The Study On The Differentially Expressed Genes In The Midgut Of Domestic Silkworms (Bombyx Mori) Exposed To Phoxim

As one of the most important economic insects, the silkworm, Bombyx mori, hasbeen bred for more than5,700years. The survival of B. mori has been significantlyaffected by the phoxim’s foray into mulberry through aerial application and watercurrents due to that B. mori has weak resistance to insecticides. The midgut in the B.mori not only is responsible for the digestion and absorption of nutrients but alsodefends the body against foreign invaders and chemical pesticides. As part of defensesystem of midgut, peritrophic membrane (PM) has multiple functions, such asprotecting intestinal epithelial cells, preventing the invasion of harmful substances andpromoting digestion.In order to study the mechanisms of metabolic resistance and injury in the midgutof B. mori exposed to phoxim, the larvaes of B. mori (Dazao strain and Jingsong×Haoyue strain) were used to investigate transcriptional characteristics of geneexpression in the midgut exposed to phoxim through gene chip and high-throughputsequencing technology. At the same time, we screened peritrophic membrane proteinsbased on the constructed cDNA library of the midgut. Main achievements weresummarized as follows:1. Transcriptional characteristics of gene expression in the midgut of B. moriexposed to phoxim through microarray.Microarray was performed to profile the gene expression changes in the midgut ofB. mori exposed to phoxim. After24h of phoxim exposure (4.0μg/mL),266genesdisplayed at least2.0-fold changes in expression levels. Among them,192genes wereup-regulated, and74genes were down-regulated. The most significant changes were14.88-fold up-regulation and23.36-fold down-regulation. According to gene ontologyannotation and pathway analysis, differentially expressed genes were mainly classifiedinto different groups based on their potential involvements in detoxification, immunne response, stress response, energy metabolism and transport. To validate the geneexpression level from the microarray,11genes of interest were selected for qRT-PCR.The qRT-PCR results indicate that the change tendency of the expression levels of10genes has similar patterns with the changes measured by the microarray analysis.2. Transcriptional characteristics of gene expression in the midgut of B. moriexposed to phoxim through high-throughput sequencing technology.Solexa sequencing technology was performed to profile the gene expressionchanges in the midgut of B. mori in response to24h of phoxim exposure and the impacton detoxification, apoptosis and immune defense were addressed. The results showedthat254genes displayed at least2.0-fold changes in expression levels, with148genesup-regulated and10~6genes down-regulated. Cytochrome P450played an important rolein detoxification. Histopathology examination and transmission electron microscoperevealed swollen mitochondria and disappearance of the cristae of mitochondria, whichare the important features in insect apoptotic cells. Cytochrome c release frommitochondria into the cytoplasm was confirmed. In addition, the Toll and immunedeficiency (IMD) signal pathways were all inhibited using qRT-PCR. Our resultsdemonstrated that phoxim exposure leads to weakened immune defense, together withincreased apoptosis in the midgut of B. mori.3. The construction and immune-screening of cDNA expression library of midgutfrom B. mori.A cDNA expression library was constructed from B. mori midgut mRNA. Thelibrary had a complexity of6.92×10~6plaques, of which over92.14%were recombinants.Titer of the amplified library was4.10×10~9pfu/mL. Based on the constructed cDNAlibrary, we used PM proteins polyclonal antibody to isolate and characterize the relatedcoding genes. Eighteen positive clones were obtained. We selected three possible targetgenes for sequence analysis.Our study provides new insights into the molecular mechanism of pesticidemetabolism in the midgut of insects, which may promote the development of highlyefficient insecticides. Our results also help better understand the impact of phoximexposure on B. mori and defense function of peritrophic membrane.