Method for creating fragrant rice by knocking out BADH2 (betaine aldehyde dehydrogenase) gene by utilizing CRISPR/Cas9 system

A systematic knockout and gene technology, applied in genetic engineering, plant gene improvement, chemical instruments and methods, etc., can solve the problem that gene functions cannot be completely removed, and achieve the effect of shortening the breeding period and simple operation of the method

Inactive Publication Date: 2018-11-30
江西省超级水稻研究发展中心
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Problems solved by technology

RNAi technology uses short double-stranded RNA synthesized in vitro to inhibit the expression of BADH2 gene in cells, and quickly obtain aroma rice [Niu X L, Tang W, Huang W Z, et al. RANi-directed downregulation of OsBADH2 results in aroma (2-acetyl-1- pyrroline)production in rice(Oryza sativa L.).BMC Plant Biol,2008,8:10; Chen Ml,Wei X J,Shao G N,Tang S Q,et al.Fragrance of the rice grain achieved viaartificial microRNA-induced down-regulation of OsBADH2.Plant Breeding,2012,131:584-590.], however, the main target of RNAi is mature RNA, which is a gene down-regulation method and cannot completely remove gene function

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  • Method for creating fragrant rice by knocking out BADH2 (betaine aldehyde dehydrogenase) gene by utilizing CRISPR/Cas9 system
  • Method for creating fragrant rice by knocking out BADH2 (betaine aldehyde dehydrogenase) gene by utilizing CRISPR/Cas9 system
  • Method for creating fragrant rice by knocking out BADH2 (betaine aldehyde dehydrogenase) gene by utilizing CRISPR/Cas9 system

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Embodiment Construction

[0032] Specific embodiments of the present invention will be described in detail below.

[0033] A method for creating fragrant rice by knocking out the BADH2 gene using the CRISPR / Cas9 system, comprising the steps of:

[0034] (1) Design the sgRNA sequence based on the BADH2 gene

[0035] Use the online public software CRISPR-P (http: / / cbi.hzau.edu.cn / crispr / ) to design the sgRNA guide sequence, select the guide sequence with a higher score and located at the appropriate position of the BADH2 gene sequence, the one selected in this example The sgRNA guide sequence is the sequence shown in SEQ ID No.2 and SEQ ID No.3.

[0036] (2) Design and synthesize sgRNA primers

[0037] According to the original sgRNA sequence and vector construction requirements designed by the online software, for the sgRNA sequence shown in SEQ ID No.2, the designed forward and reverse primers are sgU6-F

[0038] (cttgACTAGAGACGCTTGATTGT) and sgU6-R

[0039] (aaacACAATCAAGCGTCTCTAGT); for the sgRNA...

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Abstract

The invention discloses a method for creating fragrant rice by knocking out a BADH2 (betaine aldehyde dehydrogenase) gene by utilizing a CRISPR/Cas9 system, belongs to the technical field of rice breeding, and relates to a method for creating fragrant rice by applying the rice betaine aldehyde dehydrogenase (BADH2) gene. According to the method, in the fragrant rice, the function of the BADH2 (betaine aldehyde dehydrogenase) gene is lost, so that a chemical substance of 2-acetyl-1-pyrroline(2-AP) is accumulated so as to generate the fragrance, and a sgRNA guide sequence adopted by the CRISPR/Cas9 system is any one of the sequences which are as as shown in the SEQ ID NO.2 or the SEQ ID NO.3 in the figure 1 in the specification. The method has the advantages that the BADH2 (betaine aldehydedehydrogenase) gene can be successfully knocked out, so that the fragrant rice can be quickly created; and the operation is simple, time and labor are saved, and the breeding life of the fragrant riceis shortened.

Description

technical field [0001] The invention relates to the technical field of rice breeding, in particular to a method for creating fragrant rice by using the CRISPR / Cas9 system to knock out the BADH2 gene. Background technique [0002] Fragrance is one of the important quality indicators of rice, which is mainly controlled by a pair of recessive genes (BADH2) on chromosome 8. In fragrant rice, the function of BADH2 gene is missing, resulting in the chemical substance 2-acetyl-1-pyrroline (2 -AP) accumulates to produce fragrance. At present, rice aroma improvement is mainly through hybrid breeding and genetic engineering breeding. In the process of hybridization and breeding, a large number of repeated multi-generation backcrossing or selfing is required to breed aromatic rice materials with excellent comprehensive properties and pureness. The breeding cycle is long and the workload is heavy. Genetic engineering breeding has opened up a new way of rice breeding, breaking through ...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/29A01H5/00A01H6/46
CPCC07K14/415C12N15/8213C12N15/8261C12N2810/10
Inventor 唐秀英龙起樟王会民黄永兰芦明万建林曹志斌
Owner 江西省超级水稻研究发展中心
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