Gene editing method for aroma control gene of improved indica rice strain and application thereof

A gene editing and gene technology, applied in the field of crops, to achieve the effect of efficient gene editing

Active Publication Date: 2021-11-05
北大荒垦丰种业股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, how to perform multigene and multilocus efficient gene editing transformation on rice varieties by gene editing methods to obtain homozygous edited indica rice lines with fragrance, non-transgenic and stable inheritance has not been reported yet.

Method used

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  • Gene editing method for aroma control gene of improved indica rice strain and application thereof
  • Gene editing method for aroma control gene of improved indica rice strain and application thereof
  • Gene editing method for aroma control gene of improved indica rice strain and application thereof

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Experimental program
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Effect test

Embodiment 1

[0081] 1. Construction of gene editing transformation vector KF80:

[0082] Gene editing based on Streptococcus pyogenes acquired immune system CRISPR endonuclease Cas9 has been achieved in a variety of plants, and the NHEJ editing efficiency is often as high as 90%. Refer to the design of gene editing Agrobacterium transformation vector pHSE401 (Xing et al. , 2014, BMC Plant Biol 14:327), through the use of monocotyledonous plant-specific maize ubiquitin gene promoter ZmUBI1 pro and a maize U6 snRNA gene promoter ZmU6 pro, respectively for high-efficiency expression in grasses such as rice and maize Cas9 and gRNA genes, the final Agrobacterium transformation DNA binary vector for gene editing consists of three gene expression units ZmU6 pro:gRNA:AtU6-26 term, ZmUBI1 pro:SpCas9:PsE9 term, and 35S pro:HygR:35S term , There is a BsaI restriction site between ZmU6 pro and gRNA, and any gRNA sequence can be inserted using DNA ligase or Gibson cloning method after the vector is lin...

Embodiment 2

[0117] 1. Construction of gene editing transformation vector KF80:

[0118]Gene editing based on Streptococcus pyogenes acquired immune system CRISPR endonuclease Cas9 has been achieved in a variety of plants, and the NHEJ editing efficiency is often as high as 90%. Refer to the design of gene editing Agrobacterium transformation vector pHSE401 (Xing et al. , 2014, BMC Plant Biol 14:327), through the use of monocotyledonous plant-specific maize ubiquitin gene promoter ZmUBI1 pro and a maize U6 snRNA gene promoter ZmU6 pro, respectively for high-efficiency expression in grasses such as rice and maize Cas9 and gRNA genes, the final Agrobacterium transformation DNA binary vector for gene editing consists of three gene expression units ZmU6 pro:gRNA:AtU6-26 term, ZmUBI1pro:SpCas9:PsE9 term and 35S pro:HygR:35S term, There is a BsaI restriction site between ZmU6 pro and gRNA, and any gRNA sequence can be inserted using DNA ligase or Gibson cloning method after the vector is lineari...

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Abstract

The invention belongs to the field of crops, and particularly relates to a gene editing method for aroma control gene of improved indica rice strain and application thereof. Firstly, a gene editing transformation vector is disclosed, which takes pCambia3301 as a vector skeleton and comprises a ZmU6 promoter and a ZmUBI1 promoter; the gene editing transformation vector also comprises a plurality of gRNA fragments which are designed according to different exon region recognizable sites of the Badh2 gene; the gRNA fragments are located between the ZmU6 promoter and the ZmUBI1 promoter, and the gRNA fragments and the ZmUBI1 promoter are synchronously expressed through the ZmU6 promoter; the gRNA fragments are interactively connected through a tRNA technology. According to the invention, efficient gene editing transformation of multiple gene loci is carried out on a rice variety through a gene editing means, and a plurality of fragrant, non-transgenic and stably inherited homozygous edited indica rice strains are obtained.

Description

technical field [0001] The invention belongs to the field of crops, and in particular relates to a gene editing method and application of an aroma control gene of an improved strain of indica rice. Background technique [0002] The rapid development of gene editing technology in recent years uses specific endonucleases to selectively cut specific genes in cells, through two DNA repair mechanisms inherent in cells, non-homologous end joining (NHEJ) and homologous end joining (NHEJ) Rely on Homology dependent repair (HDR), repair the fracture, and ensure the survival of cells. NHEJ has high repair efficiency, does not require a template, and occupies a dominant position, but the process of cutting point linkage will introduce insertion or deletion mutations, thereby inactivating the gene; HDR needs to follow the template, although the repair is accurate, but the proportion is very small. If provided The pre-designed DNA fragment is used as a template, and the fragment sequenc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/66C12N15/53C12N15/113A01H4/00A01H5/00A01H6/46
CPCC12N15/8261C12N15/66C12N9/0008C12N15/113C12Y102/01008A01H4/008A01H5/00A01H6/4636C12N2830/34
Inventor 马瑞李忠森庄世界王明月朱婷刘丹
Owner 北大荒垦丰种业股份有限公司
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