阅读说明：本技术 一套用于水稻表观基因组甲基化/去甲基化的载体系统 (Carrier system for methylation/demethylation of rice epigenome ) 是由 谷晓峰 胡桂花 于 2020-05-21 设计创作，主要内容包括：本发明公开了一套用于水稻表观基因组甲基化/去甲基化的载体系统。本发明提供了一套用于水稻表观基因组去甲基化的载体系统。该系统通过随机去甲基化水稻基因组来快速产生DNA甲基化变异。该系统涉及水稻OsALKBH DNA 6mA去甲基化酶的表达,导致广泛的低甲基化。该“诱变”技术对于具有重要农业意义的植物,可能导致通常由DNA甲基化沉默的等位基因的差异表达,从而揭示先前隐藏的表型变异的应用具有重要意义。(The invention discloses a set of carrier system for methylation/demethylation of rice epigenome. The invention provides a set of carrier system for rice epigenome demethylation. The system rapidly generates DNA methylation variation by randomly demethylating the rice genome. The system involves expression of rice OsALKBH DNA6mA demethylase, resulting in extensive hypomethylation. This "mutagenesis" technique is of great importance to the use of plants of agronomic importance, possibly resulting in differential expression of alleles that are normally silenced by DNA methylation, thereby revealing previously hidden phenotypic variations.)

1. An expression vector comprising an expression cassette 1 and an expression cassette 2;

the expression cassette 1 is expressed by a functional fragment A which is started by a rice specific Act1 promoter;

the expression cassette 2 is expressed by a functional fragment B initiated by a rice specific Act1 promoter;

the functional fragment A comprises 10 copies of the coding sequence of GCN4 peptide and the coding sequence of dCas 9;

the functional fragment B comprises a coding sequence of sfGFP, a coding sequence of a specific enzyme and a coding sequence of a GCN4 antibody of the single-chain variable fragment scFv; the specific enzyme is rice specific methylase or rice specific demethylase.

2. The expression vector of claim 1, wherein:

the functional fragment A also comprises a3 XSV 40 NLS sequence;

and/or the functional fragment B also comprises a2 XSV 40 NLS sequence.

3. The expression vector of claim 1 or 2, wherein: the specific enzyme is rice specific DNA6mA demethylase.

4. The expression vector of claim 3, wherein: the rice specific DNA6mA demethylase is OsALKBH 1; the OsALKBH1 is (a1) or (a2) or (a3) as follows:

(a1) a protein consisting of an amino acid sequence shown in SEQ ID No. 2;

(a2) the amino acid sequence of SEQ ID No.2 is substituted and/or deleted and/or added by one or more amino acid residues, and the protein has the same function as the protein produced by SEQ ID No. 2;

(a3) and (b) a protein having homology of 75% or more than 75% with the amino acid sequence shown by SEQ ID No.2 and having the same function.

5. The expression vector of claim 3 or 4, wherein:

the expression cassette 1 is shown as the 2889-10115 th position from the 5' end of SEQ ID No. 1;

the promoter sequence in the expression cassette 2 is a reverse complementary sequence of 1 st-843 rd bits from the 5' end of SEQ ID No. 1; the reverse complement of the other elements of the expression cassette 2 is shown in SEQ ID No.1 at 20825-23917 from the 5' end.

6. The expression vector of any one of claims 3 to 5, wherein: the expression vector is a circular plasmid shown in SEQ ID No. 1.

7. Use of the expression vector of claim 1 or 2 for methylation or demethylation of the rice genome.

8. Use of the expression vector of any one of claims 3 to 6 for demethylation of the rice genome.

9. A method for demethylating genomic DNA of rice comprises the following steps: the expression vector of any one of claims 3 to 6 is introduced into recipient rice to achieve rice genomic DNA demethylation.

10. Use or method according to any of claims 7-9, wherein: the rice genomic DNA demethylation occurs within the rice nucleus.

Technical Field

The invention relates to the technical field of gene directed expression line synthesis, in particular to a set of vector system for methylation/demethylation of rice epigenome.

Background

Genetic engineering and synthetic biotechnology have become important means for studying and improving crop traits. With the development and utilization of gene editing tools Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALEs), and CRISPR/Cas9 systems, conditions for targeting and modifying specific genomic sequences to study related gene functions have become very mature. More and more research is using gene editing tools for epigenetic modification as well as gene regulation.

Rice is one of the main food crops in the world and is also a model plant for functional genome research. Rice genome-based studies have been relatively mature, however, epigenetic studies that may cause strong changes in rice phenotype are still in the infancy.

DNA methylation is important for gene regulation, transposition silencing and gene imprinting. Although the generation of specific DNA methylation patterns is important for these processes, it is unclear how methylation is regulated at each site. It has been found that methylation of plant gene promoter DNA sometimes results in transcriptional repression, and methylation deletion of methyltransferase mutant DNA results in altered gene expression and severe developmental defects. However, in many cases naturally occurring DNA methylation variations have been reported, whereby differentially methylated genes that alter expression are responsible for agronomically important traits. The ability to manipulate plant methylation to produce epigenetically distinct individuals may be invaluable for breeding and research purposes.

Disclosure of Invention

The invention aims to provide a set of vector systems for efficiently realizing methylation/demethylation of genome DNA in rice.

The invention firstly protects an expression vector, which comprises an expression cassette 1 and an expression cassette 2;

the expression cassette 1 is expressed by a functional fragment A which is started by a rice specific Act1 promoter;

the expression cassette 2 is expressed by a functional fragment B initiated by a rice specific Act1 promoter;

the functional fragment A comprises 10 copies of the coding sequence of GCN4 peptide and the coding sequence of dCas 9;

the functional fragment B comprises a coding sequence of sfGFP, a coding sequence of a specific enzyme and a coding sequence of a GCN4 antibody of the single-chain variable fragment scFv; the specific enzyme is rice specific methylase or rice specific demethylase.

The functional fragment A also comprises a3 XSV 40 NLS sequence.

The functional fragment B also comprises a2 XSV 40 NLS sequence.

The specific enzyme can be rice specific DNA6mA demethylase.

The rice specific DNA6mA demethylase can be OsALKBH1 specifically;

the OsALKBH1 is (a1) or (a2) or (a3) as follows:

(a1) a protein consisting of an amino acid sequence shown in SEQ ID No. 2;

(a2) the amino acid sequence of SEQ ID No.2 is substituted and/or deleted and/or added by one or more amino acid residues, and the protein has the same function as the protein produced by SEQ ID No. 2;

(a3) and (b) a protein having homology of 75% or more than 75% with the amino acid sequence shown by SEQ ID No.2 and having the same function.

The rice-specific Act1 promoter is (b1), (b2) or (b 3):

(b1) DNA molecule shown in the 2889-3731 th site from the 5' end of SEQ ID No. 1;

(b2) a DNA molecule which hybridizes with the DNA sequence defined in (b1) under stringent conditions and has a promoter function;

(b3) and (b) a DNA molecule having a promoter function and having 90% or more homology with the DNA sequence defined in (b 1).

The expression cassette 1 can be specifically shown as the 2889-10115 position from the 5' end of SEQ ID No. 1;

the promoter sequence in the expression cassette 2 can be a reverse complementary sequence of 1 st-843 rd bits from the 5' end of SEQ ID No. 1; the reverse complement of the other elements of the expression cassette 2 can be specified in SEQ ID No.1 at 20825-23917 from the 5' end.

The expression vector can be specifically a circular plasmid shown as SEQ ID No. 1.

In a second aspect, the present invention provides the use of an expression vector as described hereinbefore for methylation or demethylation of the rice genome.

In a third aspect, the method for demethylating genomic DNA of rice of the present invention comprises the steps of: the expression vector described above is introduced into recipient rice to achieve rice genomic DNA demethylation.

The rice genomic DNA demethylation occurs within the rice nucleus.

The recipient rice may be specifically Nipponbare rice.

The invention provides a set of carrier system for rice epigenome demethylation. The system rapidly generates DNA methylation variation by randomly demethylating the rice genome. The system involves expression of rice OsALKBH DNA6mA demethylase, resulting in extensive hypomethylation. This "mutagenesis" technique is of great importance to the use of plants of agronomic importance, possibly resulting in differential expression of alleles that are normally silenced by DNA methylation, thereby revealing previously hidden phenotypic variations.

Drawings

FIG. 1 shows the detection of protoplasts by fluorescence microscopy.

FIG. 2 shows the detection of 6mA level change in DNA by Dot Blot.

FIG. 3 shows the detection of 6mA level change in DNA by LC/MS.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.