Studies On Differentially Expressed Genes In The Silkworm Infected With Cytoplasmic Polyhedrosis Virus

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), one of the major viral pathogens for the silkworm, causes enormous damages to the sericultural industry. Studies on genes related to silkworm infected with BmCPV and understanding of the host response to Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) infection is crucial to the diagnosis of BmCPV-caused silkworm disease and the development of new control measures.Suppression subtractive hybridization method was employed to construct forward and reverse subtractive libraries from the midguts of BmCPV-infected and normal silkworm larvae. Total of 36 genes and 20 novel ESTs were identified from two reciprocal subtractive libraries. The relative transcript level of three up-regulated genes (ferritin, rpL11 and alkaline nuclease) and three down-regulated genes (serine protease, trypsin-like protease and inhibitor of apoptosis protein) at 6, 12, 24, 48 and 72h post-inoculation were analyzed by quantitative real-time PCR.Microarray analysis was employed to compare differentially expressed genes in the midguts of BmCPV-infected and normal silkworm larvae. At 24h, 48h and 72h post-inoculation, 9, 51 and 258 differentially expressed genes were identified respectively. KEGG pathways analysis indicated that at 48h and 72h post-inoculation, most genes related to metabolism pathways were down-regulated while expressions of genes involved in ribosome and proteasome pathway were all up-regulated. The expressions of several immune-related genes including serpin5, lipase, heat shock proteins, cytochrome P450s and ribosomal P0 protein were up-regulated. The expressions of genes grouped into oxidoreductases activity were all down-regulated.After the invasion of BmCPV, the function of digestion and absorption of midguts was damaged. This resulted in the decrease of the protein and amino acid metabolism in the silkworm. A large number of genes used for translation were over-expressed to meet the needs of virus replication. On the other hand, the host cells may startup apoptosis program to defense the proliferation of BmCPV. Invasion of BmCPV also induced the expressions of several immune-related genes.We firstly cloned the full length cDNA which encodes the ubiquitin-activating enzyme E1-domain containing 1 (UbE1DC1) and a hypothetic protein gene of the silkworm by using rapid amplification of cDNA ends (RACE). The full-length cDNA of UbE1DC1 gene is 1919 bp, consisting of a 100bp 5'untranslated region, a 637 bp 3'untranslated region and a 1182 bp open reading frame (ORF), encoding 393 amino acids. The protein contained the THiF_MoeB_hesA_family domain, an ATP binding site, which is belonged to the family of ubiquitin-activating enzyme E1. The full-length cDNA of hypothetic protein gene is 486 bp, consisting of a 108bp 5'untranslated region, a 153bp 3'untranslated region and a 225bp open reading frame (ORF), encoding 74 amino acids. RT-PCR analysis of the silkworm tissues silk gland, hemocyte, fat body, gonad and midgut revealed that the UbE1DC1 and hypothetic gene were expressed in all the five tissues. The quantitative real-time polymerase chain reaction analysis indicated that the relative expression of UbE1DC1 in the normal midgut was approximately as 9.78 fold of that in the BmCPV-infected midgut. It is implicated that the down-regulation of UbE1DC1 could prevent the infected cells from apoptosis. The relative transcript of hypothetic protein gene in the infected midgut was 6.28 fold than that in the normal midgut. The mechanism of its up-regulation is not clear.Our results provided not only new clues for investigating the molecular mechanism of BmCPV infection but also the theoretical basis for the furthe'[r study on the function of UbE1DC1 and hypothetic protein gene of Bombyx mori.