| Silkworm (Bombyx mori), one of the most important economic insects, has beenbred for more than5,700years. Not only is the midgut in B.mori responsible for thedigestion and absorption of nutrients, but also defends the body against foreign invadersand chemical pesticides. Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is oneof the major viral pathogens for the silkworm. To well understand the host response toBmCPV infection, high-throughput transcriptome sequencing technology (RNA-Seq)was used to study comparative transcriptome of the midgut of silkworm infected withBmCPV is this work.The resistances of four different breeds of silkworms (Lan5, Qiong10, EU17,DAZAO) to BmCPV were firstly investigated. Six different concentrations of polyhedrasuspension (108,107,106,105,104,103/ml) were respectively used to infect2nd instarsilkworms. The numbers of infected silkworms were counted at7days post infection,and then LD50was calculated with the criteria of probability analysis. The resultsshowed that the resistance to BmCPV of four different breeds from strong to weak insequence was EU17, DAZAO,Qiong10, Lan5, and their LD50s were8.03、6.68、6.23、5.60, respectively.Lan5was therefore selected as the object of the following studies. The mRNA ofmidguts of infected silkworms at36h,72h,108h,144h,180h post infection were mixed,and then subjected to RNA-Seq to compare the transcriptomes of midguts between theBmCPV-infected and non-infected silkworm (lan5). After finishing analysis of GO andKEGG of the differently expressed genes (DEGs),3up-regulated random genes and3down-regulated random genes among the all DEGs were verified by real-time PCR tocheck the consistency of the results carried out by high-throughput sequencing. Themain results are as follows:1.10812expresed genes were detected in the midgut of normal lan5. Compared with the expression level of its actin A3, there are66high expression genes (Foldchange>2). The productions of these genes are mainly involved in digestion, absorption,protein biosynthesis and innate immunity.2.11259expresed genes were detected in the midgut of infected lan5. There were387differently expressed genes, which meted obvious fold change, only expressing inone sample of lan5or lan5-cpv.264genes were up-regulated,123genes weredown-regulated. However,90DEGs were detected, which meted T-test, expressing inboth samples of lan5and lan5-cpv.66genes were up-regulated,24genes weredown-regulated.3. The results of GO analysis showed that the up-regulated genes were mainlyinvolved in binding, metabolic processes, Cellular processes, and the correspondinggenes were ATP-dependent helicase DDX18, DNA cytosine-5methyltransferase,neuropeptide receptor A33. And the down-regulated genes were mainly involved incatalytic activity, binding, metabolic processes, and the corresponding genes werepeptidoglycan-recognition protein LB, sorting nexin-8, cysteine dioxygenase type1.4.The analysis of KEGG signal pathway showed that after infection thedown-regulated genes involved protein digestion and absorption, Carbohydratemetabolism, such as serine proteinase, ethanol dehydrogenase; and the up-regulatedgenes were mainly involved in signal transduction pathway, such as MAPK signalpathway, Wnt signal pathway, Ca2+signal pathway, and pathways about transport，decomposition， such as endocytosis, lysosome. The corresponding genes werevoltage-dependent calcium channel type D, disheveled-associated activator,Ca2+/calmodulin-responsive adenylate cyclase, partitioning defective protein6, CD63antigen. In addition, the host cells may start apoptosis program to defense theproliferation of BmCPV, and its genes involved in immunity were up-regulated, such asserine protease inhibitors, lipase, cuticular proteins, peritrophic membrane chitinbinding protein, cytochrome P450, DAAM1.5.6genes (Osiris9, unknown protein1(367), unknown protein2(loc),27kDglycoprotein precursor, Esterase B1, peptidoglycan recognition protein S6) wererandomly selected to detect the changes of expression level before and after virusinfection by qRT-PCR. The result showed that expression level of Osiris9, unknownprotein1(367), unknown protein2(loc) were up-regulated by times of16.34,19.42, 41.93, and27kD glycoprotein precursor, Esterase B1, peptidoglycan recognition protein S6were down-regulated by times of36.25,15.1,66.71, respectively. The results were inline with that of high-throughput sequencing, indicating that the results ofhigh-throughput sequencing were credible.These results fully revealed the response of silkworm to BmCPV infection, andoffered new clues to understand interactions between silkworm and BmCPV.Additionally, bioinformatic analysis revealed that there was a hypothesizedoverlapping gene (ORFX) in Segment Ⅰ of the genome of Bombyx mori cypovirus(BmCPV). To confirm the hypothesis in RNA and protein level, we structured arecombinant prokaryotic expression vector containing the ORFX to prepare polyclonalantibody. Then the putative ORFX protein was checked in the posterior midgut of theinfected silkworm by western blotting and immunohistochemistry. However, theputative protein was not detected in the midgut at1-7days post-infection. Furthermore,a pairs of primers (RECPV-1/RECPV-2) to quantitate total transcriptional level ofORFX and Segment Ⅰ, and another primers (RECPV-3/RECPV-4) to quantitate thetranscriptional level of Segment Ⅰ were respectively designed based on the ORFXsequence and its downstream sequence. The relative transcriptional level of ORFX andSegment Ⅰ to actin3were estimated by qPCR with the two pairs of primers in theBmCPV-infected midgut using two types of cDNA templates which were prepared byreverse transcription with primers of Oligo (dT)/RECPV-2and Oligo (dT)/RECPV-4respectively. The results showed that the ORFX transcript was not detected in themidgut at5-7days post-infection. The ORFX was detected neither in RNA level, nor inprotein level suggesting that there might not be an overlapping gene in genomicsegment I of BmCPV. |
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