Montagem e anotação funcional do transcriptoma de prasiola crispa e bioprospecção de proteínas de ligação ao gelo

Abstract

Prasiola crispa is a green macroalga belonging to the Trebouxiophyceae class, with cosmopolitan biogeographic distribution, from Arctic to Antarctic. In Antarctic continent, P. crispa is among the most important primary producers, surviving to extreme environmental conditions such as low temperatures, osmotic stress, ultraviolet radiation and others. Due to its ability to colonize such an inhospitable environment, this alga must have adaptive mechanisms whose genes involved are of great interest in the area of Biotechnology. Among the main biomolecules of interest, the Ice-binding proteins (IBPs) stand out. IBPs are polypeptides that allow the survival of cells at low temperatures. This group of proteins has great applicability in agriculture, biomedicine and food industry. Thus, the aim of this work was sequencing, assembly and functional annotation of P. crispa transcriptome and identify genic products that are directly related to the survive ability of this organism in the Antarctic continent, mainly IBPs. Samples were collected at King George Island, Antarctic. The RNA was extracted, the library built and sequenced by Illumina HiSeq 2000, with paired-end reads strategy. Low-quality reads and adapters were removed and processed reads were subdued to the assembly by Trinity assembler. Assembled transcripts were annotated by an automatized pipeline of the MG-RAST webserver and indicated contamination with the microbiota. P. crispa transcriptome was decontaminated and subsequently functionally annotated by BLAST2GO. IBPs of P. crispa and microbiota were searched by algorithm BLASTX, aligning the transcripts against IBPs sequences in a local database. Identified transcripts through search had its amino acid sequence predicted by Transdecoder and computationally modeled by Phyre 2. The sequencing generated 31,563,740 high-quality reads. The assembly resulted in 376,797 contigs, totalizing 209,870,963 base pair (bp) and an average size of 557 bp. In the annotation by MG-RAST, 93% of the hits were characterized as belonging to bacteria. After decontamination process, 17,201 contigs were maintained as belonging to P. crispa transcriptome, of which 52.19% were identified through functional annotation. 127 transcripts were identified by BLASTX search as potential IBPs, with homology with proteins from plants, bacteria and fungi. The computational modeling of the three-dimensional structure indicated that 17 of these possible IBPs have a similar structure to other proteins in the group, of which 8 have a higher potential. In conclusion, the assembly and functional annotation was performed in a satisfactory manner and allowed the identification of potential IBPs. As a perspective, Molecular Dynamics simulations will be performed to verify the behavior of these proteins at different temperatures for the choice of which will be expressed heterologously.