Plant guide template in-situ synthesis gene editing method and application

A technology for gene editing and plant variety, applied in the field of plant-guided template in situ synthesis of gene editing

Active Publication Date: 2020-10-09
北大荒垦丰种业股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] However, the application of guided gene editing methods to rice genes to achieve precisely designed point mutations, small insertions, deletions, and substitutions has rarely been reported.

Method used

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  • Plant guide template in-situ synthesis gene editing method and application
  • Plant guide template in-situ synthesis gene editing method and application
  • Plant guide template in-situ synthesis gene editing method and application

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Experimental program
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Effect test

Embodiment 1

[0091] 1. Construction of the basic transformation vector KF120 for guiding gene editing:

[0092] (1) Cloning of the maize ubiquitin gene ZmUBI1 promoter to express Cas9: the Agrobacterium transformation vector for gene editing is based on the pCambia3301 backbone (https: / / www.addgene.org / ), using eukaryotic cell encoding optimized Streptococcus pyogenes Cas9 endonuclease and single guide RNA (sgRNA) were expressed on the same vector to achieve targeted cut-off of the target gene locus, and the transformed plants were screened by expressing the hygromycin phosphotransferase (HPT) gene of Escherichia coli. The maize genome Zea mays Ensembl-18 collected by the US Department of Energy (https: / / phytozome.jgi.doe.gov / pz / portal.html) contains two high-level constitutively expressed ubiquitin genes GRMZM2G409726 and GRMZM2G419891, whose promoters are suitable for It is used for high-level expression of Cas9 gene in grasses such as rice and corn. In order to improve gene editing eff...

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Abstract

The invention belongs to the field of crops, and particularly relates to a plant guide template in-situ synthesis gene editing method and application. The invention firstly discloses a guide gene editing basic transformation vector; the guide template editing basic vector KF120 uses pCampbias 3301 as a vector skeleton, and comprises the following three expression units from the right boundary of T-DNA: ZmU6pro: gRNA: AtU6-26 term; zmUBI1 pro:SpCas9(H840A)-MMLV- RT:PsE9 term; and 35 S pro: HYG: 35 S term.. According to the invention, a guide gene editing method is researched and created and used in plants for the first time; and precisely designed point mutation, small fragment insertion, deletion and replacement are effectively realized at two sites of the rice Waxy gene LOC _ Os06g04200.

Description

technical field [0001] The invention belongs to the field of crops, and in particular relates to a gene editing method for in-situ synthesis of a plant guidance template and its application. 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 can be inserte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/66C12N9/10A01H5/00A01H6/46
CPCC12N15/8245C12N15/8218C12N15/66C12N9/1051C12Y204/01021Y02A40/146
Inventor 李忠森马瑞王明月朱婷刘丹邓艳雪李强
Owner 北大荒垦丰种业股份有限公司
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