【For INTERNATIONAL COLLABORATIVE RESEARCH 2018】

DATE: Day 13 MonthMay Year 2019

SUMMARY of2018 RESEARCH RESULTS REPORTFor International Collaborative Research with IPR, Osaka University
Research Title
Chemical synthesis of the defensin peptide derived fromNicotiana megalosiphon to evaluate the fungal resistance insoybean transgenic lines
ApplicantName
Hilda Elisa Garay Pérez
Affiliation
Center for Genetic Engineering and Biotechnology (CIGB)
Present Title
Senior Researcher. Head of Synthetic Peptides Group
Research Collaborator (Host PI) Hironobu Hojo (Professor)
SummaryPlant diseases caused by fungi and oomycetes have an important impact for crop production in Cuba
and worldwide. Every year huge amounts of fungicides for control are applied, with the effectthereof on the environment and human health. Find alternatives with biotechnological approaches fordisease control is the biggest challenge for researchers related with this topic. Plant defensins are
small, cysteine-rich peptides, which inhibit the growth of a wide range of microorganisms and arepart of the defense mechanisms of plants against diseases. The application of the plant defensin asalternative to reduce crop losses due to the attack of pathogen constitutes an advantage with regard
to the application of chemical fungicides. First: plant defensin are derived from seeds, roots andtubers, for what they constitute nature substances that are not toxic to the host plant and neither topeople that consume the products from these plants. Second: as other protein, the defensin quickly
degrades like native substances not leaving any residual after their effectiveness expires. This
defensin was isolated from a plant and develops a strong anti- microbe activity in vitro and ex vitro
against important bacteria, fungus and oomycetes pathogens. Herewith, the constitutive expressionof this defensin in transgenic plants increased the resistance to many plant diseases with higheconomic impact. In the present work, the chemical synthesis of defensin linear peptide (45 aa) fromNicotiana megalosiphon was carried using the solid phase peptide approach. Besides, it was studied
five folding condition to identify the optimal condition that favors the presence of one major isomer(Condition I: Redox buffer (GSH/GSSG). Condition II: DMSO at 10%. Condition III: Redox buffer(GSH/GSSG) and organic solvent (Glycerol 10%). Condition IV: DMSO at 20%. Condition V:DMSO at 20% and denaturant reagent (GuHCL)). In the five conditions studied, there is notpresence of linear peptide after 24 h and various isomers of the folding peptide were formed. The useof redox buffer (GSH)/GSSG) does not promote efficiently the formation of one main isomer withadequate percentage in the isomers mixture. The addition of glycerol as organic solvent in the redox
buffer condition delays the folding reaction. The use of DMSO as oxidative reagent was the moreeffective protocol for folding the defensin peptide and the addition of GuHCL as denaturant reagent
does not enhance the folding behavior. The ESI-MS analysis demonstrated that the folded peptide
have the four disulfide bonds. This result will be included in a research paper and it will be presented
in the 5th International Symposium on Synthetic Peptide as Human and Veterinary Pharmaceutical
Products “Synthetic Peptides 2019” (June 28th – July 1st, 2019), Cuba.
*Deadline: May 17, 2019
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