium (0.43 g / L MS salts, 3 g / L sucrose, 0.8% agar, pH 5.8), and vernalized at 4°C for 3 days. Plants were grown under long-day conditions at 22°C (cool white fluorescent light on for 16 hours and off for 8 hours). Seedlings were taken for RNA-seq, ALE-seq, and ddPCR on the 10th day. Plants were grown in dark conditions at 22°C for four days and seedlings were taken for RNA degradation experiments.

[0090] 2. RT-qPCR

[0091] Total RNA was extracted using the TRIzol extraction method (TIANGEN) and reverse transcribed using ReverTra Ace qPCR RT MasterMix with gDNA Remover (TOYOBO). Quantitative PCR was performed using ChamQ Universal SYBR qPCR MasterMix (VAZYME) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Actin2 (AT3G18780) and basta were used as internal references in Arabidopsis and tobacco, respectively. Gene expression levels were calculated using the 2-ΔCt method. The primer sequences used in RT-qPCR are shown in Table 2.

[0092] Table 2

[0093]

[0094] 3. In vitro thermal denaturation experiment

[0095] The fragments containing the complete ENE and mutant ENE Mut1-7 sequences were generated by PCR amplification using the synthetic oligonucleotide sequences as templates. The PCR fragments were then transcribed in vitro using the HiScribe T7 High Yield RNA Synthesis Kit (NEB). RNA Cleanup Kit (NEB) was used to purify the transcribed RNA products. During in vitro heat denaturation, RNA and A20 were mixed in an incubation buffer [10 mM Tris-HCl (pH 7) and 10 mM MgCl2] at a 1:1 molar ratio. The mixture was then first heated at 95°C for 55 seconds and then slowly cooled to room temperature for renaturation. The mixture was then slowly denatured by slowly increasing the temperature from 60°C to 95°C. SYBR Gold (Invitrogen) was used to detect the fluorescent signal. The melting curve was detected using a qPCR instrument (CFX96 Touch Real-Time PCR Detection System, Bio-Rad). The sequences of the complete ENE sequence and the mutant ENE Mut1-7 are shown in Table 3.

[0096] Table 3

[0097]

[0098] 4. Tobacco transient expression experiment

[0099] (1) Construction of recombinant plasmid:

[0100] Using eGFP as a reporter gene, pUBQ10 as a promoter, and tHSP as a terminator, it was constructed into the pGPTVII:pUBQ10:tHSP18.2 plasmid using XmaI and XhoI enzymes to obtain the pUBQ10::eGFP:tHSP18.2 plasmid.

[0101] The ENE sequence was amplified from genomic DNA by PCR, and the ENE sequence of Evade was added to the 3'UTR end of the eGFP reporter gene using homologous recombinase (NEB). The positive clones were identified by transforming E. coli DH5α, and the plasmid DNA was extracted. After sequencing verification, the recombinant plasmid carrying the ENE sequence was obtained. The Basta gene on the same plasmid was used as the internal reference for qPCR.

[0102] (2) Instantaneous transformation of tobacco

[0103] The constructed plasmid was transferred into Agrobacterium strain GV3101, and tobacco (Nicotiana benthamiana) grown in a long-day environment at 22°C for about 4 weeks was transiently transformed by injection. The injected plants were placed in the dark for 12 hours, and then cultured at about 21°C for 3 days to observe the fluorescence of the tobacco area injected with Agrobacterium. Three biological replicates were taken from the ENE-positive transformation area (+ENE) and the ENE-negative transformation area (-ENE), and RT-qPCR was performed using eGFP-F / eGFP-R as primers.

[0104] The sequences of RT-qPCR primers are shown in Table 4.

[0105] Table 4

[0106]

[0107] 5. Tobacco dual luciferase reporter experiment

[0108] (1) Construction of recombinant plasmid:

[0109] The 3'UTR end with the ENE sequence and the 3'UTR end with the ΔENE sequence (i.e., the ENE sequence was deleted) were obtained by PCR amplification using the ENE-F / 3'LTR-R primer pair and the 3'UTR end with the ΔENE sequence (i.e., the ENE sequence was deleted), respectively. They were then ligated to the pUBQ10::fLUC:tHSP18.2 dual luciferase reporter vector using homologous recombinase (NEB) to obtain pUBQ10::fLUC:ENE:tHSP18.2 and pUBQ10::fLUC:ΔENE:tHSP18.2. Using the Evade-F / R primer pair, the Evade gene was amplified from genomic DNA by PCR and cloned into pUBQ10::fLUC:ENE:tHSP18.2 and pUBQ10::fLUC:ΔENE:tHSP18.2 by Kpn I and Pst I restriction enzyme digestion, and the positive clones were identified by transforming Escherichia coli DH5α bacteria, and plasmid DNA was extracted. After sequencing verification, pUBQ10::Evade:fLUC:ENE:tHSP18.2 and pUBQ10::Evade:fLUC:ΔENE:tHSP18.2 were obtained respectively. Among them, the Renilla luciferase (REN) gene driven by the constitutive penTCUP2 promoter was used as an internal reference gene, and tHSP was used as a terminator. The sequences of the primers are shown in Table 5.

[0110] The constructed plasmid was transferred into Agrobacterium strain GV3101. Nicotiana benthamiana grown for about 4 weeks under a long-day light environment at 22°C was used for transient transformation of tobacco plants by injection. The injected plants were cultured at about 21°C for 48 hours and then the area of ​​tobacco injected with Agrobacterium was observed for fluorescence. After observation, the leaves were collected and the LUC and REN activities were measured by Reporter Assay System (Promega), and three biological replicates were performed for ENE-positive transformed plants (+ENE) and ENE-negative transformed plants (-ENE).

[0111] Table 5

[0112]

[0113] 6. RNA degradation experiment

[0114] Four-day-old seedlings of ddm1 and ccr4a-1ddm1 double mutants were placed in an incubation buffer containing filter paper and cordycepin (1 mM PIPES, pH 6.25, 15 mM sucrose, 1 mM potassium chloride, 1 mM sodium citrate, and 1 mM cordycepin). To facilitate the penetration of cordycepin, three vacuum infiltrations were first performed, each lasting 1 minute. Samples were collected at 0, 0.5, 1, 2, and 4 hours after treatment for RT-qPCR.

[0115] 7. Droplet digital PCR (ddPCR)

[0116] In Targeting ddPCR was performed on a digital PCR system (Targeting One) according to the manufacturer's instructions. 100 ng of total DNA was digested with AluI (NEB). The reaction mixture containing 2×ddPCR Supermix (Bio-Rad), 0.8 μM primers and 0.25 μM probes, and 0.6 ng of DNA was thoroughly mixed and added to the droplet generation chip. Then, droplets were generated using a Drop maker (Targeting One), and the PCR reaction was performed in a PTC-200PCR instrument (Bio-Rad; 95°C for 10 minutes, 55 cycles, 95°C denaturation for 30 seconds, 56.8°C annealing for 1 minute, 98°C inactivation for 10 minutes, and 12°C insulation). After the PCR reaction, the PCR product was read using a Chip reader system (Targeting One). Actin2 was used as an internal single copy control. Finally, the data were analyzed for Poisson distribution using the Chip Reader R1 software to obtain the target DNA copy number. The sequences of primers and probes are shown in Table 6.

[0117] Table 6

[0118]

[0119] Example

[0120] Example 1: Evade's ENE sequence can form a triple helix structure with poly A

[0121] In order to explore how Evade escapes epigenetic transposition inhibition and achieves such strong jumping activity, the inventors analyzed the structure and sequence of Evade and found that the 3' end of Evade contains a unique T-rich repeat sequence ( Figure 1 a), namely the ENE sequence. The RNA secondary structure predicted by Mfold (http: / / www.mfold.org / ) also shows that it has a stem-loop structure formed by two U-rich segments ( Figure 1 b). The stem-loop structure formed by the U-rich repeat sequence and the polyadenylic acid tail can form an RNA triple helix structure through base pairing between UA·U (-, Watson-Crick base pairing; ·, Hoogsteen base pairing), thereby protecting the RNA end from the influence of mRNA deadenylation.

[0122] In order to detect whether the ENE sequence of Evade can form a triple helix structure, the inventors conducted an in vitro thermal denaturation experiment. This method determines the melting curve of the RNA triple helix structure by gradually increasing the temperature, using the change in fluorescence intensity when the SYBR Gold dye binds to the RNA triple helix and double helix, and detecting the fluorescence signal every time the temperature rises by 0.5°C. The melting curve of the triple helix structure formed by the U-rich segment and polyadenylic acid (Poly A) will show two different phase transition stages, and its derivative will show two different melting peaks: the first melting peak indicates the dissociation of the triple helix, and the second melting peak indicates the dissociation of the remaining double strand ( Figure 1 c). Evade RNA was mixed with in vitro synthesized poly A RNA (A20) and renatured to form a natural secondary structure. Interestingly, when ENE RNA and poly A RNA coexisted, the melting curve showed a biphasic change ( Figure 1 d), when either ENE RNA or poly A RNA is missing, the first melting peak representing the melting of the triple helix structure disappears, indicating that the ENE sequence of Evade can form a triple helix structure with poly A RNA in vitro.

[0123] In order to further characterize the formation of the triple helix structure of ENE RNA of Evade, the inventors mutated the U repeat sequence or non-stem-loop structure in the ENE sequence from U to C ( Figure 2 a). When the U in the stem-loop structure mutates to C, it represents the first melting peak of the triple helix structure ( Figure 2 c,f), while the mutation of U in the non-stem-loop structure to C does not change the melting peak of the melting curve ( Figure 2 h). The above results show that the U repeat sequence of the stem-loop structure is necessary for the formation of the RNA triple helix structure. In addition, the inventors also found that the GC base pairs adjacent to the stem-loop structure also contribute to the formation of the triple helix structure ( Figure 2 b, d, e, g).

[0124] Example 2: Evade's ENE sequence enhances gene expression

[0125] In order to study the function of the ENE sequence, the inventors added the ENE sequence of Evade to the 3'UTR end of the eGFP reporter gene and performed a tobacco transient expression experiment ( Figure 3 a). RT-qPCR analysis showed that when the ENE sequence was inserted into the 3' end, the expression level of eGFP was significantly increased ( Figure 3 b). Similar observations were also found in the transient expression experiment of tobacco with Evade ORF fused to express luciferase gene ( Figure 3 c) If Figure 3 As shown in d and 3e, the presence of the ENE sequence in the 3'UTR significantly enhanced the luciferase activity compared with the absence of ENE (-ENE). These results strongly suggest that the ENE sequence promotes gene expression in vivo.

[0126] The above data indicate that adding ENE to the end of a gene or reporter gene can enhance its gene expression activity.

[0127] Example 3: Deletion of the ENE sequence to inhibit Evade transposition activity

[0128] To further analyze the role of ENE in the transposition of Evade, the inventors used CRISPR-Cas9 technology to introduce Evade's ENE deletion mutation (evade-1, evade-2) into Arabidopsis. In order to induce the transposition of Evade, the inventors used CRISPR-Cas9 to introduce a new mutation of Decrease in Dnamethylation1 (DDM1) in both the wild type and ENE mutants. Interestingly, RT-qPCR results showed that compared with the DDM1 single mutant, the mRNA level of Evade in the evade-1 / ddm1 and Evade-2 / ddm11 double mutants was significantly reduced ( Figure 4 a). Considering the formation of RNA triple helix structure, the inventors believe that the ENE sequence of Evade may have a role similar to that of stabilizing transcripts in viruses and mammals. Therefore, the inventors conducted an in vivo RNA degradation experiment on Evade mRNA in evade-1 / ddm1 and ddm1. It was found that when ENE was missing, the degradation rate of its RNA increased significantly, indicating that its RNA stability was significantly reduced ( Figure 4 b).

[0129] Subsequently, the inventors used the ALE-seq method to further detect the level of its transposition intermediate product eclDNA and observed that when ENE was missing, the eclDNA level of Evade was significantly reduced ( Figure 4c). Finally, the copy number of Evade in DDM1 and Evade-1 / DDM1 mutants was detected by digital droplet PCR (ddPCR). Compared with ddm1, the copy number of Evade in evade-1 / ddm1 was significantly reduced ( Figure 4 d). These results suggest that ENE plays an important role in promoting the transposition activity of Evade.

[0130] The above data indicate that ENE maintains the stability of RNA by forming a triple helix structure with the poly A tail of RNA, thereby promoting its active transposition. By deleting the ENE sequence, the transposition activity of Evade can be inhibited.

[0131] Example 4: ENE sequences are conserved in different plants and transposons

[0132] The inventors found through literature review that ENE sequences are widely present in eukaryotic organisms, and there are more than 200 different types of ENE structures in plants, fungi, slime molds and fungi. These ENEs mainly appear in LTR retrotransposons, and also exist in plant DNA transposons of the hAT superfamily.

[0133] Table 7 lists the ENE sequences in some plants.

[0134] Table 7

[0135]

[0136]

[0137] In summary, in the present invention, the inventors identified the ENE sequence in the 3'UTR of the retrotransposon Evade and demonstrated that the sequence can form a triple helix structure with poly A RNA to improve the stability and transposition activity of its mRNA. Adding this sequence to the 3' end of the eGFP reporter gene can increase the mRNA level of eGFP.

[0138] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. Use of an ENE regulator for regulating the expression or activity of a transposon or gene in a plant.

2. The use according to claim 1, characterized in that The regulator includes an up-regulator and a down-regulator, wherein the up-regulator enhances the expression or activity of a transposon or a gene, and the down-regulator inhibits the expression or activity of a transposon or a gene; Preferably, the regulating the expression or activity of the transposon or gene comprises: enhancing or inhibiting the expression or activity of the ENE gene; enhancing or inhibiting the formation of a triple helix structure between the ENE gene and poly-A RNA; Enhance or inhibit the deadenylation of transposon or gene poly-A tail.

3. The use according to claim 1 or 2, characterized in that The regulator is an up-regulator or down-regulator of ENE; The ENE down-regulator includes: ENE knockout or silencing agent, ENE activity inhibitor; and / or, The ENE up-regulator includes: an ENE over-expression agent, and an ENE activation or promotion agent.

4. The use according to claim 3, characterized in that The down-regulator of ENE includes: CRISPR gene editing reagents, homologous recombination reagents or site-directed mutagenesis reagents for ENE, which cause ENE to undergo loss-of-function mutation; interfering molecules that specifically interfere with the expression of the gene encoding ENE; and / or, The ENE up-regulator includes: an expression vector or expression construct that can express ENE after being transferred into cells, a regulatory factor that specifically promotes the expression of ENE, and a CRISPR gene editing reagent or homologous recombination reagent for ENE.

5. A method for regulating transposon or gene expression or activity in a plant, the method comprising: Regulate the expression or activity of ENE sequence; regulate the formation of triple helix structure between ENE sequence and RNA; and / or, regulating transposon poly-A tail deadenylation.

6. The method for regulating transposon or gene expression or activity in plants according to claim 5, characterized in that: The method for regulating the expression or activity of the ENE sequence includes: overexpressing, knocking out or silencing ENE, or enhancing or inhibiting the activity of ENE.

7. The method for regulating transposon or gene expression or activity in plants according to claim 6, characterized in that: The method for promoting the expression or activity of the ENE sequence comprises: transferring an expression vector or expression construct capable of expressing ENE into a cell; introducing a regulatory factor that specifically promotes the expression of ENE; performing gene editing using a CRISPR system or homologous recombination reagent to introduce an ENE coding gene or ENE RNA sequence; and / or, The method for inhibiting the expression or activity of the ENE sequence includes: performing gene editing with a CRISPR system to knock out the ENE coding gene, knocking out the ENE coding gene by a homologous recombination method; silencing ENE with an interfering molecule that specifically interferes with the expression of the ENE coding gene; and performing a loss-of-function mutation on ENE in a plant containing ENE.

8. The method according to any one of claims 1 to 7, wherein: The nucleotide sequence of the ENE comprises a member selected from the group consisting of: (a) the nucleotide sequence shown in SEQ ID NO:21; (b) a nucleotide sequence derived from (a) which is obtained by substituting, deleting or adding one or more nucleotides of the nucleotide sequence shown in SEQ ID NO: 21 and has the function of (a); or (c) a nucleotide sequence derived from (a) that has more than 80% homology with the nucleotide sequence specified in (a) and has the function of (a); or (d) A fragment of the nucleotide sequence defined in (a), or a fragment thereof formed by adding or deleting one or more nucleotides at its 3' end and / or 5' end.

9. The method according to any one of claims 1 to 9, wherein: The transposon is an LTR retrotransposon; Preferably, the LTR retrotransposon includes: Copia type, Gypsy type, BEL type, DIRS type, ERV type; More preferably, the LTR retrotransposon includes: Evade and ATGP7.

10. The method according to any one of claims 1 to 9, characterized in that: The plants include: plants expressing Evade gene; plants containing ENE sequence; and / or cruciferous plants; Preferably, the plants expressing the Evade gene or containing the ENE sequence include: Arabidopsis, turnip, strawberry, rice, barley, apple, alfalfa, Sichuan mulberry, Physcomitrella patens, sorghum, tomato, potato, truffle, grape, and corn; the cruciferous plants include: Arabidopsis, turnip, watercress, rapeseed, broccoli, cabbage, white ribbon grass, wild auricularia, oil cabbage, English cabbage, curled flower, and tower vegetable.

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