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Method for improving disease resistance of rice through genome editing and sgRNA used in method

A gene editing and transgenic rice technology, applied in the biological field, can solve problems such as difficult to achieve shape-oriented improvement, difficult to achieve precise point mutation, and heavy workload

Active Publication Date: 2020-05-15
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Traditional hybrid breeding is time-consuming, heavy workload, and it is difficult to achieve shape-oriented improvement; physical, chemical, biological and other mutagenesis methods are difficult to achieve precise point mutation

Method used

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  • Method for improving disease resistance of rice through genome editing and sgRNA used in method
  • Method for improving disease resistance of rice through genome editing and sgRNA used in method
  • Method for improving disease resistance of rice through genome editing and sgRNA used in method

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Experimental program
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Effect test

Embodiment 1

[0084] Embodiment 1, preparation recombinant plasmid

[0085] Artificially synthesized recombinant plasmid pCXUN-Cas9-gRNA. The recombinant plasmid pCXUN-Cas9-gRNA is a circular plasmid.

[0086] The nucleotide sequence of the recombinant plasmid pCXUN-Cas9-gRNA is shown in SEQ ID NO:1. SEQ ID NO: 1 from the 5' end, 109-361 nucleotides are reverse complementary to the NOS terminator, 386-4516 nucleotides are reverse complementary to the coding gene of Cas9 protein, 4537-6527 The nucleotide is reverse complementary to the Ubi promoter, the 6751-7154 nucleotide is the U3 promoter, the 7155-7257 nucleotide is the coding gene of the sgRNA (the 7155-7174 nucleotide is the target sequence identification area). The recombinant plasmid pCXUN-Cas9-gRNA expresses sgRNA, and the target sequence of sgRNA is located in the rice OsVQ25 gene.

[0087] The nucleotide sequence of the rice OsVQ25 gene is shown in SEQ ID NO: 2 (genome DNA) or SEQ ID NO: 4 (coding region). From the 5' end of...

Embodiment 2

[0088] Example 2, the acquisition of gene-edited rice and the detection of its traits

[0089] 1. T 0 Acquisition of regenerated plants

[0090] 1. Introduce the recombinant plasmid pCXUN-Cas9-gRNA into Agrobacterium EHA105 to obtain recombinant Agrobacterium.

[0091] 2. Using the genetic transformation method mediated by Agrobacterium, the recombinant Agrobacterium was introduced into Nipponbare to obtain T 0 generation of regenerated plants. Specific steps are as follows:

[0092] (1) Resuspend recombinant Agrobacterium with AAM medium to obtain OD 600nm Bacterial suspension with a value of 0.3-0.5.

[0093] AAM medium: Dissolve 4.3g MS salts&vitamins salt (product of PhytoTechnology), 68.5g sucrose, 0.5g MES, 36g glucose, 500mg casamino acids, 100mL 10×AA amino acids solution and 40mg acetosyringone in deionized water , then adjust the pH value to 5.2, dilute to 1L with deionized water; sterilize at 121°C for 30min.

[0094] 10×AA amino acids solution: Dissolve 8.76...

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Abstract

The invention discloses a method for improving the disease resistance of rice through genome editing and sgRNA used in the method. The method for improving the disease resistance of rice through genome editing aims at reducing the activity and / or expression quantity of OsVQ25 protein in rice. 'Reducing the activity and / or expression quantity of OsVQ25 protein in rice' can be realized by gene editing on the OsVQ25. Designated edition of the OsVQ25 gene is achieved by utilizing the CRISPR / Cas9 technology, and the OsVQ25 gene in rice is knocked out by causing frameshift mutation to obtain a ricenew germplasm with remarkably improved resistance for rice blast and xanthomonas oryzae. The method has a great application and popularization value for rice breeding.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a method for improving rice disease resistance through genome editing and sgRNA used therefor, and in particular to a method for improving rice blast and / or bacterial blight resistance through genome editing and The sgRNA it uses. Background technique [0002] Rice (Oryza sativa L.), a diploid monocot, is one of the most important food crops in the world. Rice blast and bacterial blight are the main diseases that seriously affect the quality and yield of rice worldwide, causing heavy losses to food production and seriously threatening world food security. [0003] Rice blast is caused by the filamentous ascomycete fungus Magnaportheoryzae. It often occurs in various rice areas and has the characteristics of extremely fast epidemic speed. It is one of the most serious and harmful rice diseases in various rice areas in the world, seriously affecting High yield, quality and...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/46A01H1/02
CPCA01H1/02C12N15/113C12N15/8218C12N15/8281C12N15/8282C12N2310/20
Inventor 夏兰琴田金福郝泽芸宁约瑟王国梁
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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