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Method for applying CRISPR-Cas9 system to knockout of key gene HPT in VE synthesis pathway of barley

A systematic knockout and barley technology, applied in the field of barley transgenic material construction, can solve the problems of low efficiency of barley gene transformation and long acquisition cycle of stable transgenic materials

Pending Publication Date: 2017-06-30
ZHEJIANG UNIV
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Problems solved by technology

However, due to the low efficiency of barley gene transformation and the long period of obtaining stable transgenic materials, there are few reports on the use of CRISPR-Cas9 system to study barley genes.

Method used

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  • Method for applying CRISPR-Cas9 system to knockout of key gene HPT in VE synthesis pathway of barley
  • Method for applying CRISPR-Cas9 system to knockout of key gene HPT in VE synthesis pathway of barley
  • Method for applying CRISPR-Cas9 system to knockout of key gene HPT in VE synthesis pathway of barley

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Embodiment 1

[0054] Example 1 Obtainment and identification of the key rate-limiting enzyme gene knockout strain HPT in the barley VE synthesis pathway.

[0055] The barley variety used in the present invention is Hordeum vulgare L., cv. Golden promise, and the applicant promises that the barley variety will be released to the public within 20 years from the date of application for this patent, so as to realize and utilize the technical solution described in the present invention.

[0056] 1. Selection of gRNA target sites

[0057] Since the HPT gene is located on chromosome 7 of the barley genome, according to the target site design principle of CRISPR-Cas9 technology, the present invention designs the gRNA target site in the exon region and will be designed at the 5' end of the HPT gene (because the gene It encodes a protein, and the 5' end encodes just the functional region of the protein). Such as figure 1 shown.

[0058] 2. Cloning of gRNA fragments and vector construction

[0059...

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Abstract

The invention relates to construction of a barley transgenic material and provides a method for applying a CRISPR-Cas9 system to knockout of the key gene HPT in the VE synthesis pathway of barley. The method comprises the following steps: selection of a gRNA target site; cloning of gRNA fragments; connection of GG(gRNA-gRNA) fragments; PCR amplification of a connection product; enzyme digestion of a purified product and a target vector; a ligation reaction between the purified product and the target vector; transformation of competent Escherichia coli cells with a ligation product; preparation of Agrobacterium-mediated transgenic barley plants; screening of positive transgenic plants; and sequencing of mutants. According to the invention, the advanced gene editing technology, i.e., the CRISPR-Cas9 system, is employed for target gene editing of the key gene (HPT) in the VE synthesis pathway of barley, and effective loss-of-function mutants are obtained, so conditions are created for research on biologically-active substances in barley.

Description

technical field [0001] The present invention relates to the construction of barley transgenic materials, in particular to the application of the CRISPR-Cas9 system to knock out the HvHPT (MLOC_37476) gene. Background technique [0002] Vitamin E (vitamin E, VE) is a general term for tocopherol compounds synthesized by photosynthesis. According to whether the side chain is saturated, VE can be divided into two categories: tocopherol (tocopherol1) and tocotrienol (tocotrieno1). According to the position and number of methyl groups on the aromatic ring, each type can be divided into four forms: α, β, γ, and δ. Among them, α-tocopherol has the highest activity. In recent years, due to the superior biological characteristics of tocotrienols in some aspects, it has attracted much attention. Not only in antioxidant activity, but also in lowering cholesterol, preventing diabetes, promoting bone resorption, anti-cancer, neuroprotection, etc. [0003] Barley is one of the four majo...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H4/00A01H5/00
CPCA01H4/00C12N9/1085C12N15/8213C12Y205/01
Inventor 边红武曾章慧刘翠翠韩凝朱睦元
Owner ZHEJIANG UNIV
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