Transcriptome analysis of Spodoptera frugiperda Sf9 cells reveals putative apoptosis-related genes and a preliminary apoptosis mechanism induced by azadirachtin

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.

Figure 1

Phylogenetic relationships between caspase sequences observed in Sf9 cells transcriptome and insects from the NCBI database. Phylogenetic analyses were performed using MEGA version 5.0 based on the amino acid sequences. Values indicated at the nodes are bootstrap values based on 1000 replicates.

Figure 2

Phylogenetic relationships between IAP sequences observed in Sf9 cells transcriptome and insects from NCBI database. The tree was constructed with MEGA 5.0 using the neighbour-joining method.

Figure 3

Agarose gel of some apoptosis-related genes in S. frugiperda PCR amplified with the specific primers shown in Supplement Table 1. Lanes: M, Marker 2000, Lane 1–15, Sf-Caspase-1, Sf-Caspase-2, Sf-AIF, Sf-BI, Sf-IBM1, Sf-Grim-19, Sf-Survivin, Sf-IAP, Sf-Ras, Sf-Cyt C, Sf-Traf 6, Sf-Pcdp 5, Sf-Rptor, Sf-Pkar 1, and Sf-Buffy.

Figure 4

The qRT-PCR analysis of 9 apoptosis-related genes between controls and cells treated with azadirachtin at 12, 24 and 48 h. The GAPDH gene was used as the housekeeping gene, and the data are expressed as arithmetic mean ± SEM (n = 3). Different letters above bars indicate significant differences between different treatments (P < 0.05) using ANOVA, followed by DMRT.

Figure 5

Western blot analysis of seven apoptosis-related proteins between controls and cells treated with azadirachtin at 12, 24 and 48 h. The GAPDH was used to normalize the difference.