Magnaporthe oryzae isolated protein for improving plant resistance and inducing defense reaction of plant and gene and application of magnaporthe oryzae isolated protein

A plant resistance and protein technology, applied in the direction of plant genetic improvement, botany equipment and methods, applications, etc., to achieve the effect of low concentration, fast onset, and improved disease resistance

Inactive Publication Date: 2012-09-19
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the properties and types of elicitors produced by the bacteria are different, their protein sequences and gene sequences are rarely reported, so looking for a new protein elicitor and clarifying its sequence information are important for revealing functions and further improving plant health. Resistance is very necessary

Method used

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  • Magnaporthe oryzae isolated protein for improving plant resistance and inducing defense reaction of plant and gene and application of magnaporthe oryzae isolated protein
  • Magnaporthe oryzae isolated protein for improving plant resistance and inducing defense reaction of plant and gene and application of magnaporthe oryzae isolated protein
  • Magnaporthe oryzae isolated protein for improving plant resistance and inducing defense reaction of plant and gene and application of magnaporthe oryzae isolated protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The cultivation of embodiment 1 Magnaporthe oryzae and preparation of fermented liquid

[0030] Inoculate the streaked bacterial block of Magnaporthe oryzae strain on the tomato oatmeal medium [the preparation method of the tomato oatmeal medium is: take 30 grams of oatmeal, add 800 ml of water and boil for 30 minutes, filter off the oat residue, and Take two tomatoes, crush and squeeze the juice, take 150ml tomato juice and add it to the oat filtrate, make up water to 1000ml, add 15g agar, dissolve and aliquot, sterilize at 121°C for 30 minutes] Plate, culture at 28°C for 2 weeks , Pick the edge of the colony and inoculate in 200mL YPD liquid medium [The preparation method of YPD liquid medium is: 10g yeast extract, 20g peptone, 20g glucose (Beijing Chemical Plant, analytical grade), add deionized water to 1L] In a 500ml Erlenmeyer flask, cultured at 26°C and 130r / min for 14 days to obtain a fermented broth of Magnaporthe oryzae.

Embodiment 2

[0031] Preparation and monitoring of the crude protein elicitor of embodiment 2

[0032] Take the fermented liquid obtained in Example 1, centrifuge at 4°C and 5000rpm for 1h (or 13000rpm, 30min), collect the supernatant, and filter twice with a 0.45mm filter membrane (produced by Whatman) until there is no bacterial cell , to obtain the fermentation supernatant. Ammonium sulfate powder was added so that the final concentration of ammonium sulfate in the fermentation supernatant was 80% to precipitate the target protein, dialyzed at 4°C for 48 hours and freeze-dried to obtain extracellular protein after desalination, ie crude protein. Finally, the obtained extracellular protein was dissolved in Mes-NaOH buffer (20 mM, pH 6.0) to obtain a crude protein solution, wherein the final concentration of extracellular protein was 10 μmol / L. The protein content was determined at a wavelength of 590nm with BCATM protein assay kit (Thermo Scientific) and GF-M2000 microplate reader (Shand...

Embodiment 3

[0035] Example 3 Separation and purification of protein elicitors in crude protein solution and determination of biological activity

[0036] use The explore 10 (GE Healthcare) protein purification instrument further purified the obtained crude protein, and the sample was first passed through the HP Q HiTrap TM Anion exchange column (GE Healthcare), carried out linear elution (0%-100%, 30min) with NaCl, obtained protein elution peak (see figure 1), each protein elution peak component is carried out the detection of biological activity, makes the protein concentration be 10 μ mol / L in each component of application, the result shows, protein peak a2 (such as figure 1 shown) can strongly cause allergic reactions to tobacco; the components of the protein peak a2 are further purified by 15% Native-PAGE, rubber tapping purification and electroelution to obtain an active single protein, which requires The activity of the protein elicitor is monitored, and the monitoring method ...

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Abstract

The invention discloses a magnaporthe oryzae isolated protein and gene thereof as well as a nucleotide sequence for applying peptide with 20 amino acids and coding a peptide segment and application of the nucleotide sequence. According to the magnaporthe oryzae isolated protein, the plant resistance can be improved, the defense reaction of the plant can be induced, and the resistance of the plant can be remarkably improved by the magnaporthe oryzae isolated protein; the magnaporthe oryzae isolated protein is low in concentration and quick in onset of action; and rice leaves are treated by using a 10muMHis-MoHrip1 solution for 3 days and then the treated rice leaves are inoculated to magnaporthe oryzae, and the inhibition rate for pathogenesis can reach 63.89 percent. The amino acid sequence of the magnaporthe oryzae isolated protein is shown as SEQIDNO.2. The invention also relates to a gene for coding the protein. The amino acid sequence of the gene is shown as SEQIDNO.1.

Description

Technical field: [0001] The present invention relates to a peptide having more than 20 amino acids, a nucleotide sequence encoding the peptide and its application, in particular to an isolation method capable of improving plant resistance and inducing plant defense response from Magnaporthe oryzae Protein and the nucleotide sequence encoding the protein and its application. Background technique: [0002] Rice blast is one of the important diseases of rice, which can cause a large yield reduction, 40% to 50% yield reduction in severe cases, or even grain failure, which occurs in all rice regions in the world. In terms of taxonomic status, the causative agent of rice blast, Magnaporthe oryzae, belongs to the subphylum Pyrospora in the asexual generation, and belongs to the subphylum Ascomycota in the sexual generation. At present, the control of plant diseases mainly adopts chemical control and breeding varieties. But they often have disadvantages that cannot be eliminated....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C12N15/31C12N15/63C12N15/70C12N1/21C12N1/14C12R1/19C12R1/645
Inventor 曾洪梅邱德文杨秀芬郭立华刘峥袁京京陈铭佳
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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