Abstract
As an important botanical pesticide, azadirachtin demonstrates broad insecticidal activity against many agricultural pests. The results of a previous study indicated the toxicity and apoptosis induction of azadirachtin in Spodoptera frugiperda Sf9 cells. However, the lack of genomic data has hindered a deeper investigation of apoptosis in Sf9 cells at a molecular level. In the present study, the complete transcriptome data for Sf9 cell line was accomplished using Illumina sequencing technology, and 97 putative apoptosis-related genes were identified through BLAST and KEGG orthologue annotations. Fragments of potential candidate apoptosis-related genes were cloned, and the mRNA expression patterns of ten identified genes regulated by azadirachtin were examined using qRT-PCR. Furthermore, Western blot analysis showed that six putative apoptosis-related proteins were upregulated after being treated with azadirachtin while the protein Bcl-2 were downregulated. These data suggested that both intrinsic and extrinsic apoptotic signal pathways comprising the identified potential apoptosis-related genes were potentially active in S. frugiperda. In addition, the preliminary results revealed that caspase-dependent or caspase-independent apoptotic pathways could function in azadirachtin-induced apoptosis in Sf9 cells.
Highlights
Sf9 cells, derived from the immature ovaries of fall armyworm moth (Spodoptera frugiperda) pupae[1,2], have become one of the most widely used insect cell lines for eukaryotic protein expression with the advantages of high protein expression and easy manipulation[3]
To analyse the mechanism of apoptosis induced by azadirachtin in Sf9 cells, the present study investigated the transcriptome of Sf9 cell line using Illumina platform
A draft genome sequence and transcriptome were assembled by de novo sequencing of genomic DNA and mRNA from Sf21 cells[36,37]
Summary
Sf9 cells, derived from the immature ovaries of fall armyworm moth (Spodoptera frugiperda) pupae[1,2], have become one of the most widely used insect cell lines for eukaryotic protein expression with the advantages of high protein expression and easy manipulation[3]. Apoptosis study in Sf9 cells still stay in the level of physiological and biochemical changes and cloning and function analysis of a small amount apoptosis genes, and apoptosis mechanisms of D. melanogaster can’t fully reflect the apoptotic mechanism of Sf9 cells, comprehensive and in-depth study of apoptosis is indispensable. Several apoptotic signalling pathways have been verified as activated in apoptosis induced by azadirachtin. The lysosomal signalling pathway plays a crucial role in apoptosis induced by azadirachtin, and cathepsin L exerts its function as a prop-apoptotic factor by engaging the release of cathepsin L into the cytosol and activating caspase-329. The results of Western blotting verified roles for seven proteins in apoptosis induction by azadirachtin in Sf9 cells These results enrich the transcriptome diversity of S. frugiperda and contribute to the identification and validation of apoptosis-related genes in Sf9 cells, and reveal the preliminary apoptosis mechanism of azadirachtin
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