Site-specific insertional inactivation method and application mediated by agrobacterium tumefaciens and CRISPR/Cas9

A technology of Agrobacterium tumefaciens and genes, applied in biochemical equipment and methods, viruses/bacteriophages, DNA/RNA fragments, etc., can solve problems such as inability to insert precisely at specific sites

Active Publication Date: 2017-02-22
GUANGXI UNIV
View PDF0 Cites 39 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] So far, effective genetic manipulation cannot be carried out by transforming protoplasts in S. cane whip. It can only rely on Agrobacterium-mediated transformation

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Site-specific insertional inactivation method and application mediated by agrobacterium tumefaciens and CRISPR/Cas9
  • Site-specific insertional inactivation method and application mediated by agrobacterium tumefaciens and CRISPR/Cas9
  • Site-specific insertional inactivation method and application mediated by agrobacterium tumefaciens and CRISPR/Cas9

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0025]The Agrobacterium tumefaciens and CRISPR-Cas9-mediated gene site-directed insertion inactivation method of the present invention is through the endogenous U6 gene promoter (Ss PU6, the base sequence of SEQ.ID.No.17; Ss U6RNA, the base sequence of SEQ.ID.No.18) was fused with the target sequence of the inserted site and the sgRNA sequence by Overlapping PCR, and recombined with the endogenous gapd gene of Ustilago sativa by In-fusion technology In the binary vector of the Cas9 gene and hygromycin resistance gene driven by the subunit; then Agrobacterium tumefaciens carrying the binary vector mediated the transformation of Ustilago sativa so as to achieve the site-directed insertion of the Ustilago sativa genome .

[0026] In order to further clarify the above inventive concept, the sugarcane smut pheromone gene mfa2 is taken as an example for illustration. The experimental methods in the following examples, unless otherwise specified, are conventional methods; the experi...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

PropertyMeasurementUnit
Apertureaaaaaaaaaa
Login to view more

Abstract

The invention discloses a site-specific insertional inactivation method mediated by agrobacterium tumefaciens and CRISPR/Cas9. Endogenous snRNA promoter of Ustilago scitaminea (u6 promoter of Ustilago scitaminea) is used to drive sgRNA expression cassette. CRISPR/Cas9 system integrates with Ti plasmid of agrobacterium tumefaciens to construct the Ustilago scitaminea site-specific insertional inactivation system mediated by agrobacterium tumefaciens with hygromycin as a selection marker. Specific sequence of the objective gene is cloned into sgRNA expression cassette for transformation of Ustilago scitaminea basidiospores, thus the mobile DNA fragment is exactly inserted into the objective gene sequence of Ustilago scitaminea, achieving the goal of damage to gene function. The invention provides an important tool for study of Ustilago scitaminea functional genomics. The system has the advantages of high efficiency and accuracy, and is convenient to use to conduct gene function researches in Ustilago scitaminea.

Description

technical field [0001] The invention belongs to a molecular tool and an experimental system for fungal gene knockout in the field of microbial genetic engineering, and in particular relates to a gene-directed insertion inactivation method mediated by Agrobacterium tumefaciens and CRISPR-Cas9 and its application. Background technique [0002] Sugarcane whip smut (Sporisorium scitamineum) is the pathogenic bacterium of sugarcane smut, which belongs to Basidiomycotina smut. The sugarcane smut caused by it is a global disease, which has caused great harm to sugarcane production in my country. There are three distinct stages in the life cycle of Ustilago sativa: yeast-like haploid, dikaryotic hyphae, and diploid teliospores. [0003] Gene inactivation technology is a genetic engineering technology that changes the specific gene sequence by changing the genetic genes of organisms, resulting in gene inactivation and loss of function, and then deduces the biological function of the...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N15/113C12N15/80
CPCC07K14/37C12N15/80C12N2800/30
Inventor 陈保善卢姗吕哲姚颜萍沈笑瑞蔡晓薇
Owner GUANGXI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap