Sugarcane giant borer transcriptome analysis and identification of genes related to digestion

Abstract

Sugarcane is a widely cultivated plant that serves primarily as a source of sugar and ethanol. Its annual yield can be significantly reduced by the action of several insect pests including the sugarcane giant borer (Telchin licus licus), a lepidopteran that presents a long life cycle and which efforts to control it using pesticides have been inefficient. Although its economical relevance, only a few DNA sequences are available for this species in the GenBank. Pyrosequencing technology was used to investigate the transcriptome of several developmental stages of the insect. To maximize transcript diversity, a pool of total RNA was extracted from whole body insects and used to construct a normalized cDNA database. Sequencing produced over 650,000 reads, which were de novo assembled to generate a reference library of 23,824 contigs. After quality score and annotation, 43% of the contigs had at least one BLAST hit against the NCBI non-redundant database, and 40% showed similarities with the lepidopteran Bombyx mori. In a further analysis, we conducted a comparison with Manduca sexta midgut sequences to identify transcripts of genes involved in digestion. Of these transcripts, many presented an expansion or depletion in gene number, compared to B. mori genome. From the sugarcane giant borer (SGB) transcriptome, a number of aminopeptidase N (APN) cDNAs were characterized based on homology to those reported as Cry toxin receptors. This is the first report that provides a large-scale EST database for the species. Transcriptome analysis will certainly be useful to identify novel developmental genes, to better understand the insect's biology and to guide the development of new strategies for insect-pest control.

Fig 1. Coverage of data along the gene/transcript length suggests the presence of sequencing bias in the particular set of ESTs.

Coverage signals were extracted from BAM files and 100 quantiles were obtained from each transcript in BED files. A) All genes for which ESTs produced alignments to the reference genome of the related specie B. mori. B) Same as before considering only genes for which the ESTs coverage of data was above 0.125 RPKM. C) Assembled contigs that did not return signals of protein-coding capacities for which ESTs produced alignments.

Fig 2. E-value distribution of the top BLASTx hits.

Sequences with e-value equal to 0 are represented in the right peak. The cut-off used was 1e^-5.

Fig 3. Species distribution of the top BLAST hits for each unique sequence.

A higher similarity was observed with proteins from the lepidopteran Bombyx mori.

Fig 4. Gene Ontology (GO) assignments for SGB transcriptome.

All contigs were classified on level 2 for A) Biological Process, B) Molecular Function and C) Cellular Component.

Fig 5. Real time PCR of trypsin-like genes in three different tissues of SGB.

A) Trypsin 1, B) Trypsin 2, C) Trypsin 3, D) Trypsin 4 and E) Trypsin 5. AMID (Anterior midgut). PMID (Posterior midgut). Each bar represents the relative expression in comparison to the tissue that had the smaller expression value, arbitrarily designated as 1. Standard errors of technical triplicate are shown.

Fig 6. Real time PCR of chymotrypsin-like genes in three different tissues of SGB.

A) Chymotrypsin 1, B) Chymotrypsin 2, C) Chymotrypsin 3 and D) Chymotrypsin 4. AMID (Anterior midgut). PMID (Posterior midgut). Each bar represents the relative expression in comparison to the tissue that had the smaller expression value, arbitrarily designated as 1. Standard errors of technical triplicate are shown.

Fig 7. ClustalW2 alignment of T. licus licus trypsin-like proteins.

Many sequences preserved the characteristics of digestive serine proteases. The signal peptide is shown with a red underline. The cleavage site is shown in a green box. Red boxes indicate the active site residues. Grey boxes indicate the substrate binding region and cysteines that are possibly involved with disulfide bonds are shown in blue.

Fig 8. ClustalW2 alignment of T. licus licus chymotrypsin-like proteins.

Many sequences preserved the characteristics of digestive serine proteases. The signal peptide is shown with a red underline. The cleavage site is shown in green box. Red boxes indicate the active site residues. Grey boxes indicate the substrate binding region and cysteines that are possibly involved with disulfide bonds are shown in blue.

Fig 9. Real time PCR of APN genes in three different tissues of SGB.

A) Aminopeptidase 1, B) Aminopeptidase 2 and C) Aminopeptidase 3. AMID (Anterior midgut). PMID (Posterior midgut). Each bar represents the relative expression in comparison to the tissue that had the smaller expression value, arbitrarily designated as 1. Standard errors of technical triplicate are shown.

Fig 10. Phylogenetic analysis of representative lepidopteran APNs.

The proteins of T. licus licus are shown in green. Black spots indicate the APNs that have been reported as putative Cry toxin receptors. Red spots indicate the APNs which gene expression changed after Bt infection. Red squares indicate the APNs which silencing induced resistance of the insect to Cry1Ab. GenBank accession number is shown for each protein. Species abbreviations: Ha, Helicoverpa armigera; Hp, Helicoverpa punctigera; Hv, Heliothis virescens; Px, Plutella xylostella; Se, Spodoptera exigua; Tn, Trichoplusia ni; Bm, Bombyx mori; Ms, Manduca sexta; Ld, Lymantria dispar; Ep, Ephiphyas postvittana; Pi, Plodia interpunctella; Sl, Spodoptera litura; Cs, Chilo suppressalis; Sls, Spodoptera litoralis; Cm, Cnaphalocrocis medinalis; Ds, Diatraea saccharalis; Si, Sesamia inferens; Dp, Danaus plexippus; Os, Ostrinia nubilalis; Mc, Mamestra configurata; Aj, Achaea janata; Of, Ostrinia furnacalis.