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
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  • 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 cytotoxic

Method used

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  • 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

Example Embodiment

[0072] Example 1 Construction of vector 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 rice BADH2 gene (LOC number: LOC_Os08g32870) sequence, and design the CRISPR / Cas9-based sgRNA sequence.

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

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

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

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

Example Embodiment

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

[0089] 1. Agrobacterium transformation and colony PCR identification

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

[0091] Colony PCR: Template preparation: Take 200μL of bacterial solution from...

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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

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Application Information

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IPC IPC(8): C12N15/82A01H4/00A01H5/00
Inventor 沈倩李继明徐冉
Owner HUAZHI RICE BIO TECH CO LTD
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