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Novel protein against fungal pathogens

一种蛋白质、抗真菌的技术，应用在肽/蛋白质成分、抗体医疗成分、细菌肽等方向

Active Publication Date: 2019-03-15
NAT INST OF PLANT GENOME RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Controlling them in an environmentally friendly manner remains a challenge

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  • Novel protein against fungal pathogens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Identification of proteins involved in the antifungal and bacteriophagous activity of Burkholderia gladiolus strain NGJ1

[0069] This example provides the identification of Burkholderia gladiolus strain NGJ1 derived from healthy rice seedlings (as described by Jha et al 2015) as a potent antifungal and bacteriophagous bacterium. Initially, up to 3 dpi (days after incubation), the bacteria showed antifungal activity and prevented the growth of fungi in their vicinity. During the 8dpi of the confrontation (days after incubation), the bacteria started to feed on the fungus and displayed fungal-eating activity (e.g. figure 1 shown). Interestingly, on the minimal medium CDA (Czapek Dox agar) plates, the bacteriophagy behavior (spread of bacteria on fungi and degradation of fungal mycelia) was very prolific.

[0070] The inventors have discovered that some proteins of Burkholderia gladiolus strain NGJ1 have potential host-targeting signals and have further chara...

Embodiment 2

[0082] Example 2: Overexpression and purification of potential antifungal proteins

[0083] PCR amplification from Burkholderia gladiolus strain NGJ1 genomic DNA using gene-specific forward primer with SEQ ID No. 3 and reverse primer with SEQ ID No. 4 as disclosed in Table 2 The complete CDS of the Bg_9562 gene (333bp), and was further cloned into the pET28a bacterial expression vector (Novagen / Merck Life Science Private Limited) to obtain pET28a-9562 (eg figure 2 shown in a). Restriction sites for NdeI and HindIII have been added to the forward and reverse primer sequences, respectively.

[0084] After sequence verification, pET28a-9562 was transformed into E. coli (BL21 strain, DE3-codon+) to produce recombinant protein. protein Ni 2+ - Purify protein by NTA-agarose chromatography and analyze on SDS PAGE (eg figure 2 shown in b). It was further electroblotted onto a polyvinylidene fluoride (PVDF) membrane and probed with a mouse polyclonal antibody raised against an ant...

Embodiment 3

[0088] Example 3: Evaluation of Antifungal Activity of Purified Proteins

[0089] The antifungal activity of the protein having SEQ ID No. 2 was determined by treating the sclerotia of R. solani strain BRS1 with different concentrations (5, 10 and 15 μl / ml) of the purified protein. As controls, three different solutions were used—10 mM phosphate-buffered saline (PBS) pH 7.4, 50 mM wash solution (a component used in protein elution), and heat-inactivated Bg_9562 protein (by incubating in boiling water for 40 minutes) to process the sclerotia. After the treatment, the sclerotia were placed on a PDA (HIMEDIA, India) plate and incubated at 28°C for further growth. Results (such as image 3 Shown in a) In summary, 15 μg / ml of protein effectively prevents the growth of R. solani, while appropriate fungal growth is observed in the case of different controls. Inhibition of fungal growth by eBg_9562 protein treatment is summarized in image 3 b. The eBg_9562-treated sclerotia fail...

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Abstract

The present invention relates to a novel protein comprising novel genes that is extracted from Burkholderia gladioli strain NGJ1. A nucleotide sequence encoding the novel protein is represented by sequence SEQ ID No. 1 and amino acid sequence of the novel protein is represented by the sequence SEQ ID No. 2 is further provided. A nucleotide sequence and the amino acid sequence are obtained from genetically engineered Bg_9562 gene. The novel protein as well as encoding gene is adapted for broad spectrum anti-fungal and mycophagous activities.

Description

technical field [0001] The present invention relates to novel proteins against fungal pathogens. More specifically, the present invention relates to novel proteins from the Burkholderia gladioli strain NGJ1 that are antifungal pathogens and useful in the control of various fungal diseases. Background technique [0002] Bacteria are ubiquitous, found in soil, water, air, etc., and in multicellular organisms including insects, plants, and animals. They have very active intercellular communication systems and can sometimes behave as multicellular organisms by forming biofilms. To survive, they need to compete with other symbiotic bacteria, fungi, etc., and must live inside or with plants, animals, etc. (Frey-Klett et al, 2011; Reinhold-Hurek and Hurek, 2011). Interactions between bacteria and other organisms can be mutualistic, commensalistic, antagonistic, or parasitic (Haas and Défago, 2005; Frey-Klett et al, 2011). Usually, they directly promote plant growth by synthesizi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195A01N63/02A01N63/50
CPCC07K14/195C07K14/21A01N63/50A61K39/00A01N25/04A01N25/10A61K9/06A61K9/5161A61K38/00
Inventor 德加·马哈布·斯温苏尼尔·库马尔·亚达夫古帕吉·杰哈
Owner NAT INST OF PLANT GENOME RES
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