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Integrating transcriptomics and proteomics data for accurate assembly and annotation of genomes

ABSTRACT: Deep proteomic analysis of larvae, pupae and 15 adult tissues using high resolution Fourier transform mass spectrometry provided peptide evidence for ~9,000 computationally predicted genes accounting for 82% of total protein-coding genes in An. stephensi. Transcriptomic profiling of 4 tissues provided transcript evidence for 7,504 computationally predicted gene models and resulted in identification of 3,797 novel transcripts. We also identified protein level evidence for several novel alternative transcripts, SNPs and tissue restricted expression patterns of several proteins in An. stephensi

INSTRUMENT(S): LTQ Orbitrap Velos, LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Anopheles Stephensi (asian Malaria Mosquito)

TISSUE(S): Other Source, Testis, Brain, Head, Midgut, Malpighian Tubule, Hemolymph, Thorax, Fat Body, Salivary Gland, Leg, Pupa, Ovary, Larva, Accessory Sex Gland, Proboscis, Antenna

SUBMITTER: Akhilesh Pandey

LAB HEAD: Akhilesh Pandey

PROVIDER: PXD001128 | Pride | 2016-09-26

REPOSITORIES: Pride

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Project description:Mosquitoes possess an innate immune system that is capable of limiting infection by a variety of pathogens, including the Plasmodium spp. parasites responsible for human malaria. The Anopheles immune deficiency (IMD) innate immune signaling pathway confers resistance to Plasmodium falciparum. While some previously identified Anopheles anti-Plasmodium effectors are regulated through signaling by Rel2, the transcription factor of the IMD pathway, many components of this defense system remain uncharacterized. To begin to better understand the regulation of immune effector proteins by the IMD pathway, we used oligonucleotide microarrays and iTRAQ to analyze differences in mRNA and protein expression, respectively, between transgenic An. stephensi mosquitoes exhibiting blood meal-inducible overexpression of an active recombinant Rel2 and their wild-type conspecifics. Numerous genes were differentially regulated at both the mRNA and protein levels following induction of Rel2. While multiple immune genes were up-regulated, a majority of the differentially expressed genes have no known immune function in mosquitoes. Identified sequences were assigned putative functions and gene ontology (GO) terms based on homology to previously annotated A. gambiae gene sequences. Selected up-regulated genes from multiple GO categories were tested for both anti-Plasmodium and anti-bacterial action using RNA interference (RNAi). Based on our experimental findings, we conclude that increased expression of the IMD immune pathway-controlled transcription factor Rel2 affects the expression of numerous genes with diverse functions, suggesting a broader physiological impact of immune activation and possible functional versatility of Rel2. Our study has identified multiple novel anti-Plasmodium effectors. Midguts from midgut-specific transgenic A. stephensi at 6 and 12 hours post-blood meal and fat bodies from fat body-specific transgenic A. stephensi at 12 and 18 hours post-blood meal were compared to wild-type A. stephensi at the same time points. 3 biological replicates and 1 pseudo-replicate per array.

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Integrating transcriptomics and proteomics data for accurate assembly and annotation of genomes

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