Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Site-specific modification method for plant genome

A plant genome, site-specific modification technology, applied in the field of plant genome site-specific modification, can solve problems such as successful development and application

Active Publication Date: 2015-01-21
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
View PDF2 Cites 22 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to various unknown reasons, similar techniques have not been successfully developed and applied in plants

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 modification method for plant genome
  • Site-specific modification method for plant genome
  • Site-specific modification method for plant genome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0236] CRISPR / Cas9 using Streptococcus pyogenes SF370 causes site-directed DNA double-strand breaks in Arabidopsis protoplasts.

[0237] The result is as figure 1 shown. The oligos for constructing YFP1 target site chiRNA are YF-FP F and YF-FP R in Table 1. The results showed that when the YF-FP reporter gene and CRISPR / Cas vector were co-transfected into Arabidopsis protoplasts, a strong YFP signal could be obtained, and the gene repair efficiency based on homologous recombination was as high as 18.8% [(4.76%-0.78% ) / 21.23%]. This shows that the constructed CRISPR / Cas system functions and can efficiently cut DNA sequences in plant cells to generate double-strand breaks.

Embodiment 2

[0239] A single binary vector for Agrobacterium-mediated transformation of Arabidopsis and rice was constructed to express chiRNA and hSpCas9, and two Arabidopsis genes BRI1 and GAI and one rice gene ROC5 were selected to design target sites.

[0240] The result is as figure 2 shown. The Cas9 expression cassette of the vector is identical. For the chiRNA expression cassette, the AtU6-26 promoter was used for Arabidopsis transformation, and the OsU6-2 promoter was used for rice transformation. The oligos corresponding to the chiRNA construction of BRI1 sites 1, 2, and 3 are BRI1chiRNA1F and BRI1chiRNA1R, BRI1chiRNA2F and BRI1chiRNA2R, BRI1chiRNA3F and BRI1chiRNA3R in Table 1, respectively. The oligos corresponding to the chiRNA construction of GAI site 1 are GAI chiRNA1F and GAI chiRNA1R in Table 1. The oligos corresponding to the chiRNA construction of ROC5 site 1 are ROC5chiRNA1F and ROC5chiRNA1R in Table 1.

Embodiment 3

[0242] Generation of stable transgenic plants in Arabidopsis and rice via target loci.

[0243] The result is as image 3 shown. The PCR primers for RFLP identification of BRI1 locus 1 and 3 transgenic plants are BRI1 1F and BRI1 1R in Table 1, and the PCR primers for RFLP identification of BRI1 locus 2 transgenic plants are BRI1 2F and BRI1 2R in Table 1. The PCR primers for RFLP identification of transgenic plants at GAI locus 1 are GAI F and GAI R in Table 1. The PCR primers for RFLP identification of transgenic plants at ROC5 locus 1 are ROC5F and ROC5R in Table 1.

[0244] The results showed that a large proportion of Arabidopsis transgenic T1 plants showed phenotypes similar to the homozygous mutation of the target gene locus in the early growth stage. RFLP restriction analysis showed that the PCR products at the target sites in some transgenic plants had obvious fragments that could not be digested, indicating that the natural restriction sites at the target sites in...

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

No PUM Login to View More

Abstract

Provided is a method for plant genome site-directed modification. Specifically, a method for plant genome site-directed modification introduced by RNA is provided. By utilizing nucleic acid construct with particular structure, site-directed modification may be performed at pre-determined site in plant genome with high efficiency. Useful for screening plant with improved traits efficiently.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for RNA-guided site-directed modification of plant genomes. Background technique [0002] The invention and improvement of sequence-specific nucleases over the past decade or so have shown great power in creating site-directed mutagenesis. Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are the main representatives (Carroll et al., 2006; Christian et al., 2010 ). They are fusion proteins composed of a binding domain array that recognizes a specific nucleic acid sequence and a non-specific nuclease Fok1. After these proteins cut the nucleic acid sequence and generate double-strand breaks, organisms will repair them through two mechanisms of non-homologous end joining or homologous recombination, thereby introducing site-specific changes or modifications. The above techniques have been successfully applied in many species, including...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N5/10A01H5/00
CPCC12N15/8213C12N2800/80
Inventor 朱健康毛妍斐冯争艳张波涛
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com