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Magnaporthe oryzae avirulence gene AvrPik/kp/km/kh/7 and application thereof

A non-toxic gene and rice blast fungus technology, applied in the field of genetic engineering, can solve the problems of unstable resistance of disease-resistant varieties and unresolved susceptibility of disease-resistant varieties.

Inactive Publication Date: 2012-01-04
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the diversity and variability of the blast fungus population, the resistance of resistant varieties is unstable; so that the problem of susceptibility of resistant varieties has not been resolved

Method used

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  • Magnaporthe oryzae avirulence gene AvrPik/kp/km/kh/7 and application thereof
  • Magnaporthe oryzae avirulence gene AvrPik/kp/km/kh/7 and application thereof
  • Magnaporthe oryzae avirulence gene AvrPik/kp/km/kh/7 and application thereof

Examples

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Embodiment 1

[0043] Embodiment 1: Magnaporthe grisea avirulent gene AvrPik / kp / km / kh / 7 The cloning method and the construction of the electron physical map

[0044] Previous positioning studies have shown AvrPi7 The gene loci were finally defined within a 75 kb segment between markers MS1-21 and MS1-22, which were closely linked to the target gene and landed on the supercontig supcontig of the genome sequence of the old version of the reference strain 70-15 On 2.17 (Feng et al. 2007, Chinese Science Bulletin .2007, 52: 903-911). In July 2008, the latest version 6.0 database of the complete genome sequence of Magnaporthe grisea was released (http: / / www.broad.mit.edu / annotation / genome / magnaporthe_grisea / MultiHome.html), making the complete genome sequence of Magnaporthe grisea been supplemented and improved. Therefore, the present invention removes the molecular markers ASP-CO39, CAG2, MS1-21, MS1-25, and MS1-15 located on supercontig 2.17 according to the differences between the new a...

Embodiment 2

[0045] Embodiment 2: Magnaporthe grisea avirulent gene AvrPik / kp / km / kh / 7 Predictive annotation and sequence analysis of candidate genes

[0046] to confirm AvrPik / kp / km / kh / 7 The candidate gene of the present invention utilizes the reference sequence of bacterial strain 70-15, utilizes 2 gene annotation systems Broad Magnaporthe grisea Database Browse Regions (http: / / www.broad.mit.edu / annotation / genome / magnaporthe_grisea / Regions.html) and Softberry FGENESH 2.6 ( http: / / www.softberry.com / berry.phtml) carried out gene prediction and annotation analysis and summary on the target gene region, and initially determined AvrPik / kp / km / kh / 7 The candidate avirulent gene is a candidate gene whose encoded protein has an N-terminal secretion signal peptide, and its function is confirmed by the following gene function complementation experiment.

Embodiment 3

[0047] Embodiment 3: Magnaporthe grisea avirulent gene AvrPik / kp / km / kh / 7 Transformation and non-toxic identification of transformants

[0048] Using high fidelity (high fidelity, HF) PCR technology to amplify from the donor strain CHL346 genomic DNA (PCR amplification conditions: pre-denaturation 98 ℃ 30 sec, then 98 ℃ 10 sec, 63 ℃ 15 sec, 72 ℃ 1 min, amplified for 35 cycles, and finally extended at 72°C for 10 min.) The approximately 2.1 kb fragment of the candidate gene was cloned into the binary transformation vector pBHt2, and then introduced into the Agrobacterium strain C58C1 (Wu Yixin et al., 2008, Acta Plant Protection, 35: 421-426; Mullins et al. 2001, Phytopathology 91:173-180; Khang et al. 2007, Methods in Molecular Biology 344: 403-420). Then, through the method of Agrobacterium-mediated genetic transformation of Magnaporthe grisea, the gene was introduced into the virulent recipient strains CHL688 and CHL724, and the function gain experiment based on genetic co...

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Abstract

The invention discloses a magnaporthe oryzaea virulence gene AvrPik / kp / km / kh / 7 and an application thereof. The new magnaporthe oryzae avirulence gene AvrPik / kp / km / kh / 7 has DNA (Deoxyribose Nucleic Acid) sequences with promoters indicated by SEQ ID No. 1 and cDNA (Complementary Deoxyribose Nucleic Acid) sequences indicated by SEQ ID No. 2. The gene has three SNP (Single Nucleotide Polymorphism) haplotypes in the natural world. The invention also relates to a molecular target for designing novel pesticides according to the structure of the gene and the function thereof; the gene and a corresponding disease-resistant gene are covalently guided into oryza sativa L and other host plants to cultivate disease-resistant varieties; a specific molecular marker generated by the gene sequences is applied to the monitoring of magnaporthe oryzaea populations in a field; and the disease-resistant varieties are guided to be applied to the reasonable layout according to the monitored results.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an avirulent gene of rice blast fungus AvrPik / kp / km / kh / 7 and its application. Background technique [0002] Rice ( Oryza sativa L.) is an important food crop and an important industrial raw material for the survival of billions of people in the world, and it is also a host of various diseases and insect pests (Normile, 2008). by pathogenic fungi Magnapothe oryzae (Asexual: Pyricularia oryzae Sacc.) is one of the most devastating diseases in rice production in the world, causing 10-30% rice yield loss every year (Baker et al., 1997). Moreover, the fungus can also infect more than 50 kinds of gramineous plants such as wheat and cereals. [0003] From the point of view of environmental protection and agricultural sustainable development, the breeding and utilization of disease-resistant varieties is the safest and most effective way to control rice...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/11C12N15/63C12N1/14C12Q1/68C07K14/37A01H5/00
Inventor 潘庆华王文娟吴伟怀冯淑杰林菲王玲
Owner SOUTH CHINA AGRI UNIV
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