Pirossequenciamento do transcriptoma de folha de Lippia alba por meio da plataforma 454 GS FLX (Roche)

Abstract

Lippia alba, popularly known as erva cidreira, is widely distributed throughout the Americas and can be found through almost whole Brazil. This species is largely used in folk medicine to treat gastrointestinal and respiratory problems, especially leaves, which produce an essential oil rich in terpenes, mainly mono-and sesquiterpenes. These compounds are not only of pharmacological interest, as well as industrial. Composition of essencial oil can vary depending on the developmental stage, the plant part and other abiotic factors. However, genotypic variations also contribute to oil composition variation. Given the complexity of terpenes synthesis, including diversity of enzymes involved in these metabolic pathways, the purpose of this work was a L. alba leaf transcriptome characterization, in addition to identifying some enzymes probably involved in terpene synthesis. For that, it was made a transcriptome sequencing using 454 platform (Roche) followed by a de novo assembly. This platform, along with other NGS technologies, has been increasingly used for transcriptome sequencing in an approach known as RNA-Seq. Sequencing of a library prepared from total RNA in 1/8 plate generated 104,631 reads with average length of 184.48 bp and a total of 19,302,161 bases. Read assemblies were made using two different assemblers in order to compare them. While Newbler 2.5, proprietary software platform, assembled 2686 contigs with average length of 349bp, SeqMan2.2 generated 13,448 contigs with an average of 284bp. Then, functional annotation was performed with Blast2GO for all contigs from both assemblies; 51.49% and 30.88% of contigs, respectively, from Newbler and SeqMan were annotated. Finally, analysis of annotated sequences revealed some enzymes potentially involved in terpene synthesis. Results obtained from this pioneering study on the species show that NGS technology can be a very efficient tool for transcriptome sequencing and they will serve as reference for preparation of other more specific libraries. New sequencings should contribute to a better coverage of this transcriptome, allowing discovery of even rare transcripts.