Transcriptome Sequencing Of Antheraea Pernyi Midgut Infected With ApNPV And Related Immune Genes Research

The Chinese oak silkworm(Antheraea pernyi Guerin-Meneville,1855) which belongs to Lepidoptera:Saturniidae, is the most well-known wild silkworm for insect food and silk production with a long history of domestication. Owing to its breeding is in the wild, the larval stage would be affected by several factors, the food value of leaves, climatic factors and variety pathogens in the external environment may lead to occurrence of disease and cause huge economic loses to breeders. A. pernyi nucleopolyhedro-virus (ApNPV) is a major disease with high incidence and being infectious strongly, which pathogeny was proved as nucleopolyhedro-virus. ApNPV disease has the most extensive distribution. It can also cause considerable economic losses for breeders.ApNPV infection depends on their phenotypic differences and connected with the ability of host. Currently, the study of the ApNPV mainly focused on the pathogenesis of viral infections, which took advantages of intercellular environment of the host to assemble their own copy and released into the extracellular. A. pernyi belong to Invertabrate, they have immunologic barrier encounter with virus infection. The host initiates the immune mechanism in order to restrain the virus invasion and replication. The gene expressional levels will reflect the invasion at the beginning. Researching the NPV-infected gene expression will understand the characteristic of infection through molecular level. Studying the interaction between ApNPV and A. pernyi can provide the regulation modes of immune system. It will provide the basic foundation of molecular assistant disease-resistant breeding. The results are as follows:1. To explore the molecular basis of nucleopolyhedro-virus resistance in the Chinese oak silkworm, transcriptome analyses were carried out to evaluate the difference between virus-infected groups and the control groups. ApNPV (4.05×106/mL) was employed to infect the Chinese oak silkworm in 3rd instar, dissecting the midgut when the nucleopolyhedro-virus can be observed under the microscope. Three biological replicates for each sample were prepared for the transcriptome sequencing. The results of transcriptome analysis showed that compared with the controls a total number of 5,172 differentially expressed genes (DEGs), including 2,183 up-regulated and 2,989 down-regulated candidates were identified, of which 2,965 and 911 DEGs could be classified into GO categories, KEGG pathways by GO and KEGG analyses, respectively.We focused our attention on the DEGs that are involved molecular functions and biological process. Our analyses suggested these genes were related to enzyme activity, indicated that the enzyme system plays an important role in the anti-virus process of Chinese oak silkworm. We classified the DEGs involved in anti-virus immune system of A. pernyi into five categories:heat shock proteins, cytological apoptosis related genes, serine protease inhibitors, serine proteases and cytochrome P450. The transcriptome results will provide a theoretical basis to further research in the mechanism of the host immune response to virus infection.2. Cloning and expressional analysis of A. pernyi GTPase geneBased on the transcriptome data of ApNPV-infected migut contructed in our laboratory, genes encoding GTPase are cloned. The semi-quantitative PCR was used to analyze the profile in different development stages and tissues, and the Real-time PCR was employed to analyze the relative expression levels induced by pathogen stimulus over periods of time. The results are as follows:Sequence of ApGTPase (GenBank accessions numbers:KR821068.1) was cloned with 1194 bp open reading frame encoding 397 amino acids. The predicted molecular weight and isoelectric point of this protein was 44.6 kDa and 6.64, respectively. Using Blastp analysis, the amino acid sequence of ApGTPase shared 95% identity with Bombyx mori, the homology of other species ranged from 93% to 78%. Prokaryotic expression vector of ApGTPase was constructed and expressed in vitro by using E. coli BL21 cells. SDS-PAGE showed that target gene had been successfully expressed in the expression systems. Semi-quantitative PCR detection revealed that ApGTPase is expressed in all stages and tissues of 5th larve. The relative expressional levels of the ApGTPase gene in the midgut were increased. It indicated that ApGTPase gene was related with the immunity of A. pernyi to pathogens.3. Cloning and expressional analysis of A. pernyi lipase geneUsing RT-PCR technology, a lipase gene of A. pernyi was cloned named Aplipase (GenBank accession numbers:KR821071). The ORF is 1527 bp and encodes 508 amino acids with predicted molecular mass of 57.9 kDa and theoretical isoelectric point of 6.32. Using BLASTp analysis, the amino acid sequence of Aplipse shared 79% identity with B. mori, and the homology of Danaus plexippus and Papilio xuthus ranged from 50% to 60%. Semi-quantitative PCR detection indicated that Aplipase is mainly expressed in all stages and tissue of fatbody in 5th instar. Real-time PCR results suggested that relative expressional level of Aplipase in fatbody was increased at 48 h after ApNPV and Beauveria bassiana infection.The above researching results have provided the theoretical basis for understanding molecular changes of A. pernyi after ApNPV infection and the host-NPV interaction. It has significant meaning of study the different immunologic mechanism of A. pernyi infected with diverse pathogens. Detection the expression levels of immune-related genes can provide theoretical guidance for resistant varieties'selection.