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

Paddy rice BADH2 gene site-directed mutagenesis method through using CRISPR-CAS9 technology

A gene site-directed mutagenesis and CRI-R2 technology, which is applied in recombinant DNA technology, horticultural methods, botanical equipment and methods, etc., can solve the problems of high off-target rate, cumbersome assembly process, and a large number of sequencing tasks

Inactive Publication Date: 2017-05-17
HUAZHI RICE BIO TECH CO LTD
View PDF4 Cites 12 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the assembly process of TALEN technology modules is cumbersome, requires a lot of sequencing work, is costly and has cytotoxicity
The design of ZFN technology depends on the upstream and downstream sequences, which has a high off-target rate and is also cytotoxic

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
  • Paddy rice BADH2 gene site-directed mutagenesis method through using CRISPR-CAS9 technology
  • Paddy rice BADH2 gene site-directed mutagenesis method through using CRISPR-CAS9 technology
  • Paddy rice BADH2 gene site-directed mutagenesis method through using CRISPR-CAS9 technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Construction of carrier pHZ1-CAS9-gRNA-BADH2

[0073] 1. Selection of target sites in rice BADH2 (LOC_Os08g32870) gene

[0074] Log in to the plant transcription factor database (http: / / rice.plantbiology.msu.edu / analyses_search_locus.shtml), query the sequence of the rice BADH2 gene (LOC number: LOC_Os08g32870), and design the sgRNA sequence based on CRISPR / Cas9.

[0075] The nucleotide sequence of the sgRNA action site is 5'-GAGTGGCGCGCCCCCGCGCT-3'.

[0076] 2. Construction of the carrier pHZ1-CAS9-gRNA-BADH2

[0077] 1) Construction of OsU6-BADH2-gRNA-polyT fragment

[0078]The first round of PCR used the Cris-GL3 plasmid as a template, and used primers CRI-F1 and SQ3-R1 to amplify the 20bp target sequence of the OsU6 promoter and BADH2 gene, with a fragment size of 329bp; also using the Cris-GL3 plasmid as a template, using primer SQ3 -F2 and CRI-R2 amplify the 20bp target sequence of the BADH2 gene and gRNA-polyT, the fragment size is 180bp; the second ...

Embodiment 2

[0088] Example 2 Preparation method of rice BADH2 gene site-directed mutation plant

[0089] 1. Agrobacterium transformation and colony PCR identification

[0090] Take the competent Agrobacterium from the -80°C refrigerator and thaw it on ice, add 3-5 μL of plasmid pHZ1-CAS9-gRNA-BADH2 and mix well, put it on ice for 30 minutes, put it in liquid nitrogen for 1 minute, and then heat shock it in a 37°C water bath 1min, add 1mL of YEP medium to the aseptic operating platform, shake on a shaker at 28°C for 2-4h, take it out, centrifuge at 8000rpm / min for 1min, pour off part of the supernatant in the aseptic operating platform, and spread the remaining medium evenly with a spreader. The suspension below was mixed and spread on the YEP plate added with rifampicin and kanamycin, sealed with parafilm and placed in a 28°C incubator for cultivation. After 2 days, pick a single colony, shake the bacteria at 28°C for 24-36 hours, and perform colony PCR to obtain positive colonies.

[0...

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

The invention provides a paddy rice BADH2 gene site-directed mutagenesis method through using CRISPR-CAS9 technology. According to the fact that a paddy rice BADH2 gene is designed based on a sgRNA sequence of CRISPR / Cas9, a DNA fragment with the sgRNA sequence coded is connected to a carrier carrying CRISPR / Cas, and paddy rice is transformed, thereby achieving site-directed mutagenesis of the paddy rice BADH2 gene. The nucleotide sequence of a sgRNA acting site is shown as SEQ ID NO:1. The paddy rice endogenous gene BADH2 is edited through CRISPR-CAS9 technology to obtain a BADH2 mutant, which is convenient and efficient in creation of jasmine rice germplasm resources.

Description

technical field [0001] The invention relates to the field of plant transgenic technology and the field of crop genetic breeding, in particular to a method for site-directed mutation of rice BADH2 gene by using CRISPR-CAS9 technology. Background technique [0002] As a special member of the rice family, fragrant rice has a long history of cultivation. Almost all rice-producing countries or regions in the world have fragrant rice planted. Fragrance is an important characteristic of high-quality rice, and the breeding of fragrant rice has become one of the important contents of rice genetics and breeding. Bradbury et al. (2005) found a gene with sequence differences between basmati rice and common rice—BADH2, which encodes betaine aldehyde dehydrogenase (betaine aldehyde dehydrogenase), which may be related to the synthesis of rice fragrance. Chen et al. (2008) proved that the BADH2 gene controls rice aroma through transgene complementation experiments. There is a natural muta...

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): C12N15/82A01H4/00A01H5/00
Inventor 沈倩李继明徐冉
Owner HUAZHI RICE BIO TECH CO LTD
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