Construction Of Transcriptome Analysis Workflow Of Helicoverpa Armigera And Analysis Of Differentially Expressed Genes Involved In Bt Resistance

With the rapid development of sequencing technology,the quality and throughput of sequencing continue to increase,and the cost has rapidly declined.Transcriptomics research has entered a period of golden development.To get a further understanding of non-model organisms,this research built a transcriptome analysis process based on the latest database and high-cited tools and analyzed the differentially expressed genes of four laboratorial cotton bollworm resistant strains and one sensitive strain.The main results of the study are as follows:1.The sequencing data of all 5 strains obtained more than 20 million reads,with an average of 7.4 billion bp,and the base quality after filtration reached over Q30.2.The quality of 234808 transcripts obtained by Trinity was detected by 5 methods.The N50 was 928 bp,the ratio of Bowtie2 alignments was over 98%,Ex90N50 was 2042 bp,and 95% of insect conserved genes were detected by BUSCO.Diamond performed better than Blast in Nr,Uniprot and Pfam protein database alignment.Nr alignment result showed that 47% transcripts were aligned to Helicoverpa armigera,21% were aligned to Heliothis assulta.Besides,other lepidopteran insects such as Bombyx mori,Spodoptera litura,Chilo suppressalis were also aligned.3.The Pearson correlation coefficient of the biological repeats in one group is close to 0.9,and the hierarchical cluster with PCA analysis among groups showed no batch effect.4.The differential gene expression analysis was performed with DESeq2 and edgeR software,using the expression matrix obtained by Salmon and RSEM respectively.The combination of edgeR and Salmon detected most differentially expressed genes.5.A total of 1156 differentially expressed transcripts were obtained from the DU strain,of which 397 had RefSeq ID.GO enrichment analysis showed that 5 genes were related to aminopeptidase activity,12 genes affected protease activity and 2 genes corresponded to antibacterial immune process.KEGG pathway analysis showed that 10 genes involved in the peptidase pathway.6.A total of 995 differentially expressed transcripts were obtained from the LFC2 strain,of which 268 had RefSeq ID.GO enrichment analysis showed that 4 genes were involved in protein kinase inhibitor activity,6 genes affected transmembrane transporter activity,and 3 genes were related to proteasomes.KEGG pathway analysis showed that 5 genes were enriched in the protease-activated receptor signaling pathway,and 3 genes were involved in the proteasome pathway.7.A total of 982 differentially expressed transcripts were obtained from the XXCAD strain,of which 278 had RefSeq ID.GO enrichment analysis showed that 8 genes were related to the production of precursor metabolites,3 genes were related to proteasome and 3 genes were related to glycosyl transferase.KEGG pathway analysis showed that 6 genes were enriched in the cytochrome P450 metabolic pathway and drug metabolism pathway.8.A total of 1201 differentially expressed transcripts were obtained from the RS strain,of which 281 had RefSeq ID.GO enrichment analysis showed that 15 genes were related to ATP metabolic processes,12 genes could regulate ATP activity,7 genes were related to precursor metabolism,2 genes were involved in the cholesterol transport pathway,3 genes were involved in the fat transport pathway and 12 genes were involved in the nucleotide metabolic pathway.KEGG pathway analysis showed that 2 genes were enriched in glycosylphosphatidylinositol anchored pathway,2 genes were enriched in the proteasome pathway,and 9 genes were enriched in the peptidase pathway.