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Method for increasing rice resistance to stunt virus

A dwarf virus, rice technology, applied to other methods of inserting foreign genetic material, DNA/RNA fragments, introduction of foreign genetic material using vectors, etc., can solve problems such as undiscovered existence

Inactive Publication Date: 2007-05-02
PEKING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Gibberellic acid (GA 3 ) has been used as the most commonly used exogenous hormone to induce the internode elongation of rice. It has strong physiological activity. Although it has been found in many higher plants such as corn and barley, its existence has not been found in rice ( Kobayashi et al., The metabolism of gibberellin A 20 to gibberellin A 1 by tall and dwarf mutants ofOryza sativa and Arabidopsis thaliana, Plant Physiol., 106:1367-1372, 1994)

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  • Method for increasing rice resistance to stunt virus
  • Method for increasing rice resistance to stunt virus
  • Method for increasing rice resistance to stunt virus

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1. Yeast two-hybrid method for detection of cytokines interacting with RDV-P2 in rice

[0030] Using RDV-P2 as the bait protein, yeast two-hybrid method was used to detect cytokines interacting with RDV-P2 in rice. This example uses the MATCHMAKER system. In the MATCHMAKER system, the bait protein is fused to the DNA-binding domain (DNA-BD) of yeast GAL4, and the cDNA library is fused to the activation domain (AD) of GAL4. When the bait protein interacts with the library protein in the yeast reporter gene system, DNA-BD and AD approach each other to form a recombined transcription factor to activate the expression of the four reporter genes. Use the MatchMaker GAL4Two-Hybrid System 3 kit of Clontech Company and operate with reference to the kit instructions (the carrier and bacterial strain used in this example are provided by the kit unless otherwise specified), the specific process includes the following steps:

[0031] 1. Construction of healthy rice cDNA li...

Embodiment 2

[0040] Example 2. Cloning of the full-length sequence of OsKOS1 and further verification of the interaction between OsKOS1 and RDV-P2 by yeast two-hybrid method

[0041] 1. Cloning of the full-length sequence of OsKOS1

[0042] 1. Design the 5' end primer for cloning OsKOS1

[0043] The nucleotide sequence of the cDNA fragment of OsKOS1 screened in Example 1 was analyzed by blast (Basic Local Alignment Search Tool) on the NCBI website. As a result, the similarity between it and a cDNA fragment of rice (GenBankNo.AF088220) reached 95%. %, this fragment is a similar sequence of rice cytochrome P450. The amino acid residue sequence encoded by the retrieved cDNA fragment was compared with the amino acid residue sequence in the rice cytochrome P450 library. The amino acid residue sequences are completely identical. After searching in the rice genome database, the gene Scaffold4604 corresponding to CYP701A8 was obtained, and the amino acid residue sequence encoded by it was compa...

Embodiment 3

[0048] Example 3. Verification of the interaction between OsKOS1 and RDV-P2 by co-immunoprecipitation experiments

[0049] Mammalian cell expression system was used to verify the interaction between RDV-P2 and endogenous-kaurene oxidase-like protein OsKOS1 by co-immunoprecipitation experiments. Referring to the experimental methods of Han et al. and Huang et al. (Han et al., Mechanisms of the TRIF-induced interferon-stimulated response element and NF-κB activation and apoptosis pathways, J Biol Chem, 279:15652-15661, 2004; Huang et al., ZNF216 is an A20-like and IκB kinase γ-interacting inhibitor of NFκB activation, J Biol Chem, 279:16847-16853, 2004), the specific process includes the following steps:

[0050] 1) Construction of eukaryotic expression vectors of RDV-S2 and OsKOS1

[0051] RDV-S2 was cloned into the mammalian cell expression vector pRK-7-HA-Neo (Han et al., Mechanisms of the TRIF-induced interferon-stimulated response element and NF-κB activation and apoptosis...

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Abstract

This invention discloses a method of improving the dwarf virus resistance of rice. The rice with dwarf virus resistance is obtained by transferring the coding gene of interfering RNA of S2 gene (RDV-S2) of the rice dwarf virus into the cell or tissue infected with dwarf virus. This invention uses RNA interfering technology inhibiting the expression of RDV-S2 gene in rice infected with RDV, which made ent-kaurene oxidation enzyme gibberellin playing normal function, and then the level of gibberellin returns to normal, so the dwarfing symptoms of the rice infected with RDV will disappear and it will return to normal growth. The invention has extensive prospective in RDV prevention and cure field, and it also has positive effect in researching pathogenic mechanism of other Reovirus members.

Description

technical field [0001] The invention relates to a method for improving rice resistance to dwarf virus. Background technique [0002] Rice dwarf virus (RDV) belongs to the genus Phytoreovirus of the family Reoviridae. The RDV virus particle is an icosahedron with a diameter of about 70 nanometers, and its genome is composed of twelve double-stranded RNAs. The researchers named them according to the mobility of dsRNA in polyacrylamide gel electrophoresis, from slow to fast. S1 to S12. RDV encodes at least seven structural proteins and five nonstructural proteins. Seven structural proteins include P1, P2, P3, P5, P7, P8, and P9, encoded by S1, S2, S3, S5, S7, S8, and S9, respectively. Five nonstructural proteins include P4, P6, P10, P11, P12, encoded by S4, S6, S10, S11, S12. Among them, P2 and P8 form the virion shell, which is the coat protein of RDV. RDV can reproduce in its host plant, rice, and vector insects (Chrysantha two-spots and Leafhopper electrooptica), and sp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N15/82C12N15/11C12N15/87C12N15/113
Inventor 李毅朱士锋魏春红
Owner PEKING UNIV
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