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Method for rapidly obtaining fragrant rice material by using CRISPR/Cas9 technology

A technology of cas9-badh2 and fragrant rice, applied in the direction of recombinant DNA technology, botany equipment and methods, biochemical equipment and methods, etc.

Pending Publication Date: 2021-08-06
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The study by Chen et al. found that the recessive alleles badh2-E2 and badh2-E7 with loss of function can cause the accumulation of 2-AP and lead to the production of fragrance.

Method used

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  • Method for rapidly obtaining fragrant rice material by using CRISPR/Cas9 technology
  • Method for rapidly obtaining fragrant rice material by using CRISPR/Cas9 technology
  • Method for rapidly obtaining fragrant rice material by using CRISPR/Cas9 technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: The process of obtaining genome editing materials

[0042] 1. Design of target joints

[0043] According to the recognition characteristics of the target site recognized by CRISPR / Cas9, the 26th to 45th bp of the CDS region of the Badh2 gene was selected as the target sequence (the sequence is 5'-AGCTCTTCGTCGCCGGCGAG-3', figure 1 ), the linker primers are as follows:

[0044] Bh2-U3-F: 5'-ggcAGCTCTTCGTCGCCGGCGAG-3';

[0045] Bh2-U3-R: 5'-aaacCTCGCCGGCGACGAAGAGC-3';

[0046] The target sequence is driven by the OsU3 gene promoter.

[0047] 2. Construction of pYLCRISPR / Cas9-Badh2 gene editing vector

[0048] The construction of the gene editing vector refers to the method reported by Mao et al. (Mao Y, et al., Mol Plant, 2013, 6(6): 2008-2011.), and proceeds as follows:

[0049] (1) Target linker preparation

[0050] Dissolve linker primers (Bh2-U3-F: 5'-ggcAGCTCTTCGTCGCCGGCGAG-3'; Bh2-U3-R: 5'-aaacCTCGCCGGCGACGAAGAGC-3') with 1x TE (pH8.0) into 100 μM st...

Embodiment 2

[0079] Example 2: Obtaining of No T-DNA Plants and Analysis of Badh2 Gene Knockout

[0080] Grow T in the incubator 0Generation of genome edited plants, self-fertilization, and harvesting of seeds. After the seeds break dormancy, soak the seeds, sow them in seedling trays, and place them in the cultivation room. When the seedlings grow to two leaves and one heart, the genomic DNA of the plants is extracted by referring to the CTAB method of Murray et al. (Murray M G, et al. ., Nucleic Acids Research, 1980, 8(19): 4321-4326). Hpt gene primers encoding hygromycin (hyg283-F: 5'-TCCGGAAGTGCTTGACATT-3', hyg283-R: 5'-GTCGTCCATCACAGTTTGC-3'), Cas9 gene primers encoding Cas9 nuclease (Cas9T-F: 5' -AGCGGCAAGACTATCCTCGACT-3', Cas9T-R: 5'-TCAATCCTCTTCATGCGCTCCC-3') and sgRNA-encoding gene primers (pYLU3-F: 5'-CGTCTTCTACTGGTGCTAC-3', pYLsgRNA-R: 5'-AGTTGATAACGGACTAGCCTT-3') for detection .

[0081] The PCR reaction system consists of 2.6 μl of sterile water, 2.0 μl of primers (1.0 μl ...

Embodiment 3

[0087] Embodiment 3 fragrance phenotype identification

[0088] Theoretically, by knocking out the Badh2 gene, 2-acetyl-1-pyrroline (2-AP) can be accumulated, making rice fragrant. The present invention uses the chewing method to identify the phenotypes of genome edited plants. Dry the rice seeds, peel off the chaff, put the brown rice in your mouth, crush it with your teeth, and perceive the aroma of rice according to the sense of taste and smell. After each sample is identified, it is necessary to rinse the mouth with water to improve the accuracy of identification. After identification, the rice of BY1~BY22 strains all had fragrance.

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Abstract

The invention discloses a method for rapidly obtaining a fragrant rice material by using a CRISPR / Cas9 technology. The methodcomprises the following steps: designing a target sequence based on sequence characteristics of a Badh2 gene, constructing a CRISPR / Cas9-Badh2 gene editing carrier, converting an unfragrant rice material, identifying an editing site of the genome editing material and carrying out PCR detection on a T-DNA sequence to obtain a T-DNA-free fragrant rice material. The CRISPR / Cas9 technology is used to edit Badh2 gene to obtain a T-DNA-free fragrant rice material. The method has characteristics of simplicity, convenience, rapidness and high efficiency, and has guiding significance on improvement breeding of rice fragrance quality.

Description

technical field [0001] The invention belongs to the fields of crop genetics and breeding and innovation of new crop resources, and specifically relates to a method for quickly obtaining fragrant rice materials by using CRISPR / Cas9 technology. Background technique [0002] Aroma is an important indicator of high-quality rice. There are many kinds of aroma substances in rice, the most important of which is 2-acetyl-1-pyrroline (2-acetyl-1-pyrroline, 2-AP). The inheritance of aroma is mainly controlled by a pair of recessive genes. Bradbury et al. used map-based cloning to isolate and clone the fragrance gene fgr, which encodes betaine aldehyde dehydrogenase 2 (betaine aldehyde dehydrogenase, BAD2). The study by Chen et al. found that the recessive alleles badh2-E2 and badh2-E7 with loss of function can both cause the accumulation of 2-AP and lead to the production of fragrance. Subsequently, Kovach et al. deeply analyzed the origin and evolutionary relationship of the aroma...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/29C12N15/66A01H5/00A01H6/46
CPCC12N15/8213C12N15/66
Inventor 王芳权杨杰许扬陈智慧王军范方军李文奇蒋彦婕陶亚军
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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