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Marine penicillium citrinum antifungal protein PcPAF and preparation method thereof

An anti-fungal protein, Penicillium citrinum technology, applied in the field of anti-fungal protein, can solve problems such as loss of quality and safety of agricultural products

Inactive Publication Date: 2013-12-18
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Some crop diseases caused by viruses, bacteria and fungi have caused losses and quality and safety problems of agricultural products
At present, the prevention and control of plant diseases mainly rely on chemical pesticides, but the use of pesticides is strictly regulated and controlled

Method used

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  • Marine penicillium citrinum antifungal protein PcPAF and preparation method thereof
  • Marine penicillium citrinum antifungal protein PcPAF and preparation method thereof
  • Marine penicillium citrinum antifungal protein PcPAF and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Chromatographic separation of protein components of Penicillium citrinum (Penicillium citrinum) W1 using diethylaminoethyl (DEAE) anion-exchange chromatographic column

[0063] Use the starting buffer (20mmol / L Tris-HCl, pH8.1) to equilibrate the diethylaminoethyl (DEAE) anion-exchange chromatographic column, and prepare about 2ml of crude protein components of Penicillium citrinum W1 in diethylaminoethyl Base (DEAE) anion-exchange chromatographic column was loaded, using two column volumes of starting buffer (20mmol / L Tris-HCl, pH8.1) to wash the chromatographic column, to obtain diethylaminoethyl ether under the condition of low salt concentration The non-adsorbed component P1 on the base (DEAE) anion exchange column, and then continue to wash the diethylaminoethyl (DEAE) anion exchange with washing buffer (1mol / L NaCl, 20mmol / L Tris-HCl, pH8.1) chromatographic column to elute the rest of the adsorbed components ( figure 1 ).

Embodiment 2

[0064] Embodiment 2: Non-adsorbed component P1 uses carboxymethyl (CM) cation exchange chromatographic column to carry out chromatographic separation

[0065] Use the starting buffer (20mmol / L Tris-HCl, pH8.1) to equilibrate the carboxymethyl (CM) cation exchange chromatography column, concentrate the non-adsorbed component P1 to about 2ml in the carboxymethyl (CM) cation exchange chromatography Load the sample on the column, use two column volumes of starting buffer (20mmol / L Tris-HCl, pH8.1) to wash the chromatography, obtain the non-adsorbed component P5, and then use the washing buffer (1mol / L NaCl, 20mmol / L Tris-HCl, pH8.1) continue to wash the carboxymethyl (CM) cation exchange column, and the rest of the adsorbed components are eluted ( figure 2 ).

Embodiment 3

[0066] Embodiment 3: separation product PcPAF carries out SDS-PAGE electrophoresis

[0067] The separated component P5 was run on 15% SDS-PAGE electrophoresis at a voltage of 180V and normal temperature, and the purity of the separated component P5 was detected by the silver nitrate rapid staining method. On the electrophoresis staining map, there was only one isolated component P5 A single band was developed, and the size was around 10kDa, and the protein was named as PcPAF ( image 3 ).

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Abstract

The invention provides marine penicillium citrinum antifungal protein PcPAF and a preparation method thereof, and relates to microbial original antifungal protein. Marine penicillium citrinum W1 is stored as CCTCC (China Center for Type Culture Collection) NO: M2013205. According to the preparation method, supernatant of fermentation broth of marine penicillium citrinum W1 is separated and purified to obtain antifungal protein, naming PcPAF; the identification result shows that the obtained mass spectrometry information is failed to be matched in NCBI (National Center of Biotechnology Information) Genbank database; 15 amino acids at the N end of the protein are detected by an N end sequencing manner; by being compared with the NCBI Genbank database, the obtained amino acid sequence shows low homology, so that PcPAF can be determined to be a novel antifungal protein. By the N end sequencing, the 15 amino acids at the N end are AGNRPDFPRRHHPGG. PcPAF shows obvious effect on inhibiting pathomycete, namely, trichoderma viride and the like, of four plants, and remains the antimicrobial activity after being heated for 20 minutes at 80 DEG C.

Description

technical field [0001] The invention relates to an antifungal protein derived from microorganisms, in particular to a marine Penicillium citrinum antifungal protein PcPAF and a preparation method thereof. Background technique [0002] Some crop diseases caused by viruses, bacteria and fungi have caused losses and quality and safety problems of agricultural products. At present, the prevention and control of plant diseases mainly rely on chemical pesticides, but the use of pesticides is strictly regulated and controlled. Antifungal proteins are seen as emerging compounds that can protect plant health, as there is a need for new pesticides that not only protect plants but also comply with new regulations. Organisms are capable of synthesizing and secreting a variety of antifungal proteins either ribosomally or nonribosomally. New synthetic compounds designed based on some antifungal proteins have been expressed in transgenic plants, endowing plants with the ability to resist...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12P21/00C07K14/385C07K1/36C07K1/34C07K1/30C07K1/18C12R1/80
Inventor 陈新华郭文斌文超
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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