Method for regulating methylation level of plant genome DNA (Deoxyribose Nucleic Acid) specific region

A plant genome and specific region technology, which is applied in the field of regulating the methylation level of specific regions of plant genome DNA to achieve the effect of ensuring food security

Pending Publication Date: 2022-08-02
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Abstract
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  • Claims
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Problems solved by technology

Using the CRISPR system to specifically change the methylation level of a specific gene region is an important way to study the regulation of gene expression by DNA methylation modification, but there is no similar tool in rice and other monocotyledonous plants that can specifically regulate the target site point methylation level

Method used

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  • Method for regulating methylation level of plant genome DNA (Deoxyribose Nucleic Acid) specific region
  • Method for regulating methylation level of plant genome DNA (Deoxyribose Nucleic Acid) specific region
  • Method for regulating methylation level of plant genome DNA (Deoxyribose Nucleic Acid) specific region

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Embodiment 1

[0076] Example 1. Regulation of methylation levels in specific regions of rice genomic DNA

[0077] 1. Construct A1 and Construct A2

[0078] The plasmid map of construct A1 is as follows figure 1 As shown, construct A1 is an expression construct for reducing the methylation level of the target and nearby regions, which can express Tet1CD-dCas9 fusion protein, the nucleic acid sequence of Tet1CD-dCas9 is shown in SEQ ID No. As shown in SEQ ID No.12. Positions 1-741 of SEQ ID No. 12 are Tet1CD domains (encoding nucleic acids are positions 1-2223 of SEQ ID No. 11); positions 742-2243 are dCas9 domains (encoding nucleic acids are positions 1-2223 of SEQ ID No. 11) of bits 2224-6732). The original vector of this construct is the conventional CRISPR / Cas9 vector for rice transformation (pH-nCas9-PBE, described in "Zong, Yuan, et al." Precise base editing inrice, wheat and maize with a Cas9-cytidine deaminase fusion." Naturebiotechnology 35.5(2017):438." article), construct A1 wa...

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Abstract

The invention discloses a method for regulating the methylation level of a plant genome DNA (Deoxyribonucleic Acid) specific region. The invention provides a product for regulating and controlling the methylation level of a specific region of plant genome DNA. The product comprises 1) a methylation regulation and control fusion protein (comprising a nuclease inactivated Cas9 structural domain and a methylation regulation and control structural domain) or an expression construct of the methylation regulation and control fusion protein; and 2) a guide RNA or an expression construct thereof; the methylation regulation and control structural domain is a Tet1cd structural domain or an OsSUVH2 structural domain. The method has important significance for deeply researching different characteristics of DNA methylation between species and in the species, regulating and controlling effects of the DNA methylation on important agronomic characters, maintaining mechanisms of DNA methylation and the like. The phenotypic characteristics and stress response of crops under different environmental conditions and stress pressures can be finely regulated and controlled by regulating and controlling the spatio-temporal expression mode and level of the gene on the premise that the gene sequence is not changed, so that excellent crop varieties are cultivated, and the safety of grains in the world is favorably guaranteed.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a method for regulating the methylation level of a specific region of plant genomic DNA. Background technique [0002] Methylation is an important form of plant genome DNA modification, which plays an important role in the regulation of chromatin structure and adjacent gene expression, and is involved in silencing transposon elements to maintain genome stability, thereby affecting plant growth and development. At the same time, DNA methylation is an epigenetic modification, and part of the DNA methylation modification status can be inherited to the next generation to form stable epialleles, such as the FLOWERINGWAGENINGEN gene in Arabidopsis (Gallego-Bartolomé, Gardiner et al. al. 2018) and the Epi-d1 locus in rice (Miura, Agetsuma et al. 2009). DNA methylation is widespread in higher eukaryotes, fungi, and bacteria. It mainly exists in the form of 5mC in higher eukaryot...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/82A01H5/00A01H6/46
CPCC07K14/415C12N15/8216C07K2319/00
Inventor 李家洋宋晓光余泓孟祥兵
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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