Protein polynucleotide with separated verticillium dahliae and application thereof

A polynucleotide and protein technology, applied in the field of proteins isolated from Verticillium dahliae, can solve the problems of less protein sequence and gene sequence, and achieve the effects of quick onset, improved resistance, and improved disease resistance.

Inactive Publication Date: 2012-07-11
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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  • Protein polynucleotide with separated verticillium dahliae and application thereof
  • Protein polynucleotide with separated verticillium dahliae and application thereof
  • Protein polynucleotide with separated verticillium dahliae and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1 The cultivation of Verticillium dahliae and preparation of fermented liquid

[0027] Inoculate the streaked bacterial block of Verticillium dahliae strain on PDA [take 200 grams of peeled potatoes, cut into pieces, add 1000 ml of water and boil for 30 minutes, filter the potato pieces, make up the filtrate to 1000 ml, add Glucose 20 grams, agar 15 grams, after melting, sub-package, sterilized at 121 ° C for 30 minutes] plates, cultured at 25 ° C for 2 weeks, picked the edge of the colony and inoculated in 200 mL of Czapek-Dox plus 1% hydrolyzed tyrosine (Sigma ) [sodium nitrate 3.0g, anhydrous dipotassium hydrogen phosphate 1.0g, magnesium sulfate heptahydrate 0.5g, potassium chloride KCl 0.5g, ferrous sulfate heptahydrate 0.01g, sucrose 30.0g (Beijing Chemical Plant, analytically pure), pH 5.6, add deionized water to 1L] liquid medium in a 500ml Erlenmeyer flask, culture at 25°C and 150r / min for 14 days to obtain a fermentation broth of Verticillium dahlia...

Embodiment 2

[0028] Preparation and monitoring of the crude protein elicitor of embodiment 2

[0029] The fermentation broth obtained in Example 1 was centrifuged at 4°C and 5000rpm for 1h (or 13000rpm for 30min), and the supernatant was collected and filtered twice with a 0.45mm membrane filter (Whatman) until there were no bacteria. Ammonium sulfate powder was added so that the final concentration of ammonium sulfate in the supernatant was 80% to precipitate the target protein, dialyzed at 4°C for 48h, and finally the obtained extracellular total protein was dissolved in Tris-HCl buffer (20mM, pH 8.0), The final protein concentration was 100 μg / mL. Protein content with BCA TM Protein assay kit (Thermo Scientific) was determined at a wavelength of 590 nm by GF-M2000 microplate reader (Shandong Gaomi Caihong Analytical Instrument Co., Ltd.).

[0030] Monitoring of biological activity: Tobacco with 5-7 true leaves grown for 2 months was used as material, Tris-HCl buffer solution and BSA s...

Embodiment 3

[0032] Example 3 Separation and purification of protein elicitors and determination of biological activity

[0033] use The explore 10 (GE Healthcare) protein purification instrument further purified the obtained total protein, and the sample was first passed through the HP Q HiTrap TM Anion exchange column (GE Healthcare), linear elution with NaCl (0%-100%, 30min), obtained 6 protein elution peaks, the applied protein concentration was 50μg / mL, the results showed that the 3rd protein peak , can strongly cause an allergic reaction to tobacco; the protein peak is further purified by pheny HP hydrophobic chromatography column (GE Healthcare) and electroelution to obtain an active single protein. The monitoring of protein elicitor activity during the above purification process was carried out according to the method in Example 2. The single protein peak is detected as a single band by 15% denatured polyacrylamide gel electrophoresis, indicating that the protein is of high puri...

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Abstract

The invention provides verticillium dahliae protein capable of inducing resistance allergic reaction on tobaccos and other plants. The amino acid sequence of the protein is indicated in SEQ (sequencer) ID NO:2. One coding follows the gene of the protein in the invention, and the nucleotide sequence of the protein is indicated in SEQ ID NO:1. The protein in the invention can induce generation of defense signal molecules relative to disease resistance of the tobaccos. Under relatively low concentration, the protein can induce expression of genes relative to disease resistance of the plants; and secondary metabolites are increased to improve the capability of defending pathogenic bacteria of a cell wall. When the protein is injected into the tobacco plant, necrotic spots are formed, and simultaneously, the capability of resisting tobacco mosaic virus (TMV) of the whole tobacco is strengthened. The protein has the advantages of obviously enhancing resistance of the plants, being low in concentration, having a rapid effect, and providing a new path for improving disease resistance of the plants, so that the protein has a wide application prospect on agricultural production.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and particularly relates to a protein isolated from Verticillium dahliae, which can improve plant resistance and induce plant defense response, and a nucleotide encoding the protein and its application. Background technique: [0002] Verticillium wilt of cotton is a very harmful vascular disease. Bring great harm to the cotton production of my country every year, have a strong impact on the quality and output of my country's cotton production. Cotton Verticillium dahliae belongs to the genus Verticillium genus Leucospora in the taxonomic position of the half-knowledge fungus Hypomycetes, Hyphospora, Verticillium dahliae, and Verticillium dahliae. It is Verticillium dahliae. At present, the control of plant diseases mainly adopts chemical control and breeding varieties. But they often have disadvantages that cannot be eliminated. With the development of biotechnology in the last century, plant-in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/37C12N15/31C12N15/63C12P21/02A01N63/04A01P21/00
CPCC12N15/8279C07K14/415C12N15/8282C07K14/37
Inventor 邱德文曾洪梅杨秀芬郭立华袁京京王炳楠
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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