Isolation and sequence analysis of a β-tubulin gene from arbuscular mycorrhizal fungi
- 803 Downloads
A full-length β-tubulin gene has been cloned and sequenced from Gigaspora gigantea and Glomus clarum, two arbuscular mycorrhizal fungi (AMF) species in the phylum Glomeromyota. The gene in both species is organized into five exons and four introns. Both genes are 94.9% similar and encode a 447 amino acid protein. In comparison with other fungal groups, the amino acid sequence is most similar to that of fungi in the Chytridiomycota. The codon usage of the gene in both AMF species is broad and biased in favor of an A or a T in the third position. The four introns varied in length from 87 to 168 bp for G. gigantea and from 90 to 136 bp for G. clarum. Of all fungi in which full-length sequences have been published, only AMF do not have an intron before codon 174. The introns positioned at codons 174 and 257 in AMF match the position of different introns in β-tubulin genes of some Zygomycete, Basidiomycete, and Ascomycete fungi. The 5′ and 3′ splice site consensus sequences are similar to those found in introns of most fungi. Sequence analysis from single-strand conformation polymorphism analysis confirmed the presence of two β-tubulin gene copies in G. clarum, but only one copy was evident in G. gigantea based on Southern hybridization analysis.
KeywordsGlomeromycota Inverse PCR Beta-tubulin Arbuscular mycorrhizal fungi
The authors wish to thank Bill Wheeler for helping with the manipulation and processing of AMF cultures. We also thank Dr. Jed Doelling for reviewing the manuscript. We are grateful to one reviewer for providing an additional source of information for full-length β-tubulin sequences of the Chytridiomycota and Zygomycota. Funding was provided by a Fulbright scholarship to Zola Msiska and National Science Foundation grants DBI0650735 and DEB0649341 to Joseph Morton.
- Cleveland DW, Theodorakis NG (1994) Regulation of tubulin synthesis. In: Hyams JS, Lloyd CW (eds) Microtubules. Wiley-Liss, New York, pp 47–58Google Scholar
- Gilbert SF (ed) (2006) Developmental biology, 8th edn. Sinauer, MassachussettsGoogle Scholar
- Goldman GH, Temmerman W, Jacobs D, Contreras R, van Montagu M, Herrera-Estrella A (1993) A nucleotide substitution in one of the beta-tubulin genes of Trichoderma viride confers resistance to the antimitotic drug methyl benzimidazole-2-yl-carbamate. Mol Gen Genet 240:73–80. doi: 10.1007/BF00276886 CrossRefPubMedGoogle Scholar
- Huang S-H, Chen SHM, Jong AY (2003) Use of inverse PCR to clone cDNA ends. In: Ying S-Y (ed) Methods in molecular biology vol. 221: generation of cDNA libraries: methods and protocols. Humana, Totowa, NJ, pp 51–58Google Scholar
- Requena N, Fuller P, Franken P (1999) Molecular characterization of GmFOX2, an evolutionarily highly conserved gene from the mycorrhizal fungus Glomus mosseae, down regulated during interaction with rhizobacteria. Mol Plant Microbe Interact 12:934–942. doi: 10.1094/MPMI.19184.108.40.2064 CrossRefPubMedGoogle Scholar