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Cloning and expression analysis of an endo-1,3-β-d-glucosidase from Phytophthora cinnamomi

  • Rodrigo Costa
  • Angel Domínguez
  • Altino ChoupinaEmail author
Original Article
  • 39 Downloads

Abstract

Phytophthora is considered one of the most destructive genus for many agricultural plant species worldwide, with a strong environmental and economic impact. Phytophthora cinnamomi is a highly aggressive Phytophthora species associated with the forest decline and responsible for the ink disease in chestnut trees (Castanea sativa Miller), a culture which is extremely important in Europe. This pathogenicity occurs due to the action of several enzymes like the hydrolysis of 1,3-β-glucans at specific sites by the enzyme endo-1,3-β-d-glucosidase. The aim of this work to analyze the heterologous expression in two microorganisms, Escherichia coli and Pichia pastoris, of an endo-1,3-β-d-glucosidase encoded by the gene ENDO1 (AM259651) from P. cinnamomi. Different plasmids were used to clone the gene on each organism and the real-time quantitative polymerase chain reaction was used to determine its level of expression. Homologous expression was also analyzed during growth in different carbon sources (glucose, cellulose, and sawdust) and time-course experiments were used for endo-1,3-β-d-glucosidase production. The highest expression of the endo-1,3-β-d-glucosidase gene occurred in glucose after 8 h of induction. In vivo infection of C. sativa by P. cinnamomi revealed an increase in endo-1,3-β-d-glucosidase expression after 12 h. At 24 h its expression decreased and at 48 h there was again a slight increase in expression, and more experiments in order to further explain this fact are underway.

Keywords

Castanea sativa Endo-1,3-β-d-glucosidase Heterologous expression Homologous expression Phytophthora cinnamomi RT-qPCR 

Notes

Compliance with ethical standards

Conflicts of interest

The authors declare that there are no conflicts of interest. Also, are indebted to the careful and constructive criticisms of the reviewers.

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Centro de Investigação de Montanha (CIMO)Instituto Politécnico de BragançaBragançaPortugal
  2. 2.Departamento de Microbiología y GenéticaEdificio DepartamentalSalamancaSpain

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