Part:BBa_K2365512

MtDef4

MtDef4 is a 47-amino acid protein that is expressed constitutively and in response to biotic and abiotic stress in many tissues of a model legume, M.truncatula.Plant defensin MtDef4 is potent inhibitors of the growth of several filamentous fungi including Fusarium graminearum,and it is a nonmorphogenic defensin that does not induce hyperbranching.But it can induce plasma membrane permeabilization in Fusarium graminearum.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

The result shows the gene amplification result of MtDef4

3-dimension structure and plasmid stucture

A schematic representation of our basic plasmid types, the response construct. It includes the anti-fungal peptide coding gene (anti-fungal peptide) and the inducible promoter. This construct allows genes placed downstream of the inducible promoter to be transcribed when the yeast cells are induced with the appropriate inducer—methanol.

SDS-PAGE

HPLC Analysis Report

MS Analysis Report

The MASS spectrometry report and HPLC analysis of MtDef4, from the graph we can get the weight measured by MASS spectrometry is 5343.04U and the amino acid sequence is Arg-Thr-Cys-Glu-Ser-Gln-Ser-His-Lys-Phe-Lys-Gly-Pro-Cys-Ala-Ser-Asp-His-Asn-Cys-Ala-Ser-Val-Cys-Gln-Thr-Glu-Arg-Phe-Ser-Gly-Gly-Arg-Cys-Arg-Gly-Phe-Arg-Arg-Arg-Cys-Phe-Cys-Thr-Thr-His-Cys

Zone of inhibition

The inhibition zone results show the antifungal peptide can exhibit a great antifungal activity, and the area of the inhibition zone is dose dependent. We choose to utilize sterile water as our negative control and 100ppm carbendazim dissolved in acetic acid as positive control. MtDef4 antifungal peptide is dissolved in sterile water.

OD 595nm

We test the cell viability under the action of a series of different concentrations of antifungal peptide. MtDef4 possess great antifungal activity

We intend to use yeast system to produce a large number of antifungal peptides to kill Fusarium graminearum which causes the wheat scab. Therefore, the selection of antifungal peptide is very important. We searched for some antimicrobial peptide databases like Peptaibol Database 、Defensin Knowledgebase、MiniCOPE、APD and so on. Finally, we found 20 antimicrobial peptides that inhibit the activity of Fusarium graminearum. The ideal antifungal peptide should have the following characteristics:

• Being harmless to host cells
• Being harmless to the human body
• Having little damage microbes in the soil

Therefore, we set effective antifungal concentration、Activity object、half-life as three indexs, and established an evaluation model to evaluate if the peptide is suitable for our project.