De Novo Assembly of the Transcriptome for Oriental Armyworm Mythimna separata (Lepidoptera: Noctuidae) and Analysis on Insecticide Resistance-Related Genes

Abstract

Mythimna separata walker (Lepidoptera: Noctuidae) is a polyphagous pest of nearly 100 families of more than 300 kinds of food and industrial crops. So far, both nucleotide and protein sequence information has been rarely available in database for M. separata, strictly limiting molecular biology research in this insect species. In this study, we carried out a transcriptome sequencing for M. separata The sequencing and subsequent bioinformatics analysis yielded 69,238 unigenes, among which 45,227 unigenes were annotated to corresponding functions by blasting with high homologous genes in database, giving annotation rate of 65.32%. Several lepidopteran insects gave best matches with the transcriptome data. To gain insight into the mechanism of insecticide resistance in M. separata, 15 families of genes encoding insecticide resistance-related proteins were investigated. Substantial numbers of unigenes in these families were identified in the transcriptome data, and 17 out of 21 selected unigenes were successfully amplified. Expressions of most of these genes were detected at larval stages and in gut tissue, as was consistent with their putative involvement in insecticide resistance. Our study provides most comprehensive transcriptome data for M. separata to date, and also provides reference sequence information for other Noctuidae family insects.

Keywords: Mythimna separata; Noctuidae; insecticide resistance; transcriptome; unigene.

Fig. 1.

Electrophoresis detection of Mythimna separata total RNA. Ladder: size of the DNA band in DM2000 marker was indicated; Total RNA: 28S and 18S rRNAs were indicated by arrows.

Fig. 2.

Histogram presentation of the annotation results of Mythimna separata unigenes against different databases. The number of unigenes with annotation in different databases was shown.

Fig. 3.

Characteristics of homology search of the Mythimna separata unigenes against the non redundant (NR) protein database of NCBI. (A) e-Value distribution of the BLAST hits for the unigenes with a cut-off e-value of e⁻⁵. (B) Similarity distribution of the BLAST hits for the unigenes. (C) Species distribution of the unigene BLASTX matches against the NR protein database with a cut-off e-value of e⁻⁵. First hit of each unigenes was used for analysis and the proportions of homologous sequences in each species were shown.

Fig. 4.

Classification of the clusters of orthologous groups for the transcriptome of Mythimna separata. 13,083 (18.90%) of the total unigenes were annotated and divided into 25 specific categories based on the sequence homology.

Fig. 5.

Classification of the gene ontology for the transcriptome of Mythimna separata. 21,110 (30.49%) of the total unigenes were categorized into 59 function groups.

Fig. 6.

Expression patterns of the unigenes at different life stages. 1, egg; 2, second instar larvae; 3, third instar larvae; 4, fourth instar larvae; 5, fifth instar larvae; 6, pupa; 7, adult; 8, water as a template for negative control. Steady state level of Mythimna separata β-actin gene served as the internal control.

Fig. 7.

Expression patterns of the selected unigenes in different tissues. 1, leg; 2, gut; 3, salivary gland; 4, reproductive organ; 5, abdomen; 6, head. Steady state level of Mythimna separata β-actin gene served as the internal control.