Pathogenic related protein VdGRP1 from verticillium dahliae kleb and coded gene thereof

A technology that encodes genes and proteins. It can be used in genetic engineering, plant genetic improvement, and cells modified by introducing foreign genetic material. It can solve problems such as difficulties in genetic breeding for disease resistance, complex genetic background, and strong differentiation variability.

Active Publication Date: 2011-10-12
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The fundamental reason is that the genetic background of crops such as cotton is complex, and it is difficult to conduct in-depth research at the molecular level. In addition, the reaso...

Method used

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  • Pathogenic related protein VdGRP1 from verticillium dahliae kleb and coded gene thereof
  • Pathogenic related protein VdGRP1 from verticillium dahliae kleb and coded gene thereof
  • Pathogenic related protein VdGRP1 from verticillium dahliae kleb and coded gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, the construction of Verticillium dahliae mutant library

[0048] Verticillium dahliae strain V592 (strain V592 was collected from a cotton field in Xinjiang, and the literature recording this material is a rapid detection of the pathogenic type of Verticillium dahliae in cotton plants. Xinjiang Agricultural Sciences 2010, 47(4): 827-831, The public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences) after culturing on a PDA plate at 25°C, inoculate the spores in Cha's liquid medium (2g NaNO3, 1g KH 2 PO4, 1g MgSO 4 .7H 2 O, 1g KCl, 2mg FeSO 4 .7H 2 O and 30g sucrose / L) and cultured with shaking for 5-8 days until the spore concentration was 1.0×10 7 / mL, the spore culture solution was filtered through 4 layers of sterile gauze to remove mycelium. Centrifuge at 4000rpm for 10min to obtain spores and adjust the concentration of spores to 1.0×10 with induction medium plus AS (acetophthalate syringone) 200mM 6-7 / mL.

[0049] ...

Embodiment 2

[0050] Embodiment 2, the acquisition of mutant vdgrp1

[0051] The pathogenicity of the mutants was identified by infecting the hydroponic cotton with strains, so as to screen the mutants with altered pathogenicity. Select plump cotton seeds (Xinluzao-16, purchased from Agricultural College of Xinjiang Shihezi University), soak with 15% sodium hypochlorite for 30 minutes, rinse with sterile water for 2-3 times, then soak with sterile water to accelerate germination overnight and lay flat Moisturize in the culture box, wait for the buds to grow to 3cm, and plant them in the germination box. Drill holes with a diameter of 2 cm and a spacing of 3 cm on the foam board, wrap the root-bud junction of the germinated cotton with a sponge strip, and stuff it into the holes of the foam board. Place the foam plate on a plastic box (8-10 cm high) filled with clear water, and culture at 25° C. for 16 hours in the light and 8 hours in the dark. When the true leaves grow out, replace the c...

Embodiment 3

[0053] Embodiment 3, the acquisition of mutant gene

[0054] 1), Southern hybridization to determine the mutant T-DNA insertion copy number ( image 3 )

[0055] A, the extraction method of genomic DNA is as follows: In liquid Chapei culture medium, 200rpm shakes and cultivates mycelium. The cultured liquid was centrifuged at 10,000 rpm for 10 min and ground with liquid nitrogen. Add 500 μL of extraction buffer (100mmol / LTris-HCl, PH8.0, 100mmol / L EDTA, 250mmol / L NaCl), vortex to mix the bacterial powder and extraction buffer thoroughly, then add 50μL 20% SDS, shake the Eppendorf tube gently , mix the mixture evenly, put it in a water bath at 37°C for 1h, take it out and add 75L 5mol / L NaCl, mix gently, then add 65μL 10% CTAB and 0.75mol / LNaCl solution, mix gently, and then put it in 65°C Incubate in water for 20 min, take it out, add 700 μL of chloroform / isoamyl alcohol (24:1) solution, mix well, centrifuge at 12000 rpm for 12 min, take the supernatant, add 2 times the vol...

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Abstract

The invention discloses a pathogenic related protein VdGRP1 from verticillium dahliae kleb and a coded gene thereof. The pathogenic related protein is the protein of 1) or 2): 1) the protein consisting of the amino acid sequence shown as sequence 2 in a sequence table; and 2) the protein formed by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid residuesequence shown as sequence 2 in the sequence table, related to pathogenic fungi and derived from 1). A mutant strain vdgrpl of which the pathogenicity is weakened is obtained by screening a verticillium dahliae kleb mutant library mediated by agrobacterium tumefaciens and built through T-DNA insertion technology by the inventor. Southern hybridization proves that the mutant strain is a T-DNA single-insertion mutant. A gene causing mutant phenotype is obtained through TAIL-PCR technology by the inventor. Gene complementation tests prove that the mutation of the gene VdGRP1 weakens the pathogenicity of the strain vdgrpl which means that the gene VdGRP1 is a fungi pathogenic related gene.

Description

technical field [0001] The invention relates to a pathogenicity-related protein from Verticillium dahliae and its coding gene. Background technique [0002] Verticilliu dahliae Kleb., caused by the soil filamentous fungus Verticilliu dahliae Kleb., is a major disease that threatens cotton production. It has seriously affected the quality and yield of cotton in my country for many years. Cotton verticillium wilt was first seen in Virginia in the United States in 1914, and was subsequently discovered in other states and cotton-growing countries around the world (Shen Qiyi, 1992). It was introduced into my country with the introduction of American cotton varieties in 1935, but the damage was not serious. After the 1950s, verticillium wilt occurred successively in some cotton areas in the north and south of my country, and the speed of spread accelerated. In the late 1980s, verticillium wilt had spread to 478 cotton-growing counties (cities) across the country. Since the 1990s...

Claims

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Application Information

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IPC IPC(8): C07K14/37C12N15/31C12N15/63C12N5/10C12N1/00C12N15/80C12R1/91C12R1/645
Inventor 郭惠珊周邦军
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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