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Pyricularia oryzae resistant oryza sativa gene OsWRKY47 and application thereof

A technology of rice blast fungus and rice, applied in the field of rice gene resistant to blast fungus

Inactive Publication Date: 2012-09-19
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing transgenic results are rarely put into production applications, and the key genes involved in rice blast resistance have yet to be discovered, and how to discover these genes and identify large-scale transformed rice remains to be further studied

Method used

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  • Pyricularia oryzae resistant oryza sativa gene OsWRKY47 and application thereof
  • Pyricularia oryzae resistant oryza sativa gene OsWRKY47 and application thereof
  • Pyricularia oryzae resistant oryza sativa gene OsWRKY47 and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, the cloning of gene

[0028] (1) Cloning of OsWRKY47 gene cDNA sequence:

[0029] The genomic DNA sequence (SEQ ID No: 1), cDNA sequence (SEQ ID No: 2) and amino acid sequence (SEQ ID No: 3) of the OsWRKY47 gene were obtained from the MSU / TIGR rice genome database (http: / / rice.plantbiology .msu.edu / ).

[0030] Design gene-specific PCR primers according to the cDNA sequence of the OsWRKY47 gene:

[0031] F1: 5'-ATG GCG TCT CCT GAT GGT GG-3' (SEQ ID No: 4);

[0032] R1: 5'-TTAAGGATC GAAGCCAAACA-3' (SEQ ID No: 5).

[0033] These primers were used to clone the cDNA sequence of OsWRKY47 gene from the cDNA of the rice japonica rice (Oryza sativa L. ssp. japonica) near-isogenic line variety IRBL22 (bred by International Rice). Since the GC content in some regions of the cDNA sequence of the OsWRKY47 gene is as high as 80%, it cannot be cloned with common high-fidelity enzymes. After many experiments, the inventors used GC buffer I (Takara) and Pfu high-fideli...

Embodiment 2

[0036] Embodiment 2, the acquisition of transgenic rice constitutively expressing the OsWRKY47 gene

[0037] (1) Rice callus induction:

[0038] Select plump Taipei 309 rice seeds, peel off the seed coat, sterilize and wash, and evenly inject it into the sterilized NB solid with 2 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) Medium, 32°C continuous light for 5 days to induce callus formation.

[0039] (2) Agrobacterium transformation:

[0040] The pWM101 vector carrying the OsWRKY47 gene cDNA fragment was transformed into Agrobacterium EHA105 competent cells by electric shock method, coated with solid LB medium with 50 μg / L kanamycin, cultured in the dark at 28°C for 2 days, and then used gene-specific Positive clones were screened with primers F1 and R1. The obtained positive clones were cultured in the dark at 28° C. for 3 days in solid AB medium containing 50 mg / L kanamycin and used for rice transformation.

[0041] (3) Rice callus transformation:

[0042] Wash the Agro...

Embodiment 3

[0047] Embodiment 3, constitutively expressed OsWRKY47 gene has enhanced resistance to rice blast

[0048] (1) Detection of OsWRKY47 gene expression level in transgenic rice:

[0049] The leaves of the transgenic rice seedlings were clipped, and the total plant RNA was extracted with Trizol reagent (Invitrogen Company). After the DNA was digested with DNaseI (Takara Company), reverse transcription was performed with the reverse transcription kit of Invitrogen Company to obtain the cDNA of the transgenic plants. Gene-specific real-time quantitative PCR primers realF and realR were designed according to the cDNA sequence of the OsWRKY47 gene, and the expression level of the OsWRKY47 gene in transgenic plants was detected by real-time quantitative PCR. The internal reference gene was the UBQ gene of rice (realF2 and realR2 were used as primers). The sequences of each primer are as follows:

[0050] realF: 5'-GCG CGA GCA CAA GCA GAG TC-3' (SEQ ID No: 6);

[0051] realR: 5'-CAG TTG...

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Abstract

The invention discloses a pyricularia oryzae resistant oryza sativa gene OsWRKY47 and an application thereof. The gene is used for coding the protein with the amino acid sequence shown in SEQ IDNo:3 in a sequence table. The genomic DNA sequence and cDNA sequence of the gene are respectively shown in SEQ IDNo:1 and SEQ IDNo:2 in the sequence table. The gene OsWRKY47 has the function of improving the pyricularia oryzae resistance of the plants, is utilized to transform oryza sativa to obtain a disease resistant plant strain and enhances the resistance of oryza sativa to pyricularia oryzae.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a rice gene resistant to blast fungus and the application of the gene. Background technique [0002] Rice (Oryza sativa L.) is an important food crop in the world. Rice blast is the most serious rice disease. It occurs to varying degrees throughout the year and can lead to a 10-30% reduction in rice production. How to prevent and control rice blast becomes a problem It is a major issue of food security in all countries, especially the major rice producing countries. Selecting disease-resistant varieties is an important means of controlling rice blast: through traditional genetic breeding, some rice varieties with good blast resistance have been obtained at home and abroad, but the traditional breeding cycle is too long and inefficient, and due to the The simplification and the genetic complexity and pathogenic diversity of the physiological races of...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00
Inventor 瞿礼嘉魏桐秦跟基顾红雅
Owner PEKING UNIV
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