Expressed sequence tags (EST) analysis of a normalized full-length cDNA library from the pinewood nematode (Bursaphelenchus xylophilus)

Abstract

The pinewood nematode (Bursaphelenchus xylophilus) infects pine trees and causes pine wilt disease. To clarify the functions and subcellular localization of B. xylophilus genes/proteins transcribed and predicted from mixed stages (egg, J1, J2, J3 J4 and adult), we prepared a normalized full-length B. xylophilus cDNA library and analyzed expressed sequence tags (ESTs) using the Pendant-Pro sequence analysis suite. Most cDNAs inserted into the library ranged from 0.9 to 1.8 kb (average 1.5 kb). The 1,902 ESTs from B. xylophilus consisted of 286 clusters and 1,273 singletons. EST sizes ranged from 9 to 743 bp with a mean of 336 bp. The predicted protein length from B. xylophilus ESTs revealed that most proteins ranged from 50 to 149 amino acids. Enzyme nomenclature (EC) numbers were classified into 133 (8.5%) of 1,559 contigs using UniProt database hits by the EC numbers method. Transmembrane regions of 1559 clusters were predicted using the TMpred algorithm. The 1,559 contigs with transmembrane regions were annotated; 481 (30.8%) contigs were assigned ‘above one’ domain and 1,078 (69.1%) were assigned ‘none.’ Additionally, taxonomy was classified for 672 (43.1%) of 1,559 contigs. Of the 1,559 contigs, 685 (43.9%) were assigned gene ontology terms using the gomerger method of contigs, including singletons. Thirty-one (31) contigs of predicted proteins grouped by BLASTP identity values had significant homology to genes expressed in subcellular structures (for example, mitochondrion, plasma membrane, endoplasmic reticulum, nucleus and golgi). B. xylophilus ESTs provide the foundation for research information on related plant parasite nematodes and contribute to finding an important novel parasite control strategy.

Keywords: Pinewood nematode, Bursaphelenchus xylophilus, pine wilt disease, expressed sequence tag, Pendant-Pro Sequence Analysis Suite.

African Journal of Biotechnology, Vol 13(31) 3332-3343