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Molecular cloning and characterization of an ADP-ribosylation factor 6 gene (ptARF6) from Pisolithus tinctorius.

Authors
  • Wang, Liling1
  • Li, Haibo1
  • Zhou, Yifeng2
  • Qin, Yuchuan1
  • Wang, Yanbin1
  • Liu, Bentong1
  • Qian, Hua1
  • 1 a Zhejiang Forestry Academy, Zhejiang Provincial Key Laboratory of Forest Food, Hangzhou, 310023, People's Republic of China.
  • 2 b Zhejiang University of Science and Technology, Hangzhou, 310023, People's Republic of China.
Type
Published Article
Journal
Canadian Journal of Microbiology
Publisher
Canadian Science Publishing
Publication Date
May 2016
Volume
62
Issue
5
Pages
383–393
Identifiers
DOI: 10.1139/cjm-2015-0550
PMID: 26928195
Source
Medline
Keywords
License
Unknown

Abstract

ADP-ribosylation factor 6 (ARF6) is an evolutionarily conserved molecule that has an essential function in intracellular trafficking and organelle structure. To better understand its role during presymbiosis between plant roots and compatible filamentous fungi, the full-length cDNA sequence of ARF6 from Pisolithus tinctorius was cloned and a variety of bioinformatics analyses performed. The full-length sequence was 849 bp long and contained a 549 bp open reading frame encoding a protein of 182 amino acids. A phylogenetic analysis showed that ptARF6 was the ortholog of the ADP ribosylation factor 6/GTPase SAR1 gene from the white-rot basidiomycete Trametes versicolor. A domain architecture analysis of the ARF6 protein revealed a repeat region, which is a common feature of ARF6 in other species. Recombinant ARF6 protein was expressed with an N-terminal 6×His tag and purified using Ni(2+)-NTA affinity chromatography. The molecular mass of the recombinant protein was estimated by SDS-PAGE to be 25 kDa. The recombinant ARF6 protein bound strongly to 18:1 and 18:2 phosphatidic acids. Thus, ARF6 may participate in the signaling pathways involved in membrane phospholipid composition. The intracellular distribution of ptADP6 in HEK239T cells also indicates that ptADP6 may function not only in plasma membrane events but also in endosomal membranes events. Real-time quantitative PCR revealed that the differential expression of ptARF6 was associated with the presymbiotic stage. ptARF6 may be induced by presymbiosis during the regulation of mycorrhizal formation.

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