| Bombyx mori, has became an important lepidopteran insect with economic value through a long period of artificial domestication and breeding. Sericulture is a traditional industry in China and occupies the leading position in the world, but silkworm diseases have occurred in every sericulture area and caused the reduction in production of silk cocoons. B. mori cytoplasmic polyhedrosis virus (BmCPV) is one of the main pathogens, which often causes huge economic loss in sericulture. Currently, the primary method to keep BmCPV from silkworm is comprehensive prevention and there is no effective measures to deal with the threat of diseases unless we cultivate excellent resistance silkworm strains. In this study, the midgut and fat body of silkworm were extracted at 0,3,24, and 72 h after oral infection of BmCPV for RNA-seq. We focused on analysis of the factors that may affect BmCPV only infection of midgut and the expression pattern of viral genes, screened and preliminary identified the candidate genes that may resist to BmCPV. Finally we acquired transgenic silkworm which has the ability to resist to BmCPV via RNA interfering multiple viral genes.1. Analysis of interactions between BmCPV and silkworm through RNA-seqThe midguts and fat bodies of Dz strain which oral infected with BmCPV were collected at 0,3,24, and 72 hpi (hours post-infection), and total RNA were extracted. 24 cDNA libraries of 8 tissue samples were successfully constructed for RNA-seq, and the sequencing quality of the 24 cDNA were good, the mapping rate of each sample was above 95.3%.Bioinformatics analysis of the transcriptome data showed that the up-and down-regulated transcripts of fat body were more than that of midgut at each infection time point. One transcripts was co-upregulated and five transcripts were co-downregulated at all time point of the two tissues. And there were 100,126, and 200 transcripts only up-regulated at 3,24, and 72 hpi of midgut, respectively.GO function analysis of the differentially expressed transcripts found that the transcripts of antioxidant were up-regulated at 3,24, and 72 hpi in fat body. Then 19 transcripts with antioxidant function were screened, which belonged to BGIBMGA007453, BGIBMGA008203, BGIBMGA009576, BGIBMGA014087 and BGIBMGA000903. Bioinformatics analysis showed that 6 midgut specific genes with transmembrane transport function were induced by BmCPV, including BGIBMGA001299, BGIBMGA014248, BGIBMGA014434, BGIBMGA004804, BGIBMGA009201 and BGIBMGA012068. Some midgut specific genes may participate in defensing to BmCPV, they were BGIBMGA008167, BGIBMGA009461, BGIBMGA012452, BGIBMGA013275 and BGIBMGA007981. Sequence alignment showed that BGIBMGA008167 and BGIBMGA012452 encoded serine protease (SP), BGIBMGA009461 encoded TNF receptor-associated factor 1, BGIBMGA013275 encoded Zinc carboxypeptidase A 1, and BGIBMGA007987 encoded peptidoglycan recognition protein S2.In addition actin was induced by BmCPV, and sustained high level in midgut. Analysis of the expression pattern of BmCPV genes showed that S1,S2, S3,S4, S5, S6, S7, S8, and S10 but not S9 expressed in midgut 72 hpi, and the expression level of SI, S2, S3, S6, and S7 were very high.2. Preliminary identification of the candidate genes for resistance to BmCPV infectionRT-PCR results showed that the 5 genes with antioxidant function all expressed in entire midgut, fat body and silk gland. qPCR analysis revealed that BGIBMGA007453 and BGIBMGA008203 were induced by BmCPV in fat body, BGIBMGA014087 was induced in fat body and midgut, while BGIBMGA000903 was induced only in midgut.BGIBMGA001299, BGIBMGA014434, BGIBMGA004804 and BGIBMGA012068 were specifically expressed in entire midgut, BGIBMGA009201 only expressed in the posterior portion of midgut. BGIBMGA001299 expressed at all developmental stages of silkworm, while BGIBMGA014434 just didn’t expressed at egg-4th-d, BGIBMGA012068 and BGIBMGA009201 expressed at the developmental stages of larva, and the expression level of BGIBMGA009201 of each newly molted larva was always higher than that of moulting larva. qPCR revealed that BGIBMGA001299, BGIBMGA014434, BGIBMGA009201, and BGIBMGA012068 were induced in midgut by BmCPV.Analysis of the midgut-specific genes related with immune defence function by RT-PCR showed that BGIBMGA008167, BGIBMGA009461, BGIBMGA012452 and BGIBMGA013275 expressed in entire midgut, while BGIBMGAA007987 mainly expressed in midgut and it had also a weak expression in the silk gland. qPCR showed that BGIBMGA012452, BGIBMGA013275 and BGIBMGA007987 were up-regulated in midgut at 24 and 72 hpi, and BGIBMGA008167 was induced by BmCPV in midgut at 3 and 24 hpi.The expression of different BmAPNs subtype were detected by qPCR, the resulted showed that BmAPN1, BmAPN4 and BmAPN9 were up-regulated in midgut at 3,24, and 72 hpi. BmAPN6 was up-regulated in midgut at 3 and 24 hpi, while BmAPN7 was down-regulated at 3 hpi.3. Enhancing resistance of transgenic silkworm via RNA interfering of BmCPV genesRNAi is a widely means for antiviral, in this study, two transgenic RNAi vectors were successfully constructed, one was IE1P combined with Hr3 co-silencing the unstructured protein genes of BmCPV, including S5, S8, S9 and S10 (pb-IElP-FJG). Another was IE IP combined with Hr3 co-silencing the structured protein genes with SI, S2, S4 and the unstructured protein genes with S5, S8 (pb-IE1P-CHH). Then 2 transgentic lines with FJG-1 and FJG-2 of pb-IE1P-FJG and 2 transgenic lines with CHH-1 and CHH-2 of pb-IE1P-CHH were obtained through the embryo microinjection. Reverse PCR results showed that the exogenous fragments of 4 transgentic lines all inserted into the silkworm genome.After oral infection of BmCPV with 1×104 polyhedra per larva using third instar larvae, the mortality of FJG-1, FJG-2, CHH-1, CHH-2 and non-transgentic silkworm was 46.84%,89.90%,64.70%,43.80%, and 83.36%, respectively. The expression of viral genes were detected at 96 hpi, and the relative expression level of S1, S2, S4, S5, S8, and S10 in WT was 0.0826,0.0135,0.0138,0.0136,0.1687, and 0.0117, respectively. While only FJG-2 transgenic line was detected the expression of S8 and S10. Besides there was no significant difference in the whole cocoon weights and cocoon shell rates between transgenic lines and non-transgenic line. All results showed that it would improve the resistance but won’t affect the economic characteristics of transgenic silkworms by silencing BmCPV genes. |
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