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Rice gene OsFd2 and applications of rice gene OsFd2 in rice resistance to rice blast

A rice blast fungus and rice technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., to achieve the effect of improving the ability of rice blast resistance

Active Publication Date: 2021-05-11
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, there is no research on rice chloroplast Fd Report on application of coding gene in rice blast resistance

Method used

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  • Rice gene OsFd2 and applications of rice gene OsFd2 in rice resistance to rice blast
  • Rice gene OsFd2 and applications of rice gene OsFd2 in rice resistance to rice blast
  • Rice gene OsFd2 and applications of rice gene OsFd2 in rice resistance to rice blast

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 OsFd2 Gene acquisition

[0023] Using the protein sequence of Arabidopsis Fd2 in the NCBI database, we identified two homologous proteins of Arabidopsis Fd2 in rice with gene numbers LOC_Os08g01380 and LOC_Os03g61960, and their similarities were 72% and 68%, respectively. . The proteins encoded by the LOC_Os08g01380 and LOC_Os03g61960 genes were 139 and 153 amino acids in length, respectively, and both had chloroplast localization signal peptides at their N-terminals. According to the predicted cDNA sequence (XM_015794929.2) of the gene in the rice variety Nipponbare genome on the NCBI website, we successfully cloned it from the total cDNA of Zhonghua 11 leaves. OsFd2 The full-length open reading frame of 420 bp was found to be consistent with the predicted sequence after sequencing. clone OsFd2 The nucleotide sequences of the primers used in the open reading frame are as follows:

[0024] OsFd2ORF-F SEQ ID NO.5: 5'-ATGGCGGCGACGGCACTGAG-3',

[0025] OsFd...

Embodiment 2

[0027] Example 2 OsFd2 Construction of knockout vector

[0028] exist OsFd2 Select two target sites (sequences are shown in SEQ ID NO.3 and SEQ ID NO.4) in the coding region of the open reading frame of the gene, construct a gRNA expression cassette, and then connect the two target gRNA expression cassettes to the knockout In the vector pC1300-Cas9, obtain OsFd2 knockout vector. The detailed vector construction method refers to the patent "Construction and Application of Plant Multi-gene Knockout Vector, ZL201510485573.2", as follows:

[0029] (1) Selection of target sequence and design of primers

[0030] exist OsFd2 The coding region of the open reading frame of the gene is found to contain 5'-(N) X-The target sequence of the NGG-3' structure, wherein N represents any one of A, T, C and G, and X is 19. According to the above principles, design two target sequences, namely OsFd2-T1 and OsFd2-T2, the sequences are as follows:

[0031] OsFd2-T1 SEQ ID NO.3: 5'-TTGCCGATG...

Embodiment 3

[0056] Example 3 OsFd2 Obtaining knockout mutants

[0057] Transform the correctly sequenced binary expression vector pC1300-Cas9-SK-gRNA-1-gRNA-2 into Agrobacterium tumefaciens EHA105 by electroporation. converted to get OsFd2 Knockout mutant rice plants.

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Abstract

The invention provides a rice gene OsFd2 and applications of the rice gene OsFd2 in rice resistance to rice blast. A CRISPR / Cas9 method is adopted to knock out the rice gene OsFd2, and the phenotypic complementation of mutant plants can be performed through an overexpression vector genetic transformation method. Magnaporthe oryzae inoculation experiments are respectively carried out on wild type, knocked-out mutant and overexpression complemented plants. Results show that the knocked-out OsFd2 significantly enhances the resistance of rice plants to magnaporthe oryzae, but lethal phenotype at seedling stages is generated; and compared with mutants, the OsFd2 overexpression complemented plants is normal in growth and recovery and more susceptible to rice blast then wild types. Thus, the above results show that the OsFd2 is the gene that negatively regulates rice immunity against the rice blast and the key gene required by the normal growth of rice. The content of the invention is to provide an endogenous gene target for enhancing rice resistance through an induced silencing method when field rice blast occurs.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a rice gene OsFd2 and its application in rice blast resistance. Background technique [0002] Rice ( Oryza sativa ) is the most important food crop in my country, and the stable and high yield of rice is an important guarantee for my country's food security. by Magnaporthe grisea ( Magnaporthe oryzae ) causing rice blast has long been the most serious fungal disease that harms rice production. In my country, the rice loss directly caused by rice blast is as high as 3 billion kilograms every year. When the disease is severe, it can lead to a 40%-50% reduction in rice production, or even a total failure. [0003] Plants rely on their own immune system to produce rapid and effective defense responses to external pathogenic microorganisms. The plant innate defense system is mainly composed of two levels of immune responses, namely, the immune response triggered by pathogen-associa...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/84C07K14/415A01H4/00A01H5/00A01H6/46
CPCC07K14/415C12N15/8218C12N15/8282A01H4/005
Inventor 王莫陆民锋时华
Owner FUJIAN AGRI & FORESTRY UNIV
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